Federico Garrido

Implications for immunosurveillance of altered HLA class I phenotypes in human tumours

HLA class I downregulation is a frequent event associated with tumour invasion and development. Altered HLA class I tumour phenotypes can have profound effects on T-cell and natural killer (NK)-cell antitumour responses. Here, Federico Garrido and colleagues analyse these altered tumour phenotypes in detail, indicating their potential relevance for implementation of immunotherapeutic protocols and strategies to overcome tumour escape mechanisms.

Natural history of HLA expression during tumour development

HLA expression is frequently altered in tumours compared to the tissue from which they originate. Given the central role of MHC products in the restriction of T-cell recognition, regulation of tumour HLA expression might be a strategy for the evasion of immune surveillance by the malignant cells. Federico Garrido, Peter Stern and colleagues present data from a variety of tumour types, suggesting that HLA class I alterations may occur at a particular step between the development of an in situ lesion and an invasive carcinoma.

MHC class I antigens, immune surveillance, and tumor immune escape

Oncogenic transformation in human and experimental animals is not necessarily followed by the appearance of a tumor mass. The immune system of the host can recognize tumor antigens by the presentation of small antigenic peptides to the receptor of cytotoxic T‐lymphocytes (CTLs) and reject the nascent tumor. However, cancer cells can sometimes escape these specific T‐cell immune responses in the course of somatic (genetic and phenotypic) clonal evolution. Among the tumor immune escape mechanisms described to date, the alterations in the expression of major histocompatibility complex (MHC) molecules play a crucial step in tumor development due to the role of MHC antigens in antigen presentation to T‐lymphocytes and the regulation of natural killer cell (NK) cell function. In this work, we have (1) updated information on the mechanisms that allow CTLs to recognize tumor antigens after antigen processing by transformed cells, (2) described the altered MHC class I phenotypes that are commonly found in human tumors, (3) summarized the molecular mechanisms responsible for MHC class I alteration in human tumors, (4) provided evidence that these altered human leukocyte antigens (HLA) class I phenotypes are detectable as result of a T‐cell immunoselection of HLA class I‐deficient variants by an immunecompetent host, and (5) presented data indicating the MHC class I phenotype and the immunogenicity of experimental metastatic tumors change drastically when tumors develop in immunodeficient mice.

The selection of tumor variants with altered expression of classical and nonclassical MHC class I molecules: implications for tumor immune escape

Tumor immune escape variants can be identified in human and experimental tumors. A variety of different strategies are used by tumor cells to avoid recognition by different immune effector mechanisms. Among these escape routes, alteration of MHC class I cell surface expression is one of the mechanisms most widely used by tumor cells. In this review we focus our attention on the T-cell immune selection of MHC class I–deficient tumor variants. Different altered MHC class I phenotypes that originate from multiple molecular mechanisms can be identified in human tumors. MHC-deficient tumor clones can escape T-cell immune responses, but are in theory more susceptible to NK-cell–mediated lysis. In this context, we also review the controversial issue of the aberrant expression of nonclassical HLA class I molecules, particularly HLA-G, in tumors. This expression may be relevant in tumor cells that have lost the capacity to interact with NK inhibitory receptors—namely, those tumor cells with no HLA-B or HLA-C expression. Most published studies have not analyzed these possibilities and do not provide information about the complete HLA-A, HLA-B, or HLA-C molecule profiles of the tumors studied. In contrast, HLA-E has been reported to be expressed in some tumor cell lines with very low HLA-A, HLA-B, and HLA-C expression, suggesting that HLA-E may indeed, in some cases, play a role by inhibiting NK lysis of cells that otherwise would be destroyed by NK cells. Finally, we provide evidence that the status of the immune system in the tumor-bearing animal is capable of defining the MHC profile of the tumor cells. In other words, MHC class I–negative metastatic colonies are produced in immunocompetent animals, and MHC class I–positive colonies in T-cell immunodeficient individuals.

