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REvIEWS 
Approaches to treat immune hot, altered and cold tumours with combination immunotherapies
Jérôme Galon 
* and Daniela Bruni
Abstract | Immunotherapies are the most rapidly growing drug class and have a major impact in oncology and on human health. It is increasingly clear that the effectiveness of immunomodulatory strategies depends on the presence of a baseline immune response and on unleashing
of pre-existing immunity. Therefore, a general consensus emerged on the central part played by effector T cells in the antitumour responses. Recent technological, analytical and mechanistic advances in immunology have enabled the identification of patients who are more likely to respond to immunotherapy. In this Review, we focus on defining hot, altered and cold tumours, the complexity of the tumour microenvironment, the Immunoscore and immune contexture of tumours, and we describe approaches to treat such tumours with combination immunotherapies, including checkpoint inhibitors. In the upcoming era of combination immunotherapy, it is becoming critical to understand the mechanisms responsible for hot, altered or cold immune tumours in order to boost a weak antitumour immunity. The impact of combination therapy on the immune response to convert an immune cold into a hot tumour will be discussed.
TNM system
The tumour-node-metastasis (TNM) staging system is
a globally recognized classification of tumours based on their anatomical extent.
T refers to the size and extent of the primary tumour, N refers to the involvement of regional lymph nodes and M describes the presence of distant metastases.
INSERM, Laboratory of Integrative Cancer Immunology, Sorbonne Université, Sorbonne Paris Cité, Université Paris Descartes, Université Paris Diderot, Centre de Recherche des Cordeliers, Paris, France.
*e-mail: jerome.galon@ crc.jussieu.fr
https://doi.org/10.1038/ s41573-018-0007-y
The remarkable results achieved in the past few years with the advent of cancer immunotherapies and check-point inhibitors have revolutionized the field of oncology by putting the host immune response under the spotlight as a target for anticancer therapeutic interventions. The founding principles of the immunotherapy of cancer are threefold: first, the demonstration of immuno- surveillance using immune -deficient mouse models1,2; second, the demonstration of the major importance of pre-existing immunity and natural intratumoural T cells in humans3; and third, the unleashing of pre-existing immunity via inhibition of checkpoint receptors on
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cells in human cancers4–8. Insight knowledge of the basic mechanisms responsible for the establishment and development of tumours, on the basis of their interac-tion with and control of the host immune system, has enabled us to draw a more comprehensive picture of the possible points of intervention and has provided us with reasons that might account for therapeutic failure. An update in the current guidelines for tumour classifica-tion and subsequent treatment is therefore becoming a pressing necessity. The recent yet unofficial classifi-cation of tumours into two categories, ‘hot’ and ‘cold’, has been increasingly advocated. In this Review, we aim to suggest a more comprehensive main four-category classification of tumours — hot, altered-excluded,
altered-immunosuppressed and cold — and to provide an overview of both current and potential therapeutic strategies to best target these four categories of tumour, which in most cases involve combinatorial immunother-apy strategies. We believe that a rational, standardized and harmonized approach embracing the central role of the immune system must be adopted to guide ther-apeutic decisions. This adoption will require a general consensus and a large collective effort, as anything of great value.
Definition of hot and cold tumours
Current knowledge of the tumour–immune system interaction has already set the foundations for a rationally guided stratification of patients and ther-apeutic strategies. A powerful concept for patient stratification came with the observation that the type, density and location of immune cells within the tumour site could predict survival in colorectal cancer (CRC) more accurately than the classical TNM system for the first time in any type of cancer9. This concept led to the development and implementation of the Immunoscore3,10–13 — a robust, consensus, standard-ized scoring system based on the quantification of two lymphocyte populations (CD3 and CD8) both at the tumour centre and the invasive margin14,15.
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Pathologic T (pT) stage
The staging assigned post- surgery to guide treatment stratification, patient selection for clinical trials and prognosis prediction (as opposed to clinical staging that relies
on physical exams and imaging tests).
The Immunoscore ranges from Immunoscore 0 (I0, for low densities, such as absence of both cell types in both regions) to I4 (high immune cell densities in both locations). By classifying cancers according to their immune infiltration, the system proposed for the first time an immune-based, rather than a cancer- based, classification of tumours3, de facto introducing the notion of ‘hot’ (highly infiltrated, Immunoscore I4) and ‘cold’ (non-infiltrated, Immunoscore I0) tumours (Fig. 1). Tumour progression (T stage) and inva-sion (N stage) were dependent on the pre-existing adaptive intratumour immunity3,9. The consensus Immunoscore has been validated globally in colon cancer and has a greater relative prognostic value than pathologic T (pT) stage, pN stage, lymphovascular invasion, tumour differentiation and microsatellite instability (MSI) status16.
On the basis of these findings, the novel concept of ‘immune contexture’ was proposed13 and adopted to refer to the combination of immune variables associat-ing the nature, density, immune functional orientation and distribution of immune cells within the tumour13. These immune contexture parameters are associated with long-term survival and prediction of response to treatments10. In 2009, Camus et al. first described three major immune coordination profiles (hot, altered and cold) observed within primary CRCs, which enabled classification according to the balance between tumour escape and immune coordination17. The 2-year risk of relapse for these three types of tumour was 10%, 50% and 80%, respectively. The altered phenotype was fur-ther divided into two distinct patterns — ‘excluded’ and ‘immunosuppressed’17. In some cases, T cells are found at the edge of tumour sites (invasive margin) without being able to infiltrate them. This ‘excluded’ phenotype reflects the intrinsic ability of the host immune system to effectively mount a T cell-mediated immune response and the ability of the tumour to escape such response by physically hindering T cell infiltration (Fig. 1a). In other cases, tumour sites display a low degree of immune infiltration (Fig. 1a), which suggests the absence of physical barriers and the presence of an immuno suppressive environment that limits further recruitment17 and expansion18; this can be defined as an ‘immuno suppressed’ phenotype17. By more suitably simplifying the complexity of the tumour phenotype spectrum, these four characteristic subgroups can potentially represent a practical tool to direct therapeutic intervention (Box 1; Fig. 1b). Immunoscore proved to be a better prognostic tool for patients with CRC than MSI19, which is currently tested to predict the response of these patients to anti- programmed cell death protein 1 (PD-1) therapy20. A worldwide consensus Immunoscore study validated the prediction of risk of recurrence and survival on the basis of the three main subtypes of tumour — the immune hot, altered and cold tumours15,16.
Currently, the terms ‘hot’ and ‘cold’ are routinely used to refer to T cell-infiltrated, inflamed but non-infiltrated, and non-inflamed tumours, reflecting well the higher (I4) and lower (I0) Immunoscore categories3 (Fig.1). This immune classification has been validated in other can-cer types such as melanoma21. Apart from the presence
of tumour-infiltrating lymphocytes (TILs), additional features such as the expression of anti-programmed death-ligand 1 (PD-L1) on tumour-associated immune cells, possible genomic instability and the presence of a pre-existing antitumour immune response have been described as characteristics of hot tumours22. Conversely, apart from being poorly infiltrated, cold tumours have also been described to be immunologically ignorant (scarcely expressing PD-L1) and characterized by high proliferation with low mutational burden (low expres-sion of neoantigens) and low expression of antigen presentation machinery markers such as major histo-compatibility complex class I (MHC I)22. In an attempt to propose a more simplistic yet comprehensive classifica-tion, the four proposed types of tumour (hot, excluded, immunosuppressed and cold), based on Immunoscore, could be the first routine immune parameters to eval-uate at the time of diagnosis. Recently, a novel theory of cancer evolution at the metastatic stage was demon-strated and highlighted a model of tumour evolution in which a parallel immune selection exists, with a major role of Immunoscore and T cells in this process23. The fact that T cells are currently widely recognized as the key fighters in the antitumour battle makes the use of the Immunoscore an attractive option to help in guid-ing treatment selection. Of course, this option does not exclude the possible use of additional parameters, which in fact are required to gain further insights into the spe-cifics of each case, but its routine incorporation into clin-ical practice could definitely relieve the existing, urging need for therapeutic guidance. The limitations associ-ated with the current techniques for immune monitoring are discussed in Box 2.
Beyond hot, altered and cold tumours
The distinction between hot, altered (excluded and immunosuppressed) and cold tumours is based on the cytotoxic T cell landscape within a tumour. This power-ful simplification reflects the outcome of a tremendously complex interplay between the tumour and the immune system. For instance, apart from T cells, high expression of markers of B and follicular helper T (TFH) cells was also correlated with a significantly prolonged disease- free survival time in patients with CRCs24. By secreting
CXC-chemokine ligand 13 (CXCL13), TFH cells activate a positive loop that involves increased densities of B, TFH, T helper 1 (TH1), cytotoxic and memory T cells in breast cancer25 and CRC24. Acquisition of a memory phenotype by these adaptive immune cells long term can extend the survival of patients24.
The immune functional orientation associated with the immune contexture was first described in CRC and showed the major importance of T cells (TH1 and cyto-toxic T cells) and associated factors (interferon-γ (IFNγ), granulysin (GNLY), perforin (PRF1) and granzymes (GZMs)3). These immune signatures were associated with prolonged survival and validated in other cancer types26–31. A similar T cell-inflamed gene expression pro-file exhibited predictive utility in identifying responders to treatment with an anti-PD-1 antibody32. These obser-vations support the notion that these signatures are both prognostic and predictive10.
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a
Optimal: high Immunoscore (inflamed, hot)
Absent: low Immunoscore (non-inflamed, cold)ing ‘hot’, ‘altered’ and ‘cold’ immune tumours — immunoscore as a new approach for the classification
of cancer. a | Illustrative example of hot, altered and cold immune tumours. Brown (3,3ʹ-diaminobenzidine (DAB)) staining represents CD3+ T cells and blue (alkaline phosphatase) counterstaining provides homogeneous tissue background staining. The level and spatial distribution of CD3+ and CD8+ T cell infiltration differentiates four distinct solid tumour
phenotypes: hot (or inflamed); altered, which can be excluded or immunosuppressed; and cold (or non-inflamed). These tumour phenotypes are characterized by high, intermediate and low Immunoscore, respectively. b | Schematic representation of the four subtypes of immune tumour. Of note, in altered-excluded tumours, CD3+ and CD8+ T cell infiltrates are low at the tumour centre and high at the invasive margin, resulting overall in an intermediate Immunoscore. Altered-immunosuppressed tumours display instead a more uniform pattern of (low) CD3+ and CD8+ T cell infiltration. CT, centre of tumour; Hi, high; IM, invasive margin; Lo, low.
