The transition from HLA-I positive to HLA-I negative primary tumors: the road to escape from T-cell responses

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ScienceDirect The transition from HLA-I positive to HLA-I negative primary tumors: the road to escape from T-cell responses Natalia Aptsiauri2,3, Francisco Ruiz-Cabello1,2,3 and Federico Garrido1,2,3 MHC/HLA class I loss in cancer is one of the main mechanisms of tumor immune escape from T-cell recognition and destruction. Tumor infiltration by T lymphocytes (TILs) and by other immune cells was first described many years ago, but has never been directly and clearly linked to the destruction of HLAI positive and selection of HLA-I negative tumor cells. The degree and the pattern of lymphocyte infiltration in a tumor nest may depend on antigenicity and the developmental stages of the tumors. In addition, it is becoming evident that HLA-I expression and tumor infiltration have a direct correlation with tumor tissue reorganization. We observed that at early stages (permissive Phase I) tumors are heterogeneous, with both HLAI positive and HLA-negative cancer cells, and are infiltrated by TILs and M1 macrophages as a part of an active anti-tumor Th1 response. At later stages (encapsulated Phase II), tumor nests are mostly HLA-I negative with immune cells residing in the peri-tumoral stroma, which forms a granuloma-like encapsulated tissue structure. All these tumor characteristics, including tumor HLA-I expression pattern, have an important clinical prognostic value and should be closely and routinely investigated in different types of cancer by immunologists and by pathologists. In this review we summarize our current viewpoint about the alterations in HLA-I expression in cancer and discuss how, when and why tumor HLA-I losses occur. We also provide evidence for the negative impact of tumor HLA-I loss in current cancer immunotherapies, with the focus on reversible (‘soft’) and irreversible (‘hard’) HLA-I defects. Addresses 1 Servicio de Analisis Clinicos e Inmunologia, UGC Laboratorio Clinico, Hospital Universitario Virgen de las Nieves, 18014 Granada, Spain 2 Instituto de Investigacion Biosanitaria ibs, 18014 Granada, Spain 3 Departamento de Bioquimica, Biologia Molecular e Inmunologia III, Facultad de Medicina, Universidad de Granada, Spain Corresponding author: Garrido, Federico (federico.garrido. [email protected]) Current Opinion in Immunology 2018, 51:123–132 This review comes from a themed issue on Tumour immunology Edited by Jewett

https://doi.org/10.1016/j.coi.2018.03.006 0952-7915/ã 2018 Elsevier Ltd. All rights reserved.

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Introduction After decades of investigation of different types of MHC-I alterations in human and experimental tumors by different research groups, including ours [1–10], it has become evident that the MHC-I/HLA-I altered phenotypes frequently observed in cancer cells are not just a random epiphenomenon associated with tumor progression, but represent a crucial event that determines the fate of tumor development: either its rejection or escape from T-cell mediated response. It is well established that cytotoxic T lymphocytes play a major role in controlling and destroying nascent tumors [11,12]. These effector cells are in the focus of the clinical responses to the traditional and more recent types of immunotherapy, including monoclonal antibodies against immune checkpoint inhibitors involved in the regulation of T-cell cytotoxicity [13,14,15,16,17]. T cells require interaction with tumor MHC-I molecules to recognize tumor antigens processed and presented as small peptides. Therefore, any alteration in the expression of MHC-I undoubtedly will have a profound implication in the primary tumor growth and in the metastatic colonization. A Darwinian type of T-cell mediated immune selection is responsible for the escape of HLA-I deficient tumor cells. The classical idea of ‘Generation of Diversity [GOD] and Selection’ can be also applied to the tumor development. An explosion of MHC-I diversity takes place in the primary tumor and is followed by an immune selection and destruction of tumor cells by cytotoxic T-lymphocytes via recognition of the MHC-I/tumor peptide complex [18]. As a result, primary tumor becomes composed mostly of MHC-I negative/deficient tumor cells. During this process of cancer progression tumor tissue is undergoing remodeling and is being re-shaped by the immune microenvironment. Cancer immunotherapy is currently the most rapidly advancing area of clinical oncology due to durable responses observed in some patients treated with different types of monoclonal antibodies directed against molecules regulating T-cell activity. However, little attention is being paid to the status of tumor HLA-I expression before, during and after immunotherapy. The success of cancer immunotherapy relies on better understanding the mechanisms of cancer immune escape and the dynamic Current Opinion in Immunology 2018, 51:123–132