MHC antigens and tumor escape from immune surveillance

Publisher Summary The chapter presents that the primary and metastatic tumor cell growth results from the development of sophisticated molecular and biological mechanisms that allow tumor cells to escape immune surveillance. These escape mechanisms are selected by the cancer cells after a period of interaction with the immune system. Among these mechanisms, the MHC class I phenotypic alteration that occurs in tumor cells plays a leading role in the tumor–host scenario since these are crucial molecules for antigen presentation to T cells and modulation of NK activity. This chapter discusses the major HLA class I phenotypic alterations found in human tumors, with special attention paid to the molecular mechanisms responsible for their generation. The chapter also describes the HLA class I alterations that are detected in tumors derived from different tissues of the body, such as the bladder, breast, colon and rectum, larynx, lung, kidney, melanocytes, pancreas, and prostate. The issue of nonclassical HLA class I molecule expression in tumor cell lines and tumor tissues and its possible role in immune escape is also discussed, as are the implications of these findings for T cell-based immunotherapy. The chapter concludes with the experimental data supporting the hypothesis that the MHC class I-negative tumor variants are selected in vivo by cytotoxic lymphocyte (CTL) responses against MHC class I-positive tumor cells.

Rexpression of HLA class I antigens and restoration of antigen-specific CTL response in melanoma cells following 5-aza-2'-deoxycytidine treatment.

Cell surface expression of HLA class I/peptide complexes on tumor cells is a key step in the generation of T‐cell‐based immune responses. Several genetic defects underlying the lack of HLA class I expression have been characterized. Here we describe another molecular mechanism that accounts for the complete absence of HLA class I molecule expression in a tumor line (MSR3‐mel) derived from a melanoma patient. Hypermethylation of the MSR3‐mel DNA, specifically of HLA‐A and ‐B genes, was identified, which resulted in loss of HLA class I heavy chain transcription. Treatment of MSR3‐mel cells with the demethylating agent 5′‐aza‐2′‐deoxycytidine (DAC) allowed HLA‐A and ‐B transcription, restoring cell surface expression of HLA class I antigens and tumor cell recognition by MAGE‐specific cytotoxic T lymphocytes. The MSR3‐mel line was obtained from a metastatic lesion of a nonresponding patient undergoing MAGE‐3.A1 T‐cell‐based peptide immunotherapy. It is tempting to speculate that the hypermethylation‐induced lack of HLA class I expression is the cause of the impaired response to vaccination. This study provides the first evidence that DNA hypermethylation is used by human neoplastic cells to switch off HLA class I genes, thus providing a new route of escape from immune recognition.

Hla Class I Antigens in Human Tumors

Publisher Summary This chapter focuses on the abnormal major histocompatibility complex (MHC) expression detected in human tumors as well as on the biological role that these alterations may have in tumor development. The potential therapeutic implications of these discoveries are discussed in the chapter. MHC constitutes a set of genes that synthesize products specializing in the processing and presentation of endogenous and exogenous antigens to the immune system. Four major categories of genes are involved in this genetically controlled process: class I, class II, proteasome, and transported genes. A major characteristic of these genes is their very high degree of polymorphism. Polymorphism provides an enormous diversity of antigenic peptides that can potentially be presented, conferring to a given species the possibility of generating an immune response to a particular antigen, even though a given individual might not have the set of alleles required to do so. Human leukocyte antigen (HLA) class I molecules are ternary complexes formed by a heavy chain, a light chain of P2-microglobulin (Pam), and a peptide. Significant changes in HLA expression in tumor cells are assessed by determining the proportion of neoplastic cells that exhibit differences in immunohistochemical labeling in comparison with normal tissues from the same specimen. The role of MHC in T and NK cell recognition is discussed in the chapter. Progress in HLA class I gene transfer in cancer patients requires the precise identification of the HLA antigen losses and mechanisms responsible for HLA downregulation. Routine methods for the straightforward and accurate identification of HLA antigen losses are not yet available. The XII HLA Histocompatibility Workshop has created a new component designated “HLA and Cancer” that coordinates data from different laboratories to help achieve these aims.