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Activated lymphocytes, such as cytotoxic T cells, are
and it may exert pro-apoptotic and anti-proliferative
among the main sources of IFNγ. By increasing MHC I
effects via induction of several interferon-stimulated
and immunoproteasome expression in tumour cells, IFNγ
genes (ISGs)33,34, although these effects seem to be dose-
enhances antigen presentation and subsequent immuno-
dependent35. It should be noted that IFNγ can exert
surveillance33. IFNγ also attenuates cancer cell growth,
both antitumour and pro-tumour functions, depending
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Box 1 | T cell-based classification of tumours
characteristics of hot immune tumours
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High degree of T cell and cytotoxic T cell infiltration (high Immunoscore)
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Checkpoint activation (programmed cell death protein 1 (PD-1), cytotoxic T
lymphocyte-associated antigen 4 (CTLA4), T cell immunoglobulin mucin receptor 3 (TIM3) and lymphocyte activation gene 3 (LAG3)) or otherwise impaired T cell
functions (for example, extracellular potassium-driven T cell suppression)
Characteristics of altered-immunosuppressed immune tumours
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Poor, albeit not absent, T cell and cytotoxic T cell infiltration (intermediate Immunoscore)
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Presence of soluble inhibitory mediators (transforming growth factor-β (TGFβ), interleukin 10 (IL-10) and vascular endothelial growth factor (VEGF))
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Presence of immune suppressive cells (myeloid-derived suppressor cells and regulatory T cells)
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Presence of T cell checkpoints (PD-1, CTLA4, TIM3 and LAG3)
Characteristics of altered-excluded immune tumours
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No T cell infiltration inside the tumour bed; accumulation of T cells at tumour borders (invasive margin) (intermediate Immunoscore)
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Activation of oncogenic pathways
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Epigenetic regulation and reprogramming of the tumour microenvironment
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Aberrant tumour vasculature and/or stroma
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Hypoxia
characteristics of cold immune tumours
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Absence of T cells within the tumour and at the tumour edges (low Immunoscore)
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Failed T cell priming (low tumour mutational burden, poor antigen presentation and intrinsic insensitivity to T cell killing)
excluded or immunosuppressed) tumour. In turn, the local adaptive immunosuppression is triggered by tumour- specific T cells that are infiltrated in combinations being tested in cold immune tumou tumours. By producing IFNγ, TILs can induce immune checkpoint molecules (such as PD-L1) or immunosuppressive factors such as indoleamine-pyrrole 2,3-dioxygenase 1 (IDO1). Thus, local adaptive immunosuppression can generate hot or immunosuppressed tumours, depending on the power of the driving mechanisms.
Great diversity in the TME composition exists across different cancer types but also among patients with the same cancer and even in different tumour sites within the same patient50,51. This diversity results from the com-bination of a multitude of factors, including the occur-rence of specific driver mutations and deregulation of oncogenes in cancer cells44,52; the load and quality of passenger mutations in cancer cells19,53 ; the presence of immunosuppressive components in the TME, whether soluble (such as transforming growth factor-β (TGFβ)54) or cell-associated (such as PD-1 and/or PD-L1 (ref.55)); the presence of factors directing immune attraction24,56,57; and the presence of factors (such as interleukin 15 (IL-15)) that mediate the expansion and proliferation of cytotoxic CD8+ T cells18. This complex scenario is further complicated by the fact that the TME evolves with disease progression and recurrence24. Future stud-ies will have to reveal advanced biomarkers to groups of patients who display similar features, thereby creating
Immunoproteasome
Proteasome isoform constitutively expressed in haematopoietic cells and
induced in non-immune cells following exposure to interferon-γ (IFNγ) and other pro-inflammatory cytokines (type I interferons and tumour necrosis factor (TNF)). It is involved in antigen processing and in the expansion, maintenance and differentiation of T cell populations during an immune response.
T cell receptor (TCR)
repertoire
The variety of the TCR diversity, as generated by the somatic recombination of the germ line V, D and J gene segments and the deletion and insertion of nucleotides at the V(D)J junctions. Such variety is required to recognize a wide spectrum of antigens.
TCR Vß subfamilies
Human TCR ß locus is on chromosome 7, comprising nine multimember V subfamilies plus additional elements on chromosome 9. The presence of multiple subfamilies is due to evolutionary duplication events.
on the cellular, microenvironmental and/or molecu-lar context35–37. Furthermore, prolonged IFNγ signal-ling and enduring antigen exposure may also have an immunosuppressive role38,39, leading to T cell exhaustion and resistance to immune checkpoint blockade39.
CD4+ T cells also contribute to antitumour immu-nity. By expressing the transcription factor T-bet (also known as T-box transcription factor TBX21) and pro-ducing IFNγ, TH1 cells hinder neo-angiogenesis by inhibiting vascular endothelial growth factor (VEGF)-producing tumour-associated macrophages40 and pro-mote the recruitment of CD8+ T cells41. A fine regulation of the genetic and epigenetic networks controlling TH1 responses is required for optimal T cell functioning42.
A series of immunosuppressive mechanisms also occur in the tumour microenvironment (TME), which hinder not only the natural host immune responses but also the efficacy of cancer immunotherapies. Two types of immunosuppression mechanism operate in the TME — a tumour-intrinsic and a local adaptive immunosuppression43. The former may be induced by genetic alterations of the tumour and involves the acti-vation of various oncogenic pathways, including the WNT–β-catenin44,45 , mitogen-activated protein kinase (MAPK)46,47, Janus kinase (JAK)–signal transducer and activator of transcription 3 (STAT3)48 and nuclear factor-κB (NF-κB)49 signalling pathways. The engagement of these pathways results in the expression of cytokines and chemokines that ultimately mediate the exclusion of T cells from the TME43, or, alternatively, the repression of factors that facilitate T cell recruitment44. Depending on the specifics of the case, the tumour-intrinsic immuno suppression can result in a cold or an altered (either
specific models that can guide the choice of therapeutic regimen as well as therapeutic development. Some of the challenges associated with the approaches used to characterize the TME are discussed in Box 2.
Unleashing the pre-existing immunity
Effectiveness of immunomodulatory strategies depends on the presence of a baseline antitumour immune response, involving either tumour-associated6,7,10,58–62 or circulating immune components8. Clinical response of human melanoma following treatment with mono-clonal antibody (mAb) anti-PD-1 was dependent on immune reinvigoration of circulating exhausted CD8+
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cells (implying the pre-existence of an antitumour immune response) and pretreatment tumour burden8. This finding shows that even when in peripheral blood, pre-existing antitumour T cells can predict response. Immune checkpoint blockade inhibitors (ICIs) may trigger changes in the T cell receptor (TCR) repertoire and the expansion of specific clones of tumour-reactive T cells63. In patients with melanoma who responded to anti-PD-1 treatment, the comparison of the TCR reper-toire before and after treatment revealed the emergence of TCR Vß subfamilies — which specifically recognize the melanoma antigen recognized by T cells 1 (MART1; also known as Melan-A) — that were undetectable before treatment64. This emergence could be due to an insufficient sensitivity to detect lowly expressed clones or to a de novo second wave of immune activation, with subsequent emergence of mutant neo-epitope-tar-geting T cells65. Preclinical studies demonstrated that PD-1 blockade cannot unleash antitumour T cell responses in the absence of fully primed and committed
7,72
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Box 2 | challenges of immune monitoring
A characterization of hot, altered and cold tumours should be ideally carried out on resected tumours (primary or metastatic). However, often only biopsy specimens are available, thereby limiting the accuracy of the measurement. Although biopsy samples have been extremely valuable in providing information on the disease, they have several limitations, including being invasive, not always feasible and potentially not representative of the whole tumoural landscape. Major intermetastasis genomic and immune heterogeneity exists so that each metastasis could be considered as a separate tumour50. Furthermore, immune parameters change in the course of the disease24. Enormous effort has been put into the development of less invasive and equally informative strategies, such as liquid biopsy270. However, blood evaluation is a poor surrogate of what happens in the tumour microenvironment (TME), where ultimately the main informative and prognostic features lie. A viable alternative to biopsy has yet to be determined. Less invasive diagnostic procedures, such as immuno- positron-emission tomography (PET) imaging detecting intratumoural CD8 T cells271,
are quite promising. Apart from immunohistochemistry-based assays, current methods to characterize the immune landscape of the TME rely mostly on bulk gene expression profiling272–274. Bindea et al. first demonstrated the possibility to infer immune cell infiltration from gene expression profiles from a complex mixture of cells, revealing the immune landscape in human tumours24. Subsequently, multiple additional tools, such as CIBERSORT (which infers the relative fractions of immune subsets in the total leukocyte population)273,275, xCell (which predicts the abundance of immune cells in the overall TME)276, TIMER (which generates enrichment scores on the basis of proportions among 64 immune and stromal cell types)277 and integrated immunogenomics methods (using a CIBERSORT-based approach, which, of note, identified six immune subtypes of cancer)278 were developed to estimate the abundance of intratumoural immune infiltrates by using deconvolution of bulk gene expression data. Evident limitations of these techniques are sample variability, inconsistency in the RNA extraction step, the impossibility of univocally assigning transcripts to specific cell types and differences between the immune phenotypes from blood and TMEs belonging to distinct cancer types. Novel techniques based on single-cell approaches279 or in situ barcode sequencing280 are costly and far from large-scale diagnostic use.
antigen-specific Tcells 66, and the presence of function-ally impaired PD-1high tumour antigen-specific CD8+
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cells infiltrating the tumour has been described58. Furthermore, TCR β chain (TCRβ) deep sequencing revealed that clonally expanded tumour-reactive CD8+ TILs express PD-1 (ref.59). Altogether, this evidence strongly supports the existence of an in situ and/or peripheral antitumour immunity that confers clinical efficacy to subsequent checkpoint blockade.
There seems to be a general consensus on the cen-tral part played by effector T cells in the antitumour responses67. Upon recognition of antigenic peptides presented on the surface of cancer cells by MHC I– β2-microglobulin (β2m) complexes, CD8+ T cells kill target cells mainly by releasing cytotoxic factors such as PRF1, GNLY and GZM68. TBX21, STAT1, STAT4 and interferon regulatory factor 1 (IRF1) are among the main signalling molecules and transcription factors that regulate the production of these mediators that result ultimately in tumour rejection68,69. High T cell infil-trates in primary CRCs are associated with decreased metastatic invasion and increased overall survival70,71, and T cells are crucial for the clinical benefit of current immunotherapies. For example, the presence of CD8+ T cells at the tumour invasive margin is a prerequisite for the therapeutic success of PD-1 blockade in metastatic melanoma6, and proliferation of said CD8+ T cells in responding patients directly correlates with reduction in tumour size6. Furthermore, a high mutational burden in the TME correlated with the response to anti-PD-1 and
anti-PD-L1 in melanoma, lung, MSI-positive CRC
and several other cancer types32. By breaking tolerance, ICIs allow unleashing a pre-existing immune response to reject the tumour. However, they fail to reject the tumour in the absence of such a pre -existing response (for example, in excluded or cold tumours)6,73,74 . Of note, the ‘quality’ of this pre-existing immunity also affects the response to ICI. For instance, the upregulation of alter-native immune checkpoints (such as hepatitis A virus cellular receptor 2, HAVcr-2, also known as TIM3) fol-lowing anti-PD-1 treatment confers adaptive resistance to therapy75,76 . Indeed, in patients with renal cell carci-noma (RCC), inhibition of PD-1 alone could not rescue the functionality of CD8+ T cells that co-expressed PD-1 and TIM3 (ref.77).
As single agents, ICIs have response rates in the range 10–35%78. Indeed, at the time of diagnosis, most stage IV solid cancers are poorly infiltrated in primary tumours, if not non- infiltrated by T cells, possibly explaining the limited response to ICIs73 . When used as adjuvant therapy for high-risk stage III melanoma, the anti-PD-1 mAb pembrolizumab resulted in significantly longer recurrence-free survival79 . In an attempt to achieve increased clinical benefit of ICIs, an impressive amount of studies and clinical trials testing combinations of vari ous immunotherapy agents, as well as combinations of immunotherapy agents with standard-of-care treatments, are currently under evaluation67 (Fig. 2). These have dif-ferent likelihood of response based on pre-existing hot, altered or cold immune tumours (Fig. 1b).