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changes of the tumor microenvironment during the transition from pre-existing immune response to a therapyinduced immune response. For many years our laboratory has been actively engaged in defining altered HLA-I phenotypes in human tumors and the underlying molecular mechanisms of HLA loss or downregulation. We also have been investigating how these alterations influence the response to cancer immunotherapy by correlating them with the regression or progression of post-therapy metastatic lesions [19,20]. Recently, we observed in lung carcinoma that positive HLA-I expression on tumor cells correlate with massive diffuse infiltration of tumor mass with T-lymphocytes, while loss of tumor HLA-I expression is accompanied by the absence of CD8+ T cells within the primary tumor, which is surrounded by a fibrous stroma resembling a granuloma-like structure [21,22]. We strongly believe that it is necessary to analyze tumor HLA-I expression and monitor HLA-I changes taking place during immunotherapy to understand how, when and why the MHC/HLA alterations occur, as well as to improve the efficacy of immunotherapy and minimize resistance to treatment.

How the MHC/HLA-I alterations develop It is well established that there are multiple molecular mechanisms responsible for the generation of HLA-I altered tumor escape phenotypes. In theory, any step required for the assembly of a functional HLA-I molecule can be altered causing HLA-I loss and providing an escape route from T-cell recognition. Nevertheless, there are some common well characterized mechanisms shared by different types of malignancy and specific alterations distinctive for some particular cancers. Loss of heterozygosity (LOH) in chromosome 6 and 15 (LOH-6 and LOH-15), which include the HLA and the b2-microglobulin (b2m) regions, respectively, is the most widespread mechanism. It is responsible for HLA-I haplotype loss, which leads to expression of only three HLA alleles on the tumor cell surface. We have described this mechanism in tumors of different histological type, including lung carcinoma [21,23–28]. In some tumors (melanoma, MSI-H colorectal carcinoma and bladder cancer) this chromosome instability is a frequent and early event that can later be complemented by other abnormalities, such as HLA-I transcriptional downregulation or b2m mutation, altogether leading to a total HLA-I loss [24,29,30]. We favor the idea that the frequency of LOH-6 and LOH-15 affecting the HLA and b2m genes is greatly underestimated. Recent data obtained in our laboratory using GCH arrays indicate that tumor cells with apparently ‘normal’ HLA expression, yet harbor HLA gene microdeletions that had been previously undetected by immunohistology and classical microsatellite amplification analysis (unpublished data). In addition, the modern NGS technology that detects HLA-I and b2m mutations in recurrent metastatic lesions, is not informative in case Current Opinion in Immunology 2018, 51:123–132