''Hard'' and ''soft'' lesions underlying the HLA Class I alterations in cancer cells: implications for immunotherapy

The ability of cancer cells to escape from the natural or immunotherapy‐induced antitumor immune response is often associated with alterations in the tumor cell surface expression of Major Histocompatibility Complex (MHC) Class I antigens. Considerable knowledge has been gained on the prevalence of various patterns of MHC Class I defects and the underlying molecular mechanisms in different types of cancer. In contrast, few data are available on the changes in MHC Class I expression happening during the course of cancer immunotherapy. We have recently proposed that the progression or regression of a tumor lesion in cancer patients undergoing immunotherapy could be predetermined by the molecular mechanism responsible for the MHC Class I alteration and not by the type of immunotherapy used, i.e., interleukin‐2 (IL‐2), Bacillus Calmette‐Guèrin (BCG), interferon‐alpha (IFN‐α), peptides alone, dendritic cells loaded with peptides, protein‐bound polysaccharide etc. If the molecular alteration responsible for the changes in MHC Class I expression is reversible by cytokines (“soft” lesion), the MHC Class I expression will be upregulated, the specific T cell–mediated response will increase and the lesion will regress. However, if the molecular defect is structural (“hard” lesion), the MHC Class I expression will remain low, the escape mechanism will prevail and the primary tumor or the metastatic lesion will progress. According to this idea, the nature of the preexisting MHC Class I lesion in the cancer cell has a crucial impact determining the final outcome of cancer immunotherapy. In this article, we discuss the importance of these two types of molecular mechanisms of MHC Class I–altered expression.

Immune infiltrates are prognostic factors in localized gastrointestinal stromal tumors

Cancer immunosurveillance relies on effector/memory tumor-infiltrating CD8(+) T cells with a T-helper cell 1 (TH1) profile. Evidence for a natural killer (NK) cell-based control of human malignancies is still largely missing. The KIT tyrosine kinase inhibitor imatinib mesylate markedly prolongs the survival of patients with gastrointestinal stromal tumors (GIST) by direct effects on tumor cells as well as by indirect immunostimulatory effects on T and NK cells. Here, we investigated the prognostic value of tumor-infiltrating lymphocytes (TIL) expressing CD3, Foxp3, or NKp46 (NCR1) in a cohort of patients with localized GIST. We found that CD3(+) TIL were highly activated in GIST and were especially enriched in areas of the tumor that conserve class I MHC expression despite imatinib mesylate treatment. High densities of CD3(+) TIL predicted progression-free survival (PFS) in multivariate analyses. Moreover, GIST were infiltrated by a homogeneous subset of cytokine-secreting CD56(bright) (NCAM1) NK cells that accumulated in tumor foci after imatinib mesylate treatment. The density of the NK infiltrate independently predicted PFS and added prognostic information to the Miettinen score, as well as to the KIT mutational status. NK and T lymphocytes preferentially distributed to distinct areas of tumor sections and probably contributed independently to GIST immunosurveillance. These findings encourage the prospective validation of immune biomarkers for optimal risk stratification of patients with GIST.

High frequency of altered HLA class I phenotypes in invasive breast carcinomas

We studied 105 tumor samples obtained from patients diagnosed as having breast carcinomas for HLA class I and II (DR) antigen expression, using a panel of mAbs defining HLA-monomorphic, locus-specific and allele-specific determinants. Peripheral blood lymphocytes from patients were also typed for HLA alleles. The results indicated total HLA class I losses in 55 patients (52.3%), HLA-A locus losses in four patients (3.8%), HLA-B locus losses in eight patients (7.6%), and A, B, locus losses in 10 patients (9.5%). The remaining 28 patients whose tissues reacted positively with monomorphic- and locus-specific mAbs were tested for HLA allelic losses using several anti-HLA mAbs defining A2, A3, A9, B8, B12, etc. Of these 28 patients, 16 (57%) showed one or more losses of HLA reactivity. These results indicated that in 88.5% of patients we detected a particular HLA-altered tumor phenotype. The downregulation of HLA class I antigens in breast carcinomas may thus be more frequent than previously reported, and patients without HLA class I downregulation may be the exception rather than the rule. It cannot be ruled out that HLA alterations are present in some of the 12 patients with an apparently normal HLA phenotype, as some HLA alleles could not be studied because of the lack of appropriate mAbs. These HLA alterations could represent an important step associated with tumor invasion, conferring to the tumor cells the ability to escape from T-lymphocyte recognition.