Treating hot tumours with immunotherapy
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cell-targeting immunotherapies. By displaying a high degree of T cell infiltration, hot tumours represent a fertile ground for effective ICI-based monotherapy or combination therapy (Box 3; Fig. 3). Exhausted or dys-functional TILs express a number of inhibitory recep-tors, most notably cytotoxic T lymphocyte-associated antigen 4 (CTLA4) and PD-1. CTLA4 inhibits T cells’ early activation and differentiation (typically in the lymph nodes) whereas PD-1 modulates their effector functions (mostly within tumours), which can lead to T cell exhaustion78,80. Strategies used to target CTLA4 and PD-1 and/or PD-L1 have now been approved by the US Food and Drug Administration (FDA) for the treatment of multiple cancers (Box 3). Hot (infiltrated) TME, TH1 immune signature and PD-L1 expression are features associated with increased response to anti-PD-1 or anti-PD-L1 monotherapy6,81,82. The non-redundant nature of CTLA4 and PD-1 makes them good targets for dual checkpoint blockade; indeed, anti-CTLA4– PD-1 dual therapy has been successful in the treatment of advanced-stage melanoma83, RCC84 and non-small-cell lung cancer (NSCLC)85, resulting in regulatory approval. It can be postulated that such combinations would be effective only in the context of hot or immuno suppressed tumours as they rely on a certain degree of T cell infiltration.
Another promising target to be considered in associ-ation with anti-PD-1 and PD-L1 strategies is lymphocyte activation gene 3 (LAG3), a co-inhibitory receptor on T cells that, among other functions, enhances activity of
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a Number of trials assessing combinations with
b Number of IO agents
anti-PD-1 and/or PD-L1 therapies
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chemotherapy
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IO
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Number of trials
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c Number of IO drugs
d Major treatment combination approaches
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immunomodulator
immunomodulator
Immune
Immune
microenvironment
microenvironment
Cancer vaccine
Cancer vaccine
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bispecific antibody
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IO drugs to treat hot ( 
), altered ( 
)
and cold (
) immune tumours
Fig. 2 | overview of more than 2,000 immuno-oncology agents currently tested or in use. a | Numbers of ongoing trials
assessing therapies in combination with anti-programmed cell death protein 1 (PD-1) and/or PD-1 ligand (PD-L1). With
‘multi-combo’, we designate triple combinations, specifically, PD-1 and/or PD-L1 combined with radiochemotherapy, chemotherapy and targeted therapy, chemotherapy and immunotherapy, targeted therapy and immunotherapy or radiotherapy and immunotherapy (source: https://clinicaltrials.gov/). b | Number of immune-oncology (IO) agents currently in development, from preclinical phase to regulatory approval. c | Number of IO drugs in clinical trials sorted by mechanism of action and identifying six categories of IO agent. The ‘immune microenvironment agents’ category includes any immune modulator that does not belong to any other indicated category (for example, innate immune cell modulators). d | IO agents efficient in hot immune tumours (red circle), IO agent combinations likely efficient in altered immune tumours (yellow circle) and IO agent combinations being tested in cold immune tumours (blue circle). Nine (1–9) single or combinatorial approaches are illustrated to possibly treat hot, altered and cold tumours. Combo, combinational therapy.
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regulatory T (Treg) cells and regulates T cell proliferation,
Alternatively, ICIs could be combined with co-
differentiation and effector function78. Whereas LAG3
stimulatory checkpoint molecules, such as OX40 anti-
blockade yields only modest efficacy as a monotherapy,
gen (also known as TNFRSF4 (or CD134)), TNFRSF7
its combination with anti-PD-1 has synergistic potential
(also known as CD27), CD28, TNFRSF9 (also known as
in preclinical models78. Other likely targets to combine
4-1BB ligand receptor or CD137) and glucocorticoid-
with existing ICIs include additional co-inhibitory recep-
induced TNFR-related protein (GITR), all of which
tors such as TIM3 (a marker for exhausted T cells86);
enhance T cell expansion and effector functions while
T cell immunoglobulin and ITIM domain (TIGIT, which
controlling Treg cell suppressive functions78,91. The notion
counterbalances the co-stimulatory
function of CD226,
that CD28 conveys the second signal required to complete
that is rapidly induced following T cell activation87);
T cell activation upon TCR engagement has been known
B and T lymphocyte attenuator (BTLA; also known as
for over three decades92. Recently, CD28, and not the TCR,
CD272), which is expressed by T cells and synergizes
has been shown to be the target of PD-1 signalling93 and
with herpesvirus entry mediator (HVEM; also known
thus to be required for efficient PD-1 therapies94. Apart
as TNFRSF14), expressed on antigen-presenting cells
from enhancing effector T function, CD28 blocks the sup-
(APCs)88; V-domain Ig suppressor of T cell activation
pressive function of Treg cells92, which suggests its potential
(VISTA, which mediates a compensatory inhibitory
as an anticancer therapy95. The first-in-human clinical trial
pathway following anti-CTLA4 therapy in prostate
testing a CD28 super-agonist was associated with a life-
cancer89); and sialic acid-binding Ig-like lectin 9 (SIGLEC9),
threatening cytokine release syndrome in all six subjects
which is upregulated in TILs and possibly determines a
receiving the drug96, illustrating the need to fine-tune the
subclass of tumour-specific CD8+ TILs90.
dose and scheduling of these powerful immunotherapies.
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Box 3 | Predicting the response to anticancer therapy
Monoclonal antibodies targeting programmed cell death protein 1 (PD-1) and PD-1 ligand (PD-L1) have been approved by the US Food and Drug Administration (FDA) for the treatment of multiple cancers (melanoma; non-small-cell lung cancer; kidney, bladder, head and neck, hepatocellular, gastric and cervical cancer; Merkel cell carcinoma; Hodgkin lymphoma; and tumours with microsatellite instability (MSI)).
A considerable effort is being put into establishing valid predictors of response to cancer immunotherapies. Multiple tumour-related or immune-related predictive biomarkers have been proposed, including the expression of immunosuppressive molecules (such as PD-L1) by tumour cells281,282; the molecular profiling of the tumour microenvironment, which encompasses the expression of inflammatory genes10,283; the assessment of the mutational landscape and neoantigen load5; mismatch-repair deficiency and MSI20; tumour aneuploidy284; immune infiltration; and Immunoscore19.
A striking observation is that all of these are ultimately immune-related biomarkers, including classical tumour-related features such as somatic copy number alterations (SCNAs). High levels of SCNA are associated with a poor response to anti-cytotoxic
T lymphocyte-associated antigen 4 (CTLA4) therapy and with reduced expression of cytotoxic immune infiltration in patients with melanoma284. The immune contexture parameters act as prognostic (associated with survival), predictive (associated with response to treatment) and mechanistic (increased after treatment in responding patients) biomarkers.
Attempts to rationalize and set instrumental guidelines for personalized therapies have been made. Blank et al. introduced a dynamic model (the ‘cancer immunogram’), which required the assessment of a combination of biomarkers as a tool to guide treatment options for individual patients62. The model relies on published data from patients who responded to anti-PD-1 and/or anti-PD-L1 treatment62. An initial framework of seven parameters has been established: tumour foreignness; general immune status; immune cell infiltration; absence of checkpoints; absence of soluble inhibitors; absence of inhibitory tumour metabolism; and tumour sensitivity to immune effectors62. The evaluation of these factors can be achieved by a combination of tumour genomics, Immunoscore assay, immunohistochemistry, standard blood assays and immune gene signature, both pre-therapy and post-therapy, and could be helpful in designing possibly the most efficient therapeutic intervention for individual patients62.
Another co-stimulatory immune checkpoint molecule, inducible T cell co- stimulator (ICOS), could also be a candidate owing to its expression on activated T cells. However, despite showing a promising profile in tumour mouse models97, its concomitant expression on Treg cells98 might limit its clinical impact. On the basis of preclini-cal evidence, the requirement for combinatorial immune checkpoint blockade could be bypassed by inhibiting the chronic interferon response, shown to mediate resist-ance to ICIs39. Altogether, albeit promising, the use of co-stimulatory molecules could be limited clinically by systemic toxicity due to important off-tumour effects.
Microbiome modulation. Antibiotics can inhibit the clinical benefit of ICIs in patients with advanced-stage cancer, and a correlation between clinical responses to ICIs and the relative abundance of the Gram-negative commensal bacterium Akkermansia muciniphila has been demonstrated99. Therefore, selective modulation of gut microbiome composition might overcome resist-ance to ICIs99,100. Abundance of ‘good’ bacteria, including those belonging to the Faecalibacterium genus101, corre-lated with a higher number of effector CD4+ and CD8+ T cells in peripheral blood and with response to anti- PD-1 mAbs in patients with melanoma101. Conversely, non-responders had a higher abundance of members of the Bacteroidales order, which correlated with higher frequencies of Treg cells and myeloid-derived suppres-sor cells (MDSCs) in the systemic circulation101. Mice that received faecal microbiota transplantation from
responding patients showed an upregulation of PD-L1, which suggested that the ‘right’ microbiota could help in the development of a hot TME101. Another study on metastatic melanoma demonstrated the significant asso-ciation between commensal microbial composition and clinical response to anti-PD-1-based immunotherapy102.
It is unlikely that the modulation of the microbiota alone would work in an altered or cold tumour; none-theless, the presented evidence suggests that fairly simple lifestyle changes and/or oral supplementation of good bacteria could shape a favourable stage for subsequent immune-based therapies, which could be effective in the context of hot tumours or possibly in combination with other agents in altered or cold tumours.
Treatment of immune-altered tumours
T cell trafficking modulators. In excluded tumours, an accumulation of CD8+ T cells occurs at the tumour bor-ders. This phenomenon indicates the ability of the host to mount a T cell-mediated immune response and the physical inability of the T cells to reach the tumour bed (Fig. 1). Many explanations for this phenotype can be pro-posed. One reason for T cell exclusion could be the lack of T cell-recruiting signals, such as chemokines directing T cell trafficking, including CXCL9, CXCL10, CXCL11, CXCL13, CX3C-chemokine ligand 1 (CX3CL1), CC-chemokine ligand 2 (CCL2) and CCL5 (refs57,103). This shortage of chemokines may result from the modulation of the oncogenic, genetic and epigenetic pathways that control their expression104. Histone modification and DNA methylation can repress the expression of TH1-derived CXCL9 and CXCL10 in ovarian105 and colon cancer106. Therapeutic epigenetic modulation promoted tumour infiltration of effector T cells, slowed tumour pro-gression and improved the efficacy of PD-L1 blockade in preclinical models106,107. In metastatic melanoma, constitu-tive activation of the β-catenin pathway resulted in defec-tive recruitment of CD103+ dendritic cells (DCs) into the TME and subsequent absence of CD103+ DC -derived CXCL9 and CXCL10 in hot tumours44. Of note, the cited studies rely on the more simplistic distinction of T cell- inflamed (hot) versus non-T cell-inflamed (cold) tumours. Therefore, it can be only postulated that between these two types, there could be cases of excluded tumours; however, it is tempting to speculate that these represent the cases in which the association of T cell tracking modulators with immunotherapy yields clinical benefit.
Activation of tumour-oncogenic pathways correlat-ing with T cell exclusion has been described in muscle- invasive urothelial bladder cancer (MIUBC)108, usually characterized by poor outcome. Non-surprisingly, genes encoding PD-L1, IDO, FOXP3 (forkhead box P3; the master regulator of Treg cells), TIM3 and LAG3 were upregulated in T cell-inflamed MIUBCs, whereas an activation of β-catenin, peroxisome proliferator- activated receptor-γ (PPARγ) and fibroblast growth factor receptor 3 (FGFR3) pathways was found in the
most common non-T cell-inflamed MIUBCs108.
A ‘sufficient’ T cell infiltration in mice tumour sites, rather than a differential PD-L1 expression, is critical for the response to anti-PD-L1 therapy109. On the basis of this assumption, it can be envisaged that strategies
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that facilitate the recruitment of T cells to excluded tumours could overcome tumour resistance to check-point blockade109. TNFSF14 (also known as LIGHT, or HVEM ligand) is an activator of lymphotoxin β-receptor signalling, which triggers the production of
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cell-targeting chemokines, thereby creating a T cell- inflamed microenvironment109. Accordingly, the use of an antibody-guided LIGHT modulated the recruit-ment of T cells to the tumour site and overcame tumour resistance to ICIs109.