of macro or micro deletions of genetic material in chromosome 6 and 15. Different altered HLA-I phenotypes observed in human tumors were summarized long time ago by our group [5,7,31] and recently revisited [32]. They include: Tumor HLA phenotype no. I: HLA class I total loss. It is observed in all analyzed tumors with different frequencies and different molecular mechanisms. In melanoma and in MSI-H colorectal carcinoma it has been reported in around 15% of the tumors and is associated with b2m loss due to LOH-15 and a mutation in the second allele of the b2m gene. Similar mechanisms have been reported in B-cell lymphomas [33]. In MSS colorectal cancer the molecular mechanisms appears to be different and caused by a downregulation of the antigen presentation machinery (APM) components [34]. In breast and prostate carcinoma total HLA-I loss is observed in more than 50% of tumor samples analyzed [35,36]. HLA-I loss associated with a coordinated transcriptional downregulation of APM and HLA-I/ b2m genes has been observed in prostate, bladder and lung carcinomas [36,37,21]. Tumor HLA phenotype no. II: HLA haplotype loss. It frequently occurs as a result of the loss of genetic material at chromosome 6 (LOH-6) [28]. Often a duplication of the remaining chromosome masks the loss of heterozygosity, which in that case can be detected only by a direct HLA typing using DNA isolated from microdissected tumor tissue. This is a common molecular mechanism that occurs at early stages of tumor development and has been reported in about 30% of different types of malignancy, including melanoma, colorectal, lung, pancreas, laryngeal, breast, and bladder cancer [23–28]. These are irreversible (‘hard’) alterations, which cannot be restored with any type of conventional therapy, including immunotherapy. Tumor HLA phenotype no. III: selective HLA-A, HLA-B or HLA-C locus downregulation. It is a common mechanism observed in different human tumors and tumor cell lines [2,38]. Generally, it can be reverted by cytokines, which increase HLA-I gene transcription and lead to a subsequent upregulation on cell surface. Tumor HLA phenotype no. IV: single HLA-I allele losses. Mutations in genes coding for HLA-I alleles produce loss of the cell surface expression of one single HLA-A, HLA-B or HLA-C allele. It has been described in cervical [10], prostate [39], colorectal [40] tumors and in melanoma cell lines [41]. Tumor HLA phenotype no. V: a compound phenotype. It is a frequent phenotype observed in human tumors, when more than one molecular mechanism contribute to the generation of the aberrant HLA-I expression. In certain conditions, a combination of HLA haplotype loss with a transcriptional downregulation of HLA locus www.sciencedirect.com

The transition from HLA-I positive to HLA-I negative primary tumors: the road to escape from T-cell responses Aptsiauri, Ruiz-Cabello and Garrido 125

leaves only a single HLA-I allele on cancer cell surface [5,7]. We discovered that HLA-I haplotype loss together with a transcriptional downregulation of HLA-A, HLA-B and HLA-C genes is a common mechanism responsible for a total HLA-I loss in approximately 60% of small cell lung carcinoma samples studied [21]. Tumor HLA phenotype no. VI: Resistance to Interferon. IFN-g secretion by cytotoxic T-cells is critical for tumor rejection and can induce tumor HLA-I upregulation. Sometimes cancer cells develop resistance to IFN due to a variety of genetic lesions affecting the IFN-g or IFN-a signaling pathway. This alteration has been described in different tumor cell lines [42] and in recurrent melanoma lesions after immunotherapy [43]. Based on the underlying molecular mechanism and the ability to recover normal HLA-I expression by cytokines or after immunotherapy, these different molecular mechanisms responsible for HLA-I alterations can be classified in two major groups, namely, reversible (‘soft’) or irreversible (‘hard’) alterations. This classification has profound clinical implications in the prediction of patients’ response to cancer immunotherapy [44]. We have reported that melanoma regression was more frequent in patients with reversible regulatory tumor HLA-I alterations than in those with irreversible HLA-I gene defects [19]. Cytokine-mediated reversion of tumor HLA-I expression can restore immunogenicity and contribute to T-cell mediated rejection. In some cases interferons work together with other anti-cancer drugs in upregulating HLA-I expression. On this context, an upregulation of HLA-I expression by interferon and MEK1/2 inhibitor selumetinib was recently reported in papillary thyroid tumors with ‘soft’ mechanism of HLA loss [45]. Despite the enormous progress, there is a long way to go before the different molecular mechanisms responsible for HLA-I alterations are precisely defined in different tumor types. For example, the mechanisms responsible for total HLA-I loss [phenotype no. I] in about 60% of breast cancer patients, in 50% of prostate cancer patients, in 15% of patient with laryngeal cancer, or in 40% of patients with gastric cancer is still to be identified [5,7,32]. We have recently reported that the tumor suppressor Fhit positively regulates MHC-I expression on cancer cells [46]. Another molecule, a transcription factor NLRC5 recently reported as a candidate responsible for HLA-I expression regulation, has been identified in different tumors [47]. These transcriptional regulators could be used to correct HLA-I ‘soft’ lesions.

