Francisco Ruiz-Cabello

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.

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.

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.

Coordinated downregulation of the antigen presentation machinery and HLA class I/β2-microglobulin complex is responsible for HLA-ABC loss in bladder cancer

Downregulation of MHC class I expression is a widespread phenomenon used by tumor cells to escape antitumor T‐cell‐mediated immune responses. These alterations may play a role in the clinical course of the disease. The aim of our study was to investigate the molecular mechanism underlying the absence of HLA‐class I molecule expression in bladder cancer cells. Microdissected tumor tissues were characterized by real‐time quantitative PCR for the expression of HLA‐ABC, β2‐microglobulin and the members of the antigen processing machinery (APM) of HLA class I molecules (LMP2, LMP7, TAP1, TAP2 and tapasin). Our results showed that irreversible HLA loss by mutations in the β2‐microglobulin gene was not the cause of low HLA class I expression in bladder cancers. In contrast, we observed a coordinated transcription downregulation of HLA‐ABC and β2‐microglobulin and APM genes in microdissected tumor tissue derived from bladder carcinomas. This mechanism may represent a major factor for the downregulation of HLA class I expression and in the subsequent direct recognition of cancer cells by cytolytic T lymphocytes. Because this regulatory mechanism is frequently reversible by IFN‐gamma treatment, we conclude that HLA class I expression should be a major consideration for immunotherapeutic purposes in patients with bladder cancer.

Chromosome loss is the most frequent mechanism contributing to HLA haplotype loss in human tumors

Loss of heterozygosity (LOH) in the short arm of chromosome 6 (6p) was detected in samples obtained from colon (13.8%), larynx (17.6%) and melanoma (15.3%) tumors. The parallel study of HLA‐antigen expression in tumor tissues using locus‐ and polymorphic‐specific antibodies in combination with LOH microsatellite analysis on 6p allowed us to establish that LOH in chromosome 6 is a representative phenomenon in most tumor cells present in a given tumor tissue. In most cases, specific HLA alleles had been lost in a predominant population of tumor cells, indicating that LOH is a non‐irrelevant mutation that probably confers a selective advantage for survival of the mutant cell. We also demonstrate that LOH frequently occurred through chromosome loss rather than somatic recombination. LOH at all loci studied on the p and q arms of chromosome 6 was observed in at least 56.2% (9/17) cases. This HLA‐associated microsatellite analysis was a useful tool for classifying tumors as LOH‐positive or ‐negative, and therefore to consider a patient as a potential non‐responder or responder in a vaccination trial. Int. J. Cancer 83:91–97, 1999.

MHC Class I Antigens and Immune Surveillance in Transformed Cells

MHC class I antigens play a crucial role in the interaction of tumor cells with the host immune system, in particular, in the presentation of peptides as tumor-associated antigens to cytotoxic lymphocytes (CTLs) and in the regulation of cytolytic activity of natural killer (NK) cells. In this review we discuss the role of MHC class I antigens in the recognition and elimination of transformed cells and in the generation of tumor immune escape routes when MHC class I losses occur in tumors. The different altered MHC class I phenotypes and their distribution in different human tumors are the main topic of this review. In addition, molecular defects that underlie MHC alterations in transformed cells are also described in detail. Future research directions in this field are also discussed, including the laboratory analysis of tumor MHC class I-negative variants and the possible restoration of MHC class I expression.

Analysis of HLA expression in human tumor tissues.

Abstract. Cancer cells can be detected and destroyed by cytotoxic T lymphocytes in many experimental tumor systems, and – as has been well-documented – in some human tumors. In humans however, most diagnosed tumors are not eliminated by T cells but grow steadily, invading and metastasizing until the host is destroyed. Evidence is accumulating that progressive tumor growth occurs not because the immune system is defective or deteriorated, but because the cancer cell is capable of developing a variety of strategies to escape immune recognition. In addition, cancer cells acquire new biological properties to generate invasive capacity in order to migrate and colonize new tissues. Major histocompatibility complex (MHC) antigens are molecules that are specialized in communicating with the T cell receptor and natural killer (NK) cell ligands. With the former, they use the interaction with peptides derived from processed cellular and exogenous proteins to monitor self and non-self status. With the latter, they determine the degree of activation and killing capacity of NK cells by interacting with NK receptors. Any change in the MHC profile of tumor cells (including classical and nonclassical MHC molecules) may therefore have a profound influence on the immune recognition and immune rejection of cancer cells. We have reviewed the data from our laboratory and other groups, and have presented a standardized procedure for analyzing the MHC profile of human tumors with special emphasis on the quality and laboratory use of the material obtained from microdissected tumor samples. Appropriate tissue processing is of particular relevance, since it is not possible to obtain tumor cell lines from most patients. Oncologists require rapid information on the MHC profile of the tumor if gene therapy is envisaged to restore normal MHC class I gene expression.

Analysis of HLA class I expression in progressing and regressing metastatic melanoma lesions after immunotherapy

Despite the potential efficacy of cancer immunotherapy in preclinical studies, it did not show yet significant positive clinical results in humans with only a small number of cancer patients demonstrating objective tumor regression. This poor clinical outcome can be explained by the generation of sophisticated tumor immune escape mechanism, in particular, abnormalities in the expression of HLA class I antigens. We have studied the expression of HLA class I antigens in ten metastatic lesions obtained from a melanoma patient undergoing immunotherapy. Five lesions were obtained after Interferon-alpha-2b treatment and five after autologous vaccination plus BCG (M-VAX). Eight metastases were regressing after immunotherapy while two were progressing. The eight regressing metastases showed high level of HLA class I expression, whereas the two progressing lesions had low levels as measured by real time PCR and immunohistological techniques. These results indicate a strong association between HLA class I expression and progression or regression of the metastatic lesions. Our data support the hypothesis that the level of HLA class I expression is an important parameter of tumor immune escape that needs to be monitored.