In summary, the epigenetic modulation of TH1-derived chemokines, as well as a blockade of β-catenin signalling, could turn excluded tumours into hot tumours, thus increasing the likelihood of success of concomitant immunotherapy.
Physical barrier breakers. Another explanation for the inability of T cells to penetrate tumour sites could be the presence of physical barriers. The development of abnormal structural features is a hallmark of cancer
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Fig. 3 | The tumour–immune classification cycle as a tool to direct anticancertherapy. Tumours can be classified into four main subtypes (hot, altered-excluded,
altered-immunosuppressed and cold) according to their associated T cell (CD3+ and CD8+) presence and distribution. Hot tumours are defined by the simultaneous presence of immune contexture parameters: the cell type (CD3+, CD8+, follicular helper T (TFH),
T helper 1 (TH1), memory and exhausted T cells); the location (invasive margin, tumour core and tertiary lymphoid structures); the density (immune density and quantity); and the functional immune orientation (chemokines, cytokines, cytotoxic factors, adhesion, attraction and TH1)10. The main components, pathways and features (in green trapezoids) of the immunogram have been identified and may represent potential targets (in blue). One or more of these features can be present at once in a specific tumour.
Immunogenicity and adjuvanticity are tumour-intrinsic core characteristics that contribute to the shaping of the tumour-associated T cell landscape; we postulate that they are strikingly low or absent in cold tumours, although they may contribute to a certain extent to other subtypes. By representing a tool for the classification of a cancer into one of the four categories together with the identification of a specific defect or deregulated pathway, the spin chart could prove instrumental for building the most successful therapeutic scheme, and, conversely, narrow down the list of possible therapies by excluding potentially inefficient ones. ADORA2A, A2A adenosine receptor;
β2m, β2-microglobulin; BET, bromodomain and extra-terminal motif proteins; BTLA,
B and T lymphocyte attenuator; CAR T cell, chimeric antigen receptor T cell; CCR,
CC-chemokine receptor; CIN, chromosomal instability, CSF1R, colony-stimulating factor 1
receptor; CTLA4, cytotoxic T lymphocyte-associated antigen; CXCL, CXC-chemokine ligand; DDR, DNA damage response; ECM, extracellular matrix; EMT, epithelial– mesenchymal transition; FDA, US Food and Drug Administration; FGFR3, fibroblast
growth factor receptor 3; FOXP3, forkhead box P3; GITR, glucocorticoid-induced TNFR-
related protein; GM-CSF, granulocyte–macrophage colony-stimulating factor; HDAC, histone deacetylase; HIF1α, hypoxia-inducible factor 1-α; HLA, human leukocyte antigen; HMA, hypomethylating agent; IAP, inhibitors of apoptosis family (also known as XIAP); ICAM1, intercellular adhesion molecule 1; ICD, immunogenic cell death;
ICOS, inducible T cell co-stimulator; ICP, immune checkpoint; IDO, indoleamine
2,3-dioxygenase; IFN, interferon; IL, interleukin; LAG3, lymphocyte activation gene 3; LIGHT, tumour necrosis factor superfamily member 14; MAdCAM1, mucosal addressin cell adhesion molecule 1; MCL1, induced myeloid leukaemia cell differentiation protein Mcl1; MDSCs, myeloid-derived suppressor cells; MEK, mitogen-activated protein kinase kinase; MET, mesenchymal–epithelial transition; MSI, microsatellite instability; NK, natural killer; NOS1, nitric oxide synthase 1; PD-1, programmed cell death protein 1; PD-L1, PD-1 ligand; PI3Kγ, phosphoinositide 3-kinase-γ; PPARγ, peroxisome proliferator-
activated receptor-γ; SIGLEC9, sialic acid-binding Ig-like lectin 9; STING, stimulator of interferon genes; TDO, tryptophan 2,3-dioxygenase; TGFβ, transforming growth factor-β;
TIGIT, T cell immunoglobulin and ITIM domain; TIM3, T cell immunoglobulin and mucin
domain-containing 3; TKI, tyrosine kinase inhibitor; TLR, Toll-like receptor; Treg cells, regulatory T cells; VCAM1, vascular cell adhesion molecule 1; VEGF, vascular endothelial growth factor; VISTA, V-domain Ig suppressor of T cell activation; XCL1, lymphotactin; XCR1, chemokine XC receptor 1. Lower case i following any acronym or abbreviation indicates inhibitor; lower case a following any acronym or abbreviation indicates agonist.
progression. Many extracellular matrix proteins are sig-nificantly deregulated during the progression of cancer, causing both biochemical and biomechanical changes possibly inhibiting immunity and promoting the met-astatic cascade110. Changes in the tumour-associated vasculature (both blood and lymphatic) have been extensively described, as well as the resulting hypoxic milieu111,112 . Tumour vasculature acts as an impor-tant barrier to T cells via the deregulation of adhesion molecules (such as intercellular adhesion molecule 1 (ICAM1), vascular cell adhesion protein 1 (VCAM1) and mucosal addressin cell adhesion molecule 1 (MAdCAM1)) required for T cell extravasation113,114. Hypoxia favours the establishment of an immunosup-pressed TME, and ultimately cancer progression and treatment resistance, mostly acting through the hypoxia- inducible factor (HIF) family of transcription factors115. Among these, HIF1α not only can drive the expression of PD-L1 but also can increase adenosine generation
and signalling116. The enzymatic activity of two ecto- nucleotidases, CD39 (also known as NTPDase I) and CD73 (also known as 5ʹ-nucleotidase), is responsible for the conversion of extracellular ATP to adenosine117. Adenosine accumulation in the TME exerts a plethora of pro-cancer effects117,118. CD73 blockade in several in vivo models significantly reduced tumour growth and meta-static spread119, and combination of CD73 blockade with ICIs has been proposed as an exploitable therapeutic strategy120. In turn, CD39 blockade prolonged survival in a lethal metastatic patient-derived sarcoma model121. However, deletion of CD39 resulted in autochthonous liver cancer in mice122, suggesting caution when using CD39 blocking agents. Nonetheless, agents that tar-get CD39 are currently in the preclinical stage, whereas CD73 inhibitors are already being assessed in clinical trials123. Blocking the A2A adenosine receptor ADORA2A can restore immune competence and T cell proliferation in chronic lymphocytic leukaemia116. Therefore, HIF (mostly HIF1α and HIF2α) as well as CD73, CD39 and adenosine receptor inhibitors and/or mAbs have been developed as possible anticancer therapeutics, although thus far, no agent has reached regulatory approval123–125.
Hypoxia also exerts systemic effects via the secre-tion of growth factors and cytokines altering immune cell proliferation, differentiation and function111. One of these factors is the pro -angiogenic cytokine VEGF, which has been targeted by various pharmacological and immune-based antitumour strategies in the past decade112,126. VEGF-dependent effects extend beyond its angiogenic capabilities. For example, high expression of VEGFA inhibits the maturation of DCs, modulates TCR signalling and counteracts the beneficial effects of TH1 cells and cytotoxic T cells by suppressing the expression of IRF1 and GNLY, respectively127. Nonetheless, anti- VEGF and other angiogenesis inhibitors have not been successful as single agents, mostly owing to the devel-opment of a compensatory mechanism128. Furthermore, these inhibitors resulted in some cases in a somewhat counterintuitive increase in metastatic spread129,130. Despite these setbacks, evidence exists in support of the complementary therapeutic value of the normalization of the vascular abnormalities112, or vessel normaliza-tion. Vessel normalization is characterized by increased pericyte coverage, improved tumour vessel perfusion, reduced vascular permeability and subsequent reduced hypoxia131. Infiltration and activity of TH1 lymphocytes correlate with vessel normalization. In addition, T H1 activation by ICIs increased vessel normalization in various mouse models of breast cancer132, suggesting that a synergistic effect can be achieved by combining anti-angiogenic therapies with ICIs.
Soluble factor inhibitors. In the case of immuno suppressed tumours, tumour sites display a modest, insuf ficient degree of immune infiltration (Fig. 1), suggesting that the presence of an immunosuppressive environ-ment, rather than that of physical barriers, limits further
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cell recruitment17 and expansion18. IL-10 and TGFβ are among the best-characterized tumour-derived solu-ble factors impairing the development of an antitumour immune response133,134. As a matter of fact, IL-10 and
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TGFβ display both pro-tumour and antitumour abilities. This apparent contradiction can be explained by their numerous, yet not fully characterized, functions as well as by their different local versus systemic effects135,136. Therefore, they would not seem to constitute optimal molecular targets. Nevertheless, the recent evaluation of the combined inhibition of TGFβ and PD-L1 in multi-ple tumour types showed clinical benefit137. One of the characterized functions of IL-10 and TGFβ is their abil-ity to disrupt DC differentiation, migration and antigen presentation, some of the crucial mechanisms required to mount an effective antitumour T cell response138,139. In the presence of this type of immunosuppression, CD40-activated B cells were suggested as alternative APCs owing to their resistance to IL-10, TGFβ and VEGF138.
A new therapeutic strategy aiming at improving the effector functions of T cells is based on the ionic repro-gramming of tumour-specific T cells140. Tissue necrosis, common in solid tumours, releases potassium (usually intracellular) into the extracellular milieu, suppressing T cell effector functions141. This effect was mediated by a subsequent increase in intracellular potassium levels, inhibiting the TCR-driven AKT–mTOR pathway in a protein phosphatase 2-dependent manner140. By low-ering intracellular potassium, the overexpression of the potassium channel Kv1.3 restored T cell effector func-tions, tumour clearance and survival in a melanoma mouse model140. These results show the potential of this alternative type of intervention as a further promising anticancer strategy.
Cellular modulators of local adaptive immunity. Two key cellular mediators of local adaptive immune
suppression in the TME are MDSCs and Treg cells67. As both favour tumour progression, it is not surprising that
strategies aiming at reducing their number are currently being explored. This seems to be somewhat feasible for MDSCs and has indeed been achieved by blocking their main suppressive pathways (IDO142, arginine, trypto-phan and nitric-oxide-related pathways143–145), by regu-lating myelopoiesis or by preventing their trafficking67.
Box 4 | cancer stem cells: key mysterious players in cancer progression
Cancer stem cells (CSCs), which include mesenchymal stem cells, are a vast population of cells involved in cancer initiation and progression285. CSCs are characterized by self- renewal and differentiation capacity, similarly to their stem cell counterparts.
Importantly, their undifferentiated state makes them resistant to immune recognition, owing to the lack of expression of human leukocyte antigen (HLA) class I, as well as to chemotherapy and radiotherapy285. The epithelial–mesenchymal transition (EMT) and the mesenchymal–epithelial transition (MET) are processes of phenotypic transition between epithelial and mesenchymal states286. It is widely accepted that EMT in cancer is at least associated with, if not necessary for, the metastatic process287. The elucidation of the exact involvement of EMT and MET and CSCs in tumorigenesis and tumour aggressiveness is still an object of debate, hampered by contrasting data285. Nevertheless, targeting the EMT has been proposed as a strategy to at least partly overcome resistance to immune checkpoint blockade288. EMT might contribute to immune escape via multiple routes, including the shaping of the tumour microenvironment (through the production of soluble factors such as interleukin 10 (IL-10) and transforming growth factor-β (TGFβ)) and decreased susceptibility to immune effector cells288. CSCs express natural killer (NK) receptor ligands, implying that the exploitation of NK cells could provide a valuable strategy to counteract CSC-mediated immunosuppression285.
Nonetheless, the recent failure of the phase III, rand-omized, double-blind study involving the combination of an IDO inhibitor plus a PD-1 blocking agent to yield greater clinical benefit than anti-PD- 1 alone in unre-sectable or metastatic melanoma raises questions on the efficacy of these strategies146.