When the HLA-I alterations occur Based on our earlier observations, we believe that HLA-I loss is an early event that occurs when the primary tumor is only a few millimeters in diameter and has already www.sciencedirect.com

induced T-cell mediated immune selection. However, this hypothesis still needs a complete experimental and clinical confirmation [21,48,49]. It is becoming apparent that the immunological status of the host plays a major role in selecting the MHC/HLA-deficient tumor cells. In this context, we have data obtained in a H-2 MHC-I negative clone of the GR9 mouse fibrosarcoma indicating that spontaneous lung metastasis produce different MHC-I phenotypes depending on the host immune system: H-2-negative lesions in immune competent mice and H-2-positive metastases in mice lacking T-cells [50,51]. It is likely that recruitment of T lymphocytes and tumor infiltration is influenced by many factors, including tumor antigenicity. The co-existence of HLA-I positive, heterogeneous and completely HLA-I negative tumor lesions in the same cancer patient could represent different stages during tumor development indicating an ongoing T-cell mediated immune selection.

Why the MHC/HLA-I alterations occur: is the transition from HLA-I positive to HLA-I negative tumors during cancer progression inevitable? Generation of MHC-I deficient tumor cells is believed to be a consequence of T-cell immune selection in primary heterogeneous tumors. During this process highly antigenic MHC-I positive tumor cells are eliminated, while cells with low or negative MHC-I expression are selected for survival. We observed a diversity of MHC-I phenotypes in mouse fibrosarcomas induced by methylcholanthrene and adapted to tissue culture without a single passage [52]. This initial stage of the ‘explosion of MHC-I diversity’ shifts to the outgrowth of tumor lesions composed only of MHC-I negative cancer. This transition is directed by the T-cell mediated host immune response [18]. Similar findings were nicely demonstrated by Boesen and colleagues in T-cell deficient mice [11]. In a case of metastatic melanoma we were able to illustrate a chronological sequence of the immune escape of HLAI-negative tumor cells with LOH-15 and b2m gene mutation in several successive lesions. This case clearly demonstrates a transition from HLA-I positive/heterogeneous to HLA-I negative tumor phenotype, which was linked to the presence of tumor infiltrating CD8+ T-cells [53]. Similarly, another melanoma study also demonstrated how poorly immunogenic tumor phenotypes with HLA-I defects evolve during the disease progression due to the early emergence of an inactivating mutation in one allele of the b2m gene and the simultaneous loss of the other allele by a deletion in chromosome 15 (LOH-15) [54]. T-cell immune selection may not be always responsible for the appearance of HLA-I negative tumor cells. It is well established that different viral or cellular oncogene products can also cause HLA-I downregulation as a mechanism responsible for cancer escape and growth, Current Opinion in Immunology 2018, 51:123–132

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as it has been shown in cervical or breast carcinoma [55,56]. In these malignancies, HLA-I loss probably cannot be exclusively attributed to T-cell-mediated immune selection, and may explain the observed poor T-cell infiltration. Similarly, tumor immune escape implicated in renal cell carcinoma (RCC) is likely to be T-cell independent, since according to some reports RCC cells express HLA-I molecules while the renal tubules, normal tissue from which the tumor derive, are HLA-I negative [57]. Nevertheless, most tumors derive from HLA-I positive normal epithelia [3]. The absence of HLA-I expression in the majority of tumors can be defined as a mandatory step during tumor development since the frequency of this phenomenon is very high, reaching up to 95% of analyzed cases according to published literature [3,5,10]. It is also widely acknowledged that T-lymphocytes are playing a crucial role in destroying nascent tumors, and without MHC/HLA-I expression this destruction cannot take place [11,22].

Changes in tumor tissue architecture following anti-tumor T-cell mediated responses: from HLA-I-positive/Th1-type to HLA-I-negative/Th2-type granuloma-like tumor structure The tumor microenvironment, including the immune infiltrate and tissue organization, undergoes dynamic changes during tumor growth and progression. As a result of the T-cell anti-tumor immune response and cancer immunoediting, there is a transition from a highly immunogenic to a low immunogenic tumor phenotype. This includes a gradual loss of antigen-presentation ability of tumor cells due to the accumulation of HLA-I alterations. It is also characterized by changes in the immunological composition, the density, functional state and distribution of the leukocyte infiltrate of the tumor, all leading to certain tissue re-organization. Tumor-infiltrating lymphocytes have been associated with favorable clinical outcomes in various solid tumors [58]. We have recently reported a positive correlation between tumor HLA-I expression, immune infiltration, and tumor tissue architecture in lung cancer [21,22,32]. Figure 1 summarizes our current view on these changes. It shows a schematic representation of the process of a gradual loss of tumor HLA-I expression and tumor tissue reorganization linked to the presence of cytotoxic T-cells. At the beginning, tumor cells are MHC/HLA-I positive and are densely infiltrated by T-lymphocytes normally associated with a Th1 type of cytokine production (‘permissive’ Phase I) (Figure 2a). This immune infiltration is responsible for the destruction of HLA-I positive cancer cells and selection of MHC/HLA-I negative cells to proliferate. When T-cell response fails to destroy all tumor cells, the host tries to isolate tumor by building up a stromal barrier around it, which restrains the ‘jobless’ T-lymphocytes Current Opinion in Immunology 2018, 51:123–132

and other immune cells, such as macrophages, from entering tumor mass. This encapsulated tumor structure resembles a classical granuloma formed in response to infection with bacteria or parasites, which is characterized by a Th2 type of immune response and M1/M2 macrophage polarization (non-permissive/encapsulated Phase II) [59,60] (Figure 2b). We found a similar M1/M2 macrophage polarization in lung cancer typical for a Th2 type immune response [21,32]. Different authors, including ourselves, reported that the degree of tumor infiltration with T-lymphocytes directly correlates with the number of HLA-I positive tumor cells in lung and pancreatic cancer [21,61,62], suggesting that there is a continuous T-cell activation and selection of HLA-I negative tumor cells (Figure 3). An important question arises as how widespread among different tumors is the transition from the ‘permissive’ Phase I to the ‘encapsulated’ Phase II, or a transition from a Th1 type response in HLA-I positive tumors to a Th2 type immune landscape characterized by a formation of stromal capsule around HLA-I negative tumor nest promoting M2 polarization (Figures 1 and 2). To answer this question further studies deciphering the precise and sequential cellular and molecular events that lead to this transition are necessary.

T-cell mediated immunotherapy selects cancer cells harboring irreversible/‘hard’ HLA-I defects Cancer immunotherapy has historically used a variety of biological products that are potentially able to boost antitumor T-cell mediated immune responses, which include BCG, IL-2, autologous tumor vaccines, dendritic cells loaded with peptides, tumor peptides alone, polysaccharide K[PSK], adoptive cell therapy with TILs, or checkpoint inhibitors [63,64,65]. All these therapies have demonstrated various degrees of clinical benefit in metastatic cancer patients, including complete and partial responses with both regressing and progressing lesions. In most cases it is not possible to define why some metastatic lesions progress and others regress. We analyzed in detail the HLA-I expression in progressing and regressing melanoma metastatic lesions in patients treated with autologous vaccination [19,20] and found a strong and direct correlation between high tumor HLA-I expression and regression of the metastasis, whereas the progressing lesions were harboring irreversible HLA-I defects. Furthermore, genome-wide gene expression analysis of regressing and progressing melanoma lesions showed that only 146 genes were differentially expressed in both types of lesions. These genes include IFN and HLA genes suggesting the crucial role they play in tumor rejection as a part of an immunological constant of rejection [20,66]. The predictive value of the molecular mechanisms leading to HLA-I aberrant www.sciencedirect.com