The selective targeting of the γ-isoform of phospho-inositide 3-kinase (PI3Kγ), highly expressed in myeloid cells, successfully reshaped the TME and promoted cytotoxic-T cell-mediated tumour regression in preclini-cal mouse models for several cancers147. The combination of this pharmacological approach with PD-1 blockade is currently under investigation in clinical trials147.
Blockade of colony -stimulating factor 1 receptor (CSF1R) could deplete the pro-tumoural macrophage population, in particular the M2 subtype, thus favouring the induction of a cytotoxic antitumour T cell response following PD-L1 blockade148. Clinical trials are ongo-ing to test the clinical activity of a combined treatment associating antibodies against CSF1R andanti-PD-L1 in patients with metastatic cancers148. Another example of modulation of tumour-associated myeloid cells can be found in the study by Nakhlé et al. on bladder tumours. In mouse models, targeting S100A9 — a zinc and cal-cium protein with a prominent role in the regulation of inflammatory processes and immune response — with tasquinimod, a regulator of MDSC accumulation, resulted in a re-education of tumour-infiltrating mye-loid cells from a pro -tumoural M2 phenotype towards an antitumoural M1 phenotype149. The parallel increase in PD-L1 expression could explain the lack of tumour growth inhibition following treatment with tasquini-mod as single therapy149 while simultaneously providing a solution to overcome this obstacle. Indeed, the com-bination of tasquinimod with an anti-PD-L1 antibody enhanced the antitumour immune response in preclini-cal bladder tumour models149. Along the same lines, the depletion of MDSCs induced a CD8+ Tcell-dependent tumour rejection in mouse models of head and neck squamous cell carcinoma (HNSCC) when combined with anti-CTLA4 therapy150. The same study revealed the existence of an MDSC-rich gene expression profile with a T cell-inflamed phenotype in > 60% of patients in an HNSCC cancer cohort150. This observation reit-erates the idea that a comprehensive characterization of the TME and its key components could prove to be tremendously useful in pinpointing the most promising therapeutic strategy.
The targeting and reduction of tumour-associated Treg cells remain quite challenging to achieve. Impor tantly, Treg cells express high levels of PD-1, which acts as a stimulatory receptor, rather than inhibitory, in these cells67, which may therefore reduce the benefits of an eventual anti–PD-1 antibody-based therapy67. Clinical approaches for Treg cell depletion have also not been very successful, having the downside of also reducing
the tumour-suppressive Treg cells, critical for preventing autoimmunity. Crucially, Treg cells in the TME are not dysfunctional, in contrast to other TILs67.
A third, less characterized population of pro-cancer cells is represented by cancer stem cells, which are discussed in Box 4.
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Tumour-specific antigen
(TSA). Antigen not encoded in the normal genome, expressed exclusively by tumour cells.
Antigenicity
Presence of tumour-associated antigens (TAAs) capable of engaging with T cell receptors or antibodies (B cell receptors), thereby driving adaptive immunity.
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Innate immune response modulators. The lack of ade- |
immune tolerance160. It should be noted that also |
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quate innate immune response could constitute a lim- |
persistent type I interferons, just as IFNγ, can confer |
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iting factor restraining the development of an effective |
resistance to immune checkpoint blockade39, thereby |
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adaptive, antitumour response, therefore originating an |
suggesting possible setbacks of this therapeutic |
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immunosuppressed tumour or contributing to the estab- |
strategy. |
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lishment of a cold tumour. One of the first indications of |
Apart from the DNA sensor cGAS, various other |
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the activation of innate immune pathways in tumour set- |
pattern-recognition receptors, such as Toll-like recep- |
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tings came with the correlation between the expression |
tors (TLRs), RNA helicase RIG-I-like receptors and |
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of ISGs and T cell-associated transcripts in metastases |
NOD-like receptors have a role in endogenous stress |
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from melanoma151. Indeed, a type I interferon signature |
signal recognition occurring in tumours161,162. Therefore, |
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predicted favourable clinical outcome in breast carci- |
they are being investigated for their anticancer poten- |
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noma following treatment with cancer vaccines152 and |
tial. TLRs are highly expressed by tumour-infiltrating |
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classic chemotherapy153. Type I interferons in tumours |
immune cells, notably APCs, leading to their activation |
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derive mostly from the activation of the stimulator of |
upon ligand stimulation. The upregulation of MHC II, |
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interferon genes (STING) pathway by cytosolic tumour- |
CD80 and CD86 by TLR-stimulated APCs and their |
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derived DNA within conventional, basic leucine zipper |
conversion from tolerogenic to immunogenic have |
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ATF-like transcription factor 3 (BATF3)-expressing, |
been demonstrated in murine and human TMEs163,164. |
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tumour-associated DCs. The pattern-recognition |
TLRs can also be expressed by tumour cells, in which they |
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receptor cyclic GMP-AMP |
synthase (cGAS) recognizes |
can exert direct cytotoxic effects upon stimulation165. |
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cytosolic DNA, thereby stimulating the formation of the |
Intratumoural injection of a TLR9 agonist reverted |
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STING ligand cyclic-GMP-AMP154. These events culmi- |
resistance to PD-1 blockade, resulting in durable and |
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nate in the promotion of tumour-specific antigen (TSA) |
systemic tumour rejection in mouse models166. Similarly, |
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cross-presentation and T cell cross-priming by these |
intratumoural injection of TLR7 and TLR9 agonists, in |
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cells155–157. Regression of established tumours and sys- |
combination with PD-1 blockade, suppressed primary |
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temic antitumour responses were observed in preclinical |
tumour growth and prevented metastatic spread in |
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models following intratumoural injection of a STING |
HNSCC models167. Finally, intratumoural injection of |
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agonist158. Therefore, STING agonists are currently being |
a TLR9 ligand, combined with administration of an |
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tested in clinical trials159. |
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anti-OX40 antibody, successfully mediated regression |
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The modulation of the innate immune landscape |
even of a variety of histological tumour types, includ- |
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within the TME represents a possible point of inter- |
ing that of spontaneous breast cancers168. The efficacy of |
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vention in the case of T cell-infiltrated tumours that |
TLR agonists has been investigated in multiple clinical |
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do not respond to ICI (such as immunosuppressed |
trials169–173. When used as monotherapies, these agents |
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tumours). The local injection of a STING agonist in |
yielded limited success174; however, combination strate- |
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the tumour site, followed by treatment with an anti- |
gies (for example, with anticancer vaccines) seem to be |
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PD-L1 antibody, was able to control, or completely |
quite promising175–177. |
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reject, T cell-inflamed tumours in a mouse model of |
Modulation of APC activation can be achieved by |
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HNSCC149. Again, as the study refers to only hot and |
using agonist anti-CD40 mAbs. CD40 is a TNFRSF |
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cold tumours, we can only assume that this case may |
member mostly expressed on APCs. By interacting with |
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fall into the category of an immunosuppressed tumour |
its ligand on activated TH cells, it triggers APC activa- |
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rather than that of a hot tumour. STING activation trig- |
tion and subsequent induction of adaptive immunity178. |
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gered the production of type I and II interferons and |
Agonistic anti-CD40 antibodies performed remark- |
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the accumulation of DCs in tumour-draining lymph |
ably well in preclinical models of B cell lymphomas179 |
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nodes, boosting the T cell-mediated response; this |
and bladder tumours180. There are multiple ongoing |
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came with a concomitant increase in the expression of |
clinical trials involving CD40 agonists, and potential |
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PD-L1 in the TME149. The subsequent addition of an |
combinations with ICIs are being evaluated181. |
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anti-PD-L1 mAb successfully removed the immuno- |
The chemokine XC receptor 1 (XCR1) and its ligand |
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suppressive status, thereby allowing the establishment |
lymphotactin (XCL1) regulate migration and function |
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of a local, as well as systemic, T cell-mediated anti |
of CD103+CD11b− DCs182–184. Intratumoural natural |
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tumour activity149. Of note, the STING-agonist-mediated |
killer (NK) cells produce CCL5 and XCL1, thereby pro- |
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induction of type I interferon and accumulation of DCs |
moting DC recruitment in multiple human cancers and |
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in draining lymph nodes were not enough to mount an |
affecting patient survival184. Future anticancer strate- |
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adaptive immune response in a parallel model of non- |
gies exploiting these newly discovered axes could prove |
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T cell-inflamed (cold) tumour149. Poor antigenicity or |
successful. |
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an intrinsic insensitivity to T cell killing in this model |
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were proposed as possible explanations for these find- |
The treatment of immune cold tumours |
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ings149. The potential of STING as an immune adjuvant |
Cold tumours, characterized by low Immunoscore, |
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that promotes the priming of tumour antigen-specific |
are the most challenging to eradicate and are invar- |
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CD8 T cells has also been shown in murine tolerogenic |
iably associated with poor prognosis. A proposed |
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HER2+ breast tumours160. In this study, the injection of |
approach to overcome the lack of a pre-existing immune |
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a STING agonist in combination with PD-L1 blockade |
response — and ultimately to turn cold tumours into |
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and OX40 activation resulted in tumour regression by |
hot tumours — is to combine a priming therapy that |
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providing priming and overcoming antigen-enforced |
enhances T cell responses (such as vaccines, adoptive |
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Abscopal effect
Phenomenon characterized by the regression of metastases outside the field of radiation after irradiation of one tumour site. Although rarely detected, it is well documented in patients with more immunogenic tumours.
Adjuvanticity
Presence of damage- associated molecular patterns (DAMPs) and stress signals driving the innate immunity.
Genotoxic chemotherapies
Chemical agents that cause DNA damage, such as single- strand and double-strand breaks, loss of excision repair, crosslinking, alkali-labile sites, point mutations and structural and numerical chromosomal aberrations.