The transition from HLA-I positive to HLA-I negative primary tumors: the road to escape from T-cell responses Aptsiauri, Ruiz-Cabello and Garrido 127

Figure 1

Transition from HLA-I-positive to HLA-I-negative pattern during tumor developement PERMISSIVE PHASE I

Tumor Initiation

T cell infiltration

T cell immuno-selection of HLA-I-negative tumor cells

Th-1 like Response HLA-I-positive

HLA-I positive Tumor Cell

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ENCAPSULATED PHASE II

Th-2 like Response HLA-I-heterogeneous HLA-I + / –

HLA-I-negative Granuloma-like tumor structure

CTL Current Opinion in Immunology

Tumor tissue reorganization during T-cell mediated immune response. In phase I the tumor is heavily infiltrated by T-cells, including CD4+, CD3+, CD8+ lymphocytes and CD206+ M1 macrophages. Tumor is heterogeneous with both HLA-I positive and HLA-negative cells, and has a diffused structure of the parenquima and stroma typical for a Th1-type immune response and T-cell mediated cytotoxicity. In phase II the process of T-cell mediated destruction of HLA-I positive tumor cell finishes and the ‘jobless’ T-cells reside in the stroma that surrounds the tumor nest. In this phase the escaped tumor cells are all HLA-I negative and are encapsulated by the stroma. This structure resembles a granuloma that is formed around pathogens (bacteria or parasites) as a part of a Th2-type of immune response.

expression becomes essential for circumventing cancer resistance to immunotherapy. Interestingly, we have just found that opposite to tumor HLA-I phenotype, PDL-1 expression in cancer cells is not guided by T-cell immunoselection, and its expression is random in the analyzed lung cancer tissue samples [unpublished data]. HLA-I expression may be upregulated by immunomodulating treatment only in the case of reversible/‘soft’ molecular HLA-I aberrations [67]. In this context, we have recent indications that antibodies against EGFR used in experimental tumor models can upregulate MHC-I molecules [68]. In contrast, HLA-loss variants with irreversible/ ‘hard’ defects escape from T-cell recognition and develop progressing therapy-resistant metastasis. This assumption has been recently confirmed in a study in which b2m mutation was identified among frequent mutations associated with acquired resistance to PD-1 blockade [43]. Considering that NK cells are capable of killing MHC-I negative tumors, a question arises ‘why NK cells are not www.sciencedirect.com

killing these MHC-I-negative targets?’ This question still does not have an answer. We know that NK cells do not infiltrate MHC-I negative tumors; they remain outside the tumor nest, which suggests that these tumors develop inhibitory mechanisms that block NK cell activation and migration [22]. Most NK cells are defective in cancer patients, even at early stages of tumorigenesis, which implies that MHC-I-negative tumor clones are not able to be fully eliminated. Loss of both NK and T cell functions which occurs during the cancer progression will likely contribute to the survival of tumors with no/low MHC-I. The common ‘hard’ alterations include HLA-I haplotype loss associated with LOH-6 or LOH-15 together with mutations/deletions in the remaining copy of b2m gene, or mutations in IFN signaling pathways [44]. The recovery of MHC/HLA-I expression in immunotherapy-resistant recurrent tumors with ‘hard’ HLA-I Current Opinion in Immunology 2018, 51:123–132

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Figure 2

(a)

HLA-I

HLA-I positive tumor

CD3

(b)

HLA-I

CD45

CD8

HLA-I negative tumor

CD3

CD45

CD8

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Immunohistochemistry of a lung carcinoma tissue immunostained with anti-HLA-I, anti-CD45, anti-CD3 and anti-CD8 antibodies: (a) HLA-I positive tumor with diffused tumor architecture and tumor infiltrating T-cells (Permissive Phase I). (b) HLA-I negative encapsulated tumor (Phase II) with tumor T-cells in the stroma surrounding tumor nests.

alterations or the development of novel strategies to target HLA-I deficient cancer cells is a major challenge for the future of cancer treatment. One possibility could be gene therapy aimed to restore tumor HLA-I expression and T-cell recognition by replacing defective b2m of HLA-I heavy chain genes [69–71].

Current Opinion in Immunology 2018, 51:123–132

Conclusions Tumor immunotherapy has reached fascinating times, similar to when BCG was first locally instilled in 1976 to treat superficial bladder tumors [72], or when IL-2 was used for the first time in 1987 in metastatic melanoma [63], or when the first tumor peptide was systemically injected in melanoma patients alone or as www.sciencedirect.com

The transition from HLA-I positive to HLA-I negative primary tumors: the road to escape from T-cell responses Aptsiauri, Ruiz-Cabello and Garrido 129

Figure 3

Correlation between HLA class I expression and tumor infiltrating T Lymphocytes in tumor tissue nests 100 90 80

Tumor infiltrating T lymphocytes

70 60 50 40

(mean cells/field) 30 20 10 0

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0

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Correlation between tumor HLA-I expression and infiltration with T-lymphocytes. HLA-I antigen expression in tumors directly correlates with the degree of tumor T-cell infiltration inside the tumor nests.

a part of a dendritic cell vaccine [13,14]. Current immune checkpoint therapies against cancer are in the center of attention of oncologists and immunologist due to their clear clinical benefit and positive responses in patients [15,16,17], although relapses occur and the reason for that is still a ‘black box’ in many clinical trials. A growing awareness exists of the fact that the success of immunotherapy depends on the induction of a strong and longlasting anticancer immune response leading to tumor eradication. However, tumor cells can evade immune surveillance and destruction by cytotoxic T-cells by various strategies, including the aberrant expression of HLAI antigens (due to accumulating mutations in HLA-I/b2m genes) and defective IFN signal transduction pathway [44,53,54]. In order to eliminate cancer, novel immunotherapy strategies must correct these defects and restore T-cell recognition and elimination of the tumor cells. Importantly, all types of immunotherapy, including checkpoint inhibitors, depend on the level of HLA-I expression on cancer cells, but the majority of current cancer immunotherapy protocols do not include routine examination of tumor HLA-I expression and anti-tumor activated T-cells remain unable to see HLA-I negative tumor targets. It is difficult to understand why HLA-I molecules are not properly analyzed in post-therapy recurrent cancer lesions, since it is already accepted that HLA-I-negative tumor cell variants are T-cell immunoselected and that irreversible/‘hard’ HLA-I alterations accumulate in the recurrent progressing metastases www.sciencedirect.com

[43,44,53,54]. This paradox is hard to explain considering the amount of resources that are dedicated to improve our understanding of the tumor immune escape mechanisms [73,74]. HLA analysis in tumor tissues is a missing parameter that should be implemented [75]. Cooperation of pathologists with clinical oncologists, surgeons and immunologist is necessary to explore the immune contexture of tumors and to analyze tumor HLA-I defects in order to have reliable predictive biomarkers that tailor individual cancer treatment strategies and to monitor a response to anticancer therapies. However, it must be acknowledged that this is not an easy task due to the complexity of the HLA system and difficulties to collect fresh frozen tumor samples before, during and after the therapy. We should remember that MHC was first discovered in mice immunized with allogenic tumors in 1937 [76]. Now, 80 years later, the role of MHC in cancer is being revisited and requires urgent attention in order to override immune escape mechanisms and improve clinical efficacy of cancer immunotherapy.

Acknowledgements We would like to thank Dr Monica Bernal for helping to prepare the figures, and Dr Francisco Perea for providing the images of tumor tissue immunolabeling. This work was supported by grants from the Instituto de Salud Carlos III co-financed by FEDER funds (EU) (PI11/01386, PI14/ 01978, PI 16/00752, RETIC RD 06/020, RD09/0076/00165, PT13/0010/ 0039] and Junta de Andalucı´a in Spain [Group CTS-143]. Current Opinion in Immunology 2018, 51:123–132

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