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T cell transfer (ACT) or strategies that turn the tumour |
stimulates the formation of the STING ligand cyclic- |
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into a vaccine) with the removal of co-inhibitory signals |
GMP-AMP154. The revised model adds an extra level of |
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(through approaches such as ICIs or MDSC depletion) |
complexity by showing that pattern recognitions occur |
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and/or the supply of co-stimulatory signals (such as |
within cGAS-containing micronuclei, which form |
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anti-OX40 or anti-GITR)67. The concern with this type |
and accumulate only following cell cycle progression |
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of approach would be the concurrent increase in unde- |
through mitosis188. This dependence on actively cycling |
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sired side effects67, which is the case of most combina- |
cells could possibly explain the observed delayed onset |
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torial therapies, surely requiring careful evaluation. In |
of inflammatory signalling upon radiotherapy and other |
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principle, it would make sense that a priming therapy |
DNA double-stranded |
-breaking therapies188. |
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would be beneficial in the case of non-inflamed, cold |
By being readily available and free from patent rights, |
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tumours, whereas inflamed, hot tumours would benefit |
radiotherapy presents the risk of being used as a sim- |
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more from immune interventions that counteract the |
ple add-on to any immunotherapy, without a rationale |
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tumour-induced T cell dysfunctions22,67. However, this |
defining dose, fractionation, sequencing and timing. A |
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black and white distinction of hot versus cold tumour is |
deeper knowledge on the molecular mechanisms trig- |
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an oversimplification of an incredibly complex scenario. |
gered by different regimens of radiotherapy within the |
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The introduction of the altered categories (excluded and |
TME needs to be gained to carefully design efficient |
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immunosuppressed) may more suitably represent inter- |
therapeutic schemes73. There are evident limitations in |
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mediate phenotypes. Hence, it is possible that some of |
studying the effect of radiotherapy in mouse models, |
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the proposed approaches could in fact be effective only |
including the intrinsic radiosensitivity and immuno- |
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in the case of altered tumours. It is likely that the paral- |
genicity differences and the choice of tumour implanta- |
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lel occurrence of multiple pro-tumour mechanisms ulti- |
tion site73. Nonetheless, abscopal effects were observed |
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mately leads to the establishment of a cold tumour and |
in patients treated with anti-CTLA4 mAbs and under- |
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combinatorial approaches are likely needed to achieve |
going radiation regimens similar to those regimes used |
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clinical benefit. |
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in mice, highlighting the translational potential of mice |
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studies in this context73. As tumours can develop differ- |
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Radiotherapy. A promising priming therapy to be |
ent immune-evasion strategies, the choice of radiation |
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associated with subsequent immunotherapy is ioniz- |
regimen should be made according to the specifics of |
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ing radiotherapy67,73. The currently achievable precise |
the case. For example, as a low dose of radiation pro- |
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delivery of radiotherapy and the resulting induction |
moted vascular normalization, such an approach could |
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of immunogenic cell death (ICD) pathways can poten- |
be useful in excluded tumours73. |
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tially convert the tumour into an in situ vaccine67. The |
A promising strategy to promote T cell infiltration |
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consequences of this approach not only involve the irra- |
into a cold tumour and convert it into a hot one came |
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diated tumour site itself but can possibly contribute |
from a study by Zheng et al. on a pancreatic cancer |
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to the achievement of systemic tumour control through |
mouse model189. Patients with CD8+Tlo PD-L1hi pan- |
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the so-called ‘abscopal effect’67. The DNA released fol- |
creatic cancer respond poorly to treatment with ICIs, |
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lowing the radiation-induced cell damage might trig- |
vaccine or their combination, as well as to radiother- |
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ger a STING-mediated type I interferon production, |
apy. The development of a murine model that mim- |
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possibly by tumour-infiltrating CD8α+CD103+ DCs in |
icked CD8+ Tlo PD-L1hi pancreatic tumours enabled the |
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mice (or their human counterpart CD141+ DCs185), a |
demonstration that the sequential combination of radio |
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DC subset specialized in antigen cross-presentation186; |
therapy, vaccination (with live cells expressing a model |
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this will then fuel a T cell-mediated antitumour immune |
immunodominant antigen) and checkpoint inhibition |
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response186,187. This process relies on a certain degree of |
(anti-PD-L1 mAb) resulted in tumour regression and |
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infiltration of CD103+ DCs, which could be a limiting |
improved survival compared with individual treatments |
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factor in cold tumours but may be effective in excluded |
or radiotherapy plus vaccination189. It is likely that the |
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tumours. A recently developed mouse melanoma model |
radiation enabled the recruitment of vaccine-primed |
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that lacks intratumoural CD8α+CD103+ DCs44 could |
T cells, which could exert their antitumour effects in a |
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shed light onto the ability of radiotherapy to prime T cell |
no-longer |
immunosuppressive microenvironment. |
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responses in non-infiltrated tumours. The combination |
Despite the growing amount of information, the lack |
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of radiotherapy with further anti-immunosuppressive |
of sufficient preclinical data impedes a guided design of |
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strategies could potentially increase the frequency of the |
clinical trials, the results of which are often inconclusive |
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abscopal effect. Indeed, combining radiotherapy with |
in attributing a therapeutic benefit to radiotherapy. It |
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anti-CTLA4 mAbs in melanoma and NSCLC (which |
would be challenging, yet crucial, to demonstrate une- |
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normally does not respond to anti-CTLA4) |
significantly |
quivocally the contribution of radiation to immuno- |
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improved the therapeutic outcome67. Additionally, maxi- |
therapy response. An optimal integration of radiation |
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mal anti-CTLA4 therapy-driven abscopal responses in a |
biology with tumour immunology could give rise to |
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mouse melanoma model was dependent on STING sig- |
potentially great clinical benefits190. |
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nalling188, further supporting the key role of STING and |
Chemotherapy. Anticancer agents can augment the |
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type I interferons in this context. The same study |
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revealed a possible reason for the observed time frame |
immunogenicity of tumour cells through two main |
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(days rather than minutes) in which the radiation- |
routes — the antigenicity191 and the adjuvanticity191,192 |
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induced inflammatory responses occur. In a classi- |
(Fig. 4). Genotoxic chemotherapies can induce mutations |
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cal model, the cGAS recognizes cytosolic DNA, then |
leading to the generation of neo-epitopes (therefore |
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tumour-
194,195
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tolerance |
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Fig. 4 | schematic representation of treatments of immune cold tumours. The key factors or processes to be tackled to
achieve clinical benefit are the following: priming the immune response (for example, with a neo-epitope cancer vaccine) (a); modulating the immune response (for example, with transforming growth factor-β (TGFβ) inhibitors) (b); expanding cytotoxic T cell proliferation (for example, with interleukin-15 (IL-15)) (c); inducing recruitment of cytotoxic immune cells (for example, by epigenetic modulation or the use of dendritic or T cell-recruiting chemokines) (d); breaking tolerance (using agents such as anti-programmed cell death protein 1 (PD-1) and/or PD-1 ligand (PD-L1) therapy) (e); inducing immunogenicity (for example, with an instability inducer, such as an inhibitor of MutL homologue 1 (MLH1)) (f); inducing immunological cell death (for example, by means of chemotherapy) (g); and inducing adjuvanticity (with an oncolytic virus) (h). CTL, cytotoxic T lymphocyte; MDSC, myeloid-derived suppressor cell; NK, natural killer; TAA, tumour-associated
antigen; TFH, follicular helper T cell; TLS, tertiary lymphoid structure; Treg cell, regulatory T cell.
Damage-associated molecular patterns
(DAMPs). Intracellular molecules that are hidden from immune recognition under physiological conditions. These molecules are secreted, exposed or released upon cellular stress or tissue injury and recognized by pattern- recognition receptors expressed on innate immune cells.
Tumour-associated antigens
(TAAs). Antigens that are preferentially expressed by tumour cells but they can also be found in normal tissues (except for the TSAs, which are exclusively expressed by tumour cells). They can be broadly categorized into aberrantly expressed self- antigens, mutated self-antigens and TSAs.
increasing the antigenicity). However, such neoantigens may be lowly expressed on tumour cells, thus having a modest impact on the immune response193. Nonetheless, chemotherapies that trigger an ICD — such as anthra-cyclines, cyclophosphamide, oxaliplatin and taxanes — can concomitantly increase the adjuvanticity by releasing
damage-associated molecular patterns (DAMPs) and activating apoptotic or necroptotic pathways196. The calreticulin exposure at the membrane provides an ‘eat-me’ signal that favours the transfer of
associated antigens (TAAs) to DCs197. Dying tumour cells can also stimulate a TLR3-dependent, cancer-cell-autonomous type I interferon response, which induces the local production of CXCL10, attracting T cells and memory T cells to the tumour bed56,153,198. Even if these ICD pathways have been elucidated only in mouse mod-els, they might be clinically relevant. Indeed, chemother-apy modified the local immune microenvironment in patients with breast cancer199. Following neoadjuvant chemotherapy (NAC), these patients had an increased ratio of intratumoural CD8+ T cells to FOXP3+ cells200. This event was accompanied by a clonal expansion of antitumour T cells that correlated with response to NAC,
followed by a complete pathological response200. After chemotherapy, the absence of autophagy-related protein LC3B (LC3B+) puncta and a low CD8+ to FOXP3+cells ratio were associated with a bad prognosis in patients with breast cancer201,202. Low calreticulin levels on tumour cells correlated with a weaker immunosurveil-lance in ovarian cancer204 and NSCLC203,204. Low levels of antigen -specific circulating T cells were associated with poor clinical outcome in patients with acute mye-loid leukaemia205,206. In CRC, neoadjuvant chemotherapy increased the adaptive immune response, and there was a significantly higher frequency of high Immunoscore metastases in patients achieving pathological and radiological responses50. Chemotherapy might also stimulate the activation of immune effectors through off-target effects, resulting in general immune stimu-lation207 . Agents such as 5-fluorouracil deplete intra tumour MDSCs208, whereas cyclophosphamide depletes Treg cells209 and triggers the translocation of immuno stimulatory bacteria from the gut lumen to the damaged epithelium210. Effector T cells abrogate stroma-mediated chemoresistance in ovarian cancer211. IFNγ-producing CD8+ T cells may block cysteine and glutathione (both
R e v i e w s
(LC3B+) puncta
LC3 is a protein involved in the formation of autophagosomes and autolysosomes. Punctate (as opposed to diffused) LC3 staining indicates autophagy, as determined by fluorescence microscopy.
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of which confer resistance to platinum-based chemo- |
treatments may sensitize patients to further immuno- |
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therapy) synthesis by fibroblast-associated tumour |
therapies. This approach might be a well-suited option |
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cells211. Altogether, these observations support the ever- |
in the case of immunosuppressed tumours, which |
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growingly endorsed hypothesis that chemotherapy is |
show potential for infiltration (no physical barriers) |
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not simply tumour suppressive but is also involved in |
but insufficient T cell response. However, it may be not |
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the positive modulation of the immune system. It could |
the most suitable choice in the case of cold tumours. |
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be postulated that the beneficial effect of chemotherapy |
In support of this hypothesis, a study by Spranger et al. in |
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depends on the presence of an adaptive pre-existing |
patients with melanoma demonstrated that no dif- |
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immunity. In such a case, hot and altered immune |
ference exists between inflamed and non-inflamed |
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tumours as measured by Immunoscore may be more |
tumours in terms of antigenic load and/or mutational |
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susceptible to chemotherapy (as well as radiotherapy) |
burden108. In this particular study, the lack of BATF3- |
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than cold tumours212. Furthermore, this would constitute |
lineage DCs may have been the cause for the ‘cold’ |
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the rationale for coupling chemotherapy with ICIs. The |
TME. An assessment of the mutational load within the |
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success of the combination of anti-PD-1 therapy plus |
tumour could provide a rational basis for the use of |
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chemotherapy in metastatic NSCLC213 demonstrates the |
antigenicity-enhancing therapies. |
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strength of this dual approach. |
The proteasomal machinery, which leads to the pro- |
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Targeted therapies. An insufficient TAA load could |
cessing of peptides and their presentation on human leu- |
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kocyte antigen (HLA) class I (MHC I) molecules, has |
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in principle impair the mounting of an efficient T cell- |
been pinpointed as a novel exploitable point of inter- |
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mediated immune response. Therefore, therapies that |
vention to increase antigenicity224. The set of epitopes |
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increase the antigenicity could prove beneficial in pro- |
presented for CD8 recognition are collectively termed |
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moting the recruitment of T cells to tumour sites and |
the ‘immunopeptidome’. However, compelling evidence |
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the subsequent elimination of tumour cells (Fig. 4). |
indicates that the proteasome hosts a more complex |
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Tumour antigenicity can be enhanced by therapies that |
mechanism of epitope splicing, defined as proteasome- |
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favour the re-expression of TAAs, for example, DNA- |
catalysed peptide splicing, by which the fusion of two |
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demethylating agents (such as 5-azacytidine (AZA)) |
non-contiguous peptidic segments generates novel |
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or epidermal growth factor receptor (EGFR) and MEK |
epitopes224. The proteasome-generated spliced peptide |
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inhibitors214. AZA is a cytosine analogue and a potent |
pool accounts for one-third of the entire HLA class I |
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DNA methyltransferase inhibitor that has been used for |
immunopeptidome in terms of diversity and one-fourth |
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many years to treat myelodysplastic syndromes. The fact |
in terms of abundance224. Intriguingly, this newly iden- |
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that it induced a late clinical response in some patients |
tified pool represents a unique set of antigens that bear |
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suggested the possible implication of the immune sys- |
distinguishing immunological characteristics224, which |
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tem in its mode of action215. In fact, AZA upregulated |
makes it an attractive target for future therapeutic |
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MHC I, β2m and cancer testis antigen genes, as well as |
manipulations. |
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genes involved in IFNγ signalling216. In ovarian cancer, |
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AZA also induces a cytosolic double-stranded RNA- |
DNA-repair-based therapy. High mutational and |
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dependent type I interferon response by increasing the |
putative neoantigen load correlate with clinical bene |
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expression of DNA hypermethylated endogenous retro- |
fit from immune checkpoint blockade therapy in |
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viruses (ERVs)217. AZA-induced ERV transcripts were |
lung cancer and melanoma225. Therefore, strategies |
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found in melanoma218 and endometrial cancer cells219. |
that increase the burden of neoantigens in tumour |
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Patients can be stratified according to their basal ERV |
cells could in principle be used in combination with |
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levels and antiviral gene. A high antiviral gene signa- |
subsequent checkpoint inhibition. This notion is sup- |
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ture was significantly associated with durable clini- |
ported by a recent mouse study by Germano et al. that |
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cal responses in patients with melanoma treated with |
involved the genetic inactivation of DNA mismatch |
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anti-CTLA4 therapy217. |
repair (MMR) protein MutL homologue 1 (MLH1) in |
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Inhibitors of EGFR, RET kinase and MEK are |
colorectal, breast and pancreatic cancer cells, ultimately |
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broadly used in the current clinical routine, despite the |
inactivating the cellular DNA MMR mechanisms, thus |
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lack of knowledge on their detailed molecular mech- |
inducing genomic instability and triggering immune |
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anisms of action. Their role as negative regulators of |
surveillance226. Apart from highlighting the importance |
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MHC I expression and antigen presentation machinery |
of neoantigen burden, this study points out the poten- |
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in multiple cancer types was revealed by a pooled short |
tial impact of strategies inhibiting the DNA damage |
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hairpin RNA interference-based analysis of the human |
response (DDR) in tumour cells226. The inhibition of |
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kinome214. In vivo studies demonstrated that activated |
the DDR has been previously proposed to sensitize |
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MAPK signalling inhibited components of MHC I and |
cervical cancer cells to subsequent chemotherapy |
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the antigen presentation machinery214. The use of MAPK |
and/or radiotherapy, thereby contributing to the suc- |
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inhibitors indeed enhanced the T cell-mediated killing |
cess of such treatments227. Numerous agents block- |
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of tumour cells214. |
ing DDR components, such as ATR serine/threonine |
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Proteins that prevent tumour cell apoptosis, includ- |
kinase, ATM (ataxia telangiectasia mutated), check- |
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ing members of the inhibitor of apoptosis protein |
point kinase 1 (CHK1), DNA-dependent protein |
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family220, MCL1 (ref.221) and mTOR222 (as well as IL-6 |
kinase (DNA-PK), p38 MAPK and MAPK-activated |
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(ref.223)), represent further possible targets. Overall, |
protein kinase 2 (MK2), are currently being evaluated |
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given their ability to increase tumour antigenicity, these |
preclinically and clinically228. |
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R e v i e w s
Adoptive cell therapy. Engineered T cells that express artificial chimeric antigen receptors (CARs) targeting a tumour cell surface molecule were first produced in 1993 (ref.229), but only since 2010 have they revealed their true potential as anticancer agents230. CAR-T cell-based therapies rely on the isolation, ex vivo manipulation and expansion of antigen-specific T cells, which are subsequently transferred to the same patient (through adoptive cell therapy)231. This method has shown sev-eral potential advantages over conventional therapies, including specificity, rapidity, high success rate and long- lasting effects232. The two presently approved therapies, as well as most of the current clinical trials based on this technology, are directed against CD19, a classical B cell malignancy-associated antigen.
Typically, CARs are transduced into T cells from the patient using randomly integrating vectors233, which is a limitation of this system as it may lead to undesired effects such as oncogenic transformation, variable expression levels and transcriptional silencing234,235. To circumvent these limitations, Eyquem et al. directed
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CD19-specific CAR to the TCR α constant (TRAC) locus by using CRISPR–Cas9-mediated genome editing, yielding a uniform CAR expression in human peripheral blood T cells236. By enhancing tumour rejection, such optimization of the CAR-T cell-based technology is an attractive option to adopt for future therapeutic designs.
A further optimization strategy to enhance the efficacy of adoptive T cell therapy was suggested by Hervas-Stubbs et al., and it relies on priming of naive CD8+ T cells with type I interferon237. Specifically, IFNα-primed CD8+ T cells show enhanced ability to persist and to mount a robust recall response compared with naive CD8+ T cells while preserving a low differ-entiation profile; in addition, they display heightened responsiveness to IL-15 and IL-7, which mediate T cell expansion and activation237. It could be envisaged that combing IFNα-primed ACT with subsequent cytokine (IL-15 and/or IL-7) administration might constitute a successful combinatorial therapy. Despite most studies on CAR T cells focusing on targeting CD19 (that is, B cell malignancies), a significant effort is being directed at identifying alternative candidates given the more lim-ited success against solid tumours. TSAs would consti-tute ideal targets because their cognate T cells, unlike T cells targeting TAAs, would not trigger undesirable autoimmune reactions238. Epitopes carrying driver somatic mutations would be the best candidates as they are TSAs and are critically involved in the process of malignant transformation238. However, their limited mutation frequency and the restrictions offered by all the steps of the antigen presentation pathway make
these tumour-specific mutated epitopes hard to target. The enlarged immunopeptidome spectrum recently revealed with the existence of spliced epitopes might therefore have a profound impact on the adoptive T cell therapy field238.
In a study by Verdegaal et al. that included two patients with stage IV melanoma, tumour-specific T cells were expanded by repeated stimulation with cell lines established from the resected lesion239. Albeit labo-rious, ACT proved to be effective, as both patients were
long-term survivors. It should be noted that melanoma represents the solid cancer type with the best response to ACT thus far43. The ACT was also useful to analyse the stability of neoantigen-specific T cell responses and the antigens they are directed against239. The study revealed a highly dynamic environment, in which the
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cell-recognized neoantigens selectively disappeared from the tumour cell population, with a concomitant development of neoantigen-specific T cells among the TILs239. Importantly, the authors suggested the occur-rence of T cell-mediated neoantigen immunoediting; therefore, a broad neoantigen-specific T cell response should be sought to avoid tumour resistance239. In fact, this is a concept that should be considered in all strat-egies aiming at inducing neoantigen-specific immune responses — the broader, the better.
The crucial question at this point is whether ACT therapies will be able to overcome failed spontaneous T cell priming and convert cold into hot tumours. This question was addressed in a non-T cell-inflamed β-catenin-expressing murine melanoma model by Spranger et al.240. The study revealed failed trafficking of tumour-specific effector T cells that had been adop-tively transferred into tumours owing to the absence of the T cell-recruiting chemokines CXCL9 and CXCL10 (ref.240). The same question was also addressed in patients with non-Hodgkin lymphoma undergoing anti-CD19 CAR-T cell therapy in a multicentre trial (ZUMA-1) and revealed that a stronger immune contexture predicted increased likelihood of response, supporting the notion that CAR-T cell therapy may not be enough by itself to treat patients with cold tumours241.
Taken together, these results further reiterate the idea that a deeper analysis of the TME and a consequent tumour classification are required before and/or during any therapeutic intervention.
Oncolytic therapy. Despite being known for nearly a century, the ability of viruses to kill tumour cells, and hence their therapeutic benefit in cancer patients, has been documented only recently in several clinical tri-als242 . Oncolytic viruses are native or genetically mod-ified viruses that selectively infect and replicate within tumour cells, eventually leading to tumour cell lysis242. Alongside this direct and local antitumour activity, oncolytic viruses can also induce a potent, systemic and potentially durable antitumour immunity242. The dying tumour cells release TAAs and additional DAMPs, thereby eliciting an efficient antitumour immune response242. In fact, the power of this approach lies in the ultimate engagement of systemic immunity, which results in therapeutic responses not only at the site of injection but also at distant tumour sites242.
Cancer cells represent a fertile environment for viral replication owing to their intrinsic abnormalities in the signalling pathways involved in cell stress and homeo-stasis and, possibly, in the antiviral machinery. The lat-ter should be carefully considered in individual patients as disease-induced deregulation of the host-antiviral mechanisms can influence the therapeutic activity of the oncolytic viruses. For example, protein kinase R (PKR), which helps in the clearing of intracellular viruses, may
R e v i e w s
Differentiation antigens
Antigens derived from proteins that are expressed in a given type of tumour and the corresponding healthy tissue, often in lower amounts.
be differentially expressed and/or activated in different cancer types242,243.
In order to be used therapeutically, oncolytic viruses have to be devoid of virulence factors yet retain their immunostimulatory abilities. Many viruses have been engineered accordingly and assessed in clinical trials, including adenoviruses, poxviruses, herpes simplex virus type 1 (HSV-1), coxsackieviruses, poliovirus, measles virus, Newcastle disease virus (NDV) and reovirus242. Many of the currently evaluated oncolytic viruses have a natural tropism for cell surface proteins overexpressed by cancer cells. Two examples are CD46 and HVEM, which are cell entry receptors for the Edmonton strain of measles virus and HSV-1, respectively242.
Talimogene laherparepvec (T-VEC) represents the first FDA-approved virotherapeutic approach for the localized treatment of patients with unresectable melanoma. As mentioned above, a potentially thera-peutic strategy to ‘heat up’ cold tumours could lie in the use of an oncolytic virus as priming therapy, combined with the removal of co-inhibitory signals (Fig. 4). Indeed, T-VEC administration in combination with ipilimumab (an anti–CTLA4 antibody) in patients with unresecta-ble stage IIIB–IV melanoma yielded greater antitumour activity than ipilimumab alone244. A phase Ib study using T-VEC followed by the anti-PD-1 antibody pembroli-zumab in patients with advanced melanoma resulted in an outstanding response rate of 62%245. Patients who responded to treatment with T-VEC displayed increased CD8+ T cells, high mutational burden and high expres-sion of PD-L1 protein and IFNG in several cell subsets in tumours245, suggesting the need for a pre- existing tumour-specific T cell pool for the anti-PD-1 therapy to be effective246. Importantly, this improved anti tumour activity did not come at the expense of the safety profile244,245. Despite being highly encouraging, the per-centage of responders to the combination treatment shows still room for improvement and further points of interventions should be envisaged. A possible rea-son for therapeutic failure could lie in the possible poor antigenicity (low TAAs) of cold tumours; in this case, virion-mediated lysis of cancer cells may not release enough TAAs to prime antitumour T cell responses. Vaccine-based therapy (discussed in the next section) may represent a more suitable option in this context.
Another intratumour therapeutic approach to achieve an abscopal effect is offered by the use of oncolytic pep-tides. The nine amino acid residue (9-mer) oncolytic peptide LTX-315 acts on both drug-resistant and drug- sensitive cancer cells, rather than healthy cells, thereby causing the lysis of their plasma and organelle mem-branes, which act as danger signals initiating an innate and subsequent systemic adaptive immune response247. The intratumour injection of LTX -315 resulted in a complete rejection of fibrosarcomas established in a rat model. This tumour rejection relied on T cell infiltration in both injected and distal tumour sites247.
Physical barriers (including necrosis, calcification, hypoxia, acidosis, increased proteolytic activity, high interstitial pressure, poor vascularization and/or dense extracellular matrix) may reduce the spread of oncolytic viruses, limiting their biodistribution and consequent
therapeutic efficacy242. Although this limitation can be overcome in physically accessible tumours by intra tumoural injections, the same factors could limit T cell trafficking and the establishment of a successful immune response. In other words, it is reasonable to hypothesize the existence of cases in which such priming therapy only ‘promotes’ a tumour to an altered (excluded in this example) phenotype; this will then additionally require not only an ICI but also an additional strategy (such as anti-VEGF) to become hot.
Vaccine-based therapy. After an initial identification of neoantigens from tumour cells, putative antigen or antigenic epitope or epitopes can be presented through a variety of platforms, such as whole tumour cell prepara-tions, through MHC-specific peptides, whole or partial proteins encoded by RNA or DNA, or in recombinant viral or bacterial vectors expressed in DCs248 . The use of additional vaccine adjuvants, such as TLR agonists, could boost the immune response against TAAs. Despite seemingly valuable, anticancer vaccines turned out to be quite disappointing, as proved by the low overall objec-tive response rate obtained in numerous clinical trials248(Fig. 2c). However, this lack of success translated into a quest for the reasons underlying this ineffectiveness. A key aspect to consider is that anticancer vaccines were tested in patients with established cancers, in which immunosuppressive mechanisms were already in place. Therefore, the increase in ICIs brought along a renewed interest in these therapeutic approaches248. Indeed, stud-ies on ICIs highlighted the positive correlation of the somatic mutation burden and consequent emergence of neoantigens with clinical benefit225,249, providing a rationale for combination therapies involving ICIs and T cell priming anticancer vaccines (Fig. 4). ICIs would de facto play the part of vaccine adjuvants. It is tempting to speculate that future studies involving the combina-tion of T cell boosting tumour vaccines with the T cell suppression-preventing ICIs may translate into clinical benefit in patients with cold tumours.
A challenging aspect of anticancer vaccination is finding the optimal antigen for vaccination. Among the possible candidates are overexpressed self-proteins (such as prostate-specific antigen (PSA)), differentiation antigens (such as protein Melan-A or gp100 (ref.250)) and mutated antigens (neoantigens)248. Thus far, there is only one FDA-approved vaccine, sipuleucel-T, for the treatment of asymptomatic or minimally symptomatic hormone- refractory prostate cancer. Sipuleucel- T consists of autologous DCs loaded with recombinant human fusion protein encoding the prostatic acid phosphatase (PAP) antigen (the expression of which increases with pros-tate cancer progression) and granulocyte–macrophage colony-stimulating factor (GM-CSF) to sustain DC maturation.
The selective detection of cytomegalovirus (CMV) antigens in certain types of tumour (such as glioblas-toma (GBM)) but not in normal tissue suggested the opportunity to use immunological interventions that target these viral proteins. Accordingly, vaccination with CMV pp65 RNA-pulsed DCs was developed to treat GBM251. Unfortunately, this approach did not
R e v i e w s
Neoantigen fitness
The likelihood of a peptide to be immunogenic, as measured by its binding affinity to major histocompatibility complex (MHC) and subsequent recognition by T cells.
translate into higher clinical benefit than the stand-ard of care251,252 . However, a clear clinical benefit was observed when preconditioning the vaccine site with tetanus–diphtheria (Td) toxoid; such a potent recall anti-gen significantly improved lymph node homing and the efficacy of tumour antigen-specific DCs251. Interestingly, the blinded interim data of the overall patient popula-tion enrolled in a phase III randomized, double-blind, placebo-controlled clinical trial of an autologous tumour lysate- pulsed DC vaccine (DCVax-L) for newly diag-nosed GBM revealed extended survival253. Saying that tumour vaccines could per se provide sufficient basis to convert a cold into a hot tumour is an overstatement, as this example clearly shows. Notwithstanding, it shows once again the power of a multifactorial approach and opens the intriguing perspective of exploiting past vaccinations against infectious diseases.
Other vaccines targeting defined tumours or TAAs are currently being evaluated as they present obvious advantages, including the possibility of mass production, easy monitoring of immune responses and a generally tolerable safety profile248.
The broad range of neoantigens and their positive association with improved clinical responses suggested their possible use for vaccination. Furthermore, as new epitopes appear continuously during tumour progres-sion, immune editing and T cell suppression seem unlikely as they require time248. A neoantigen fitness model, which measured the likelihood of neoantigen presentation by the MHC and subsequent recognition by T cells, predicted survival in patients with melanoma treated with anti-CTLA4 therapy and patients with lung cancer treated with PD-1 blockade254. Newly identified low-fitness neoantigens could constitute ideal targets for developing novel cancer vaccines254. A difficulty of this approach is its intrinsic personalized nature — hence the bench-to-bedside time frame. Nonetheless, the development and optimization of high-throughput screening techniques and epitope-predicting algorithms may allow for such strategy. The recently reported RNA-based poly-neo-epitope approach used in 13 patients with melanoma made the concept of indi-vidualized mutanome vaccines a promising reality255. All patients completed treatment with a maximum of 20 neo-epitope vaccine doses, which were well tolerated, and developed T cell responses against at least three mutations, resulting in sustained progression-free sur-vival255. This strategy identified a patient in which the combination of the vaccine and PD-1 blockade yielded a complete response. Another study demonstrated the feasibility, safety and immunogenicity of a vaccine that targets up to 20 predicted personal tumour neoantigens. Of six vaccinated patients, four had no recurrence at 25 months after vaccination, whereas two with recur-rent disease were subsequently treated with anti-PD-1 treatment and experienced complete tumour regression, with expansion of the repertoire of neoantigen-specific
-
cells256. Apart from the undoubted merit of paving the way to personalized vaccine-based immunother-apy, this study supplied evidence of the potential of the combinatorial therapeutic approach with current immunotherapies.
T cell immunomodulators. Several cytokines (such as IL-2, IL-7, IL-15, IL-21, IL-12, GM-CSF and IFNα) are known to modulate T cell expansion, survival and/or function and were or are being tested in clinical trials as potential anticancer agents, in monotherapy or in combi-nation257. IL-2 was the first cytokine to be characterized as a ‘T cell growth factor’ (TCGF) in 1976 (ref.258). IL-2 became an early candidate for cancer immunotherapy and showed promising results as a single agent in met-astatic RCC and melanoma258. However, its pleiotropic effects on the immune system (notably, the targeting of Treg cells) as well as the severe associated side effects greatly reduced its importance as a single anticancer agent258. Combinations with traditional anticancer ther-apies as well as immunotherapies, including ACTs, have since been investigated but displayed similar limitations to the monotherapy. A renewed interest in IL-2 came with the development of a strategy to redirect the speci-ficity of IL-2 towards adoptively transferred T cells259. By using orthogonal IL-2 cytokine–receptor pairs (mutant IL-2 and IL-2 receptor (IL-2R)), this approach enables selective expansion of desired T cells, with negligible
off-target effects and toxicity259.
The IL-2 -related cytokine IL-15 has lately attracted attention in the cancer immunotherapy field as it does not target Treg cells but NK cells and lacks other unde-sired features of IL-2 (ref.260). Reduced IL-15 expression due to chromosomal instability impaired the intratu-moural T and B cell proliferation and correlated with higher risk of tumour recurrence and reduced survival of patients with CRC18 , providing a clear indication of its key anticancer role. Current efforts aim at ensuring sufficient bioavailability, which is low at present owing to rapid renal clearance260. ALT-803 is a super-agonist of the IL-15–IL-15Rα complex that successfully pro-longed the half-life of IL-15 and promoted enhanced immune activation in vivo261. ALT-803 was well tolerated and resulted in clinical responses in patients with hae-matological malignancies who relapsed after allogeneic haematopoietic cell transplantation261.
IL-7 is yet another potent growth, activation and survival factor for CD8+ T cells that could improve anti-tumour CD8+ T cell responses262. Owing to its features, it is not surprising that recombinant IL-7 (rIL-7) was considered as a tool to improve ACT263. Indeed, an ACT mouse model demonstrated the efficacy of rIL-7 in inducing CD8+ T cell proliferation and differentiation, and tumour rejection, in an IL-7R-dependent manner263. Combining T cell-modulating cytokines with other approaches could improve the therapeutic success. This possibility was demonstrated by an in vivo study that featured a modified NDV that co- expressed IL15 and IL7 (ref.264). Such a tumour vaccine displayed improved antitumour activity compared with a non-modified vaccine264.
IL-21 is a promoter of T cell responses265 and could be exploited for its potential antitumour activity. IL-21 counteracts the in vitro TGFβ1-induced FOXP3 expres-sion in purified naive CD4 T cells, confirming its known additional ability to alleviate Treg -mediated immuno-suppression in several cancers266 . Apart from targeting T cells, IL-21 also regulates TFH (ref.267) and B cells265.
R e v i e w s
Together with CXCL13, IL-21 is a crucial element reg-ulating the TFH–B cell axis within the TME in CRC, and its presence correlated with increased patient survival24. In addition, Lewis et al. demonstrated the synergistic antitumour activity of IL-21 and anti- CTLA4 and/or anti-PD-1 therapy in various tumour models268. The decreased percentages of intratumour CD4+CD25+FOXP3+ T cells were accompanied by increased CD8+ T cell infiltrates, CD8+ T cell prolifer-ation, increased levels of effector memory T cells and overall enhanced antitumour activity268. Combining the adoptive transfer of IL-21-primed, melanoma-reactive CD8+ T cells with anti-CTLA4 therapy controlled refrac-tory metastatic melanoma in a patient269, showing the potential of IL-21 in combinatorial anticancer therapies.
Concluding remarks
The concept of personalized cancer immunotherapies has been ever-growingly advocated in recent years. Possibly the biggest challenge impeding the achieve-ment of such an ambitious approach lies in the lack of a comprehensive knowledge of the cancer–immune interaction parameters; even when this knowledge
is available, standardized methods of measuring most parameters are lacking. Accurate measurement of parameters is quite crucial, as their relative ‘weight’ varies considerably among individual patients62. It is clear that the identification of key features, immune- related or tumour-related, at the moment of diagnosis is needed to build a solid classification strategy supporting subsequent therapy. Here, we provide a panel of thera-peutic strategies to be deployed or developed (if not yet available) against hot, altered and cold tumours. It is reasonable to assume that the colder the tumour is, the more approaches are needed. Interestingly, all proposed therapeutic designs ultimately involve combinations with immunotherapies to achieve maximal efficiency. Given the pivotal role of T cells against cancer, the care-ful assessment of the pre-existing T cell landscape and of the immune microenvironment should be routinely used to differentiate specific cases, not only in the clin-ical setting but also at the preclinical stage. This infor-mation could help in the translation of study findings in clinical indications.
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Acknowledgements
The authors thank the following institutions for their financial support: the National Cancer Institute of France (INCa), the Plan Cancer, the Canceropole Ile de France, INSERM, Cancer Research for Personalized Medicine (CARPEM), the Paris Alliance of Cancer Research Institutes (PACRI), H2020 PHC-32-2014 APERIM grant number EEAA15006DDA, MedImmune (grant number RVE15004DSA) and LabEx Immuno-oncology.
Author contributions
The authors contributed equally to all aspects of the article.
Competing interests
Immunoscore is a registered trademark from INSERM. J.G. is co-founder and chairman of the scientific advisory board of HalioDx. J.G. has patents associated with an ‘in vitro method for the prognosis of progression of a cancer’ (PCT/ IB2006/003168 and PCT/EP2013/062405). J.G. estab-lished Collaborative Research Agreement (grants) with Perkin-Elmer, IO Biotech, MedImmune, Astra Zeneca, Janssen, Imcheck Therapeutics. J.G. participated to Scientific Advisory Boards of BMS, MedImmune, Astra Zeneca, Novartis, Definiens, Merck Serono, IO Biotech, ImmunID, Nanostring, Illumina, Northwest Biotherapeutics, Actelion, Amgen, Kite Pharma and Merck MSD. J.G. was a consultant for BMS, Roche, GSK, Compugen, Mologen and Sanofi.
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