Ignacio Algarra

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.

DETECTION OF BREAST CANCER CELLS IN THE PERIPHERAL BLOOD IS POSITIVELY CORRELATED WITH ESTROGEN-RECEPTOR STATUS AND PREDICTS FOR POOR PROGNOSIS

We investigated whether detection of cytokeratin‐positive (CK+) cells in the peripheral blood (PB) of breast cancer patients before chemotherapy could be a prognostic factor. Blood from a total of 92 breast cancer patients was evaluated for the presence of CK+ cells. Blood samples were collected before chemotherapy. Patients entered in the study included: neoadjuvant (n = 25), adjuvant (n = 42) and metastatic (n = 25). Blood samples (10 ml) were centrifuged using a double density‐gradient to recovering the mononuclear cell (MNC) and granulocyte cell (GC) fractions. Subsequently, positive immunomagnetic cell separation was carried out to isolating CK+ cells. The enriched cell fraction was cytocentrifuged and then immunocytochemically labeled using an anti‐cytokeratin antibody. Our results indicated that breast tumor cells sediment with both MNC and GC fractions. We therefore recommend examination of both fractions in all enrichment protocols. CK+ cells in PB were identified in 57 of 92 (62%) patients when MNC and GC fractions were assessed (range = 1–61 cells, median = 8). No CK+ cells were detected in blood samples of 16 healthy donors. There were significant differences in the presence of CK+ cells according to estrogen receptor expression (p = 0.049), and lymph node status (p = 0.033), but not to the age, menopausal status, type of patient (neoadjuvant, adjuvant or metastatic), TNM stage, histological type, progesterone receptor expression, c‐erbB2 expression, p53 expression or Ki67 expression. Regarding the relationship between tumor size (T) and the presence of CK+ cells, a borderline significant trend was observed (p = 0.07). The median follow‐up of the patients was 21 months and statistical analysis (Kaplan‐Meier analysis) showed that using the method we present, the detection of CK+ cells in PB before starting the chemotherapy in breast cancer patients was significantly correlated with both progression‐free survival (p = 0.058) and overall survival (p = 0.003). In conclusion, the present study suggests that detection of CK+ cells in PB before chemotherapy might identify breast cancer patients with poor prognosis.

MHC class I‐deficient metastatic tumor variants immunoselected by T lymphocytes originate from the coordinated downregulation of APM components

Previous reports from our group indicated that the MHC class I phenotype of metastatic lung colonies produced by a mouse fibrosarcoma tumor clone (B9) were, depending on the immune status of the host, MHC class I negative in immunocompetent mice and MHC class I positive in immunodeficient athymic nude/nude mice. Now we report the identification of the molecular alterations responsible for the changes of MHC class I molecules in both situations. Metastatic nodes were analyzed for the mRNA level of H‐2 class I and β2‐microglobulin genes, and several gene components of the major histocompatibility complex (MHC) class I antigen‐processing machinery (APM). These included the genes coding for the low‐molecular‐weight proteins LMP2, LMP7, LMP10, the transporter associated with antigen processing (TAP‐1, TAP‐2), and calnexin, calreticulin, tapasin, PA‐28‐α, PA‐28‐β, ERP‐59 and ER‐60. Analyses with RT‐PCR showed that TAP‐1, TAP2, LMP‐2, LMP7, LMP10, tapasin and calnexin mRNA specific for these genes was absent in metastases produced in immunocompetent mice. In contrast, similar techniques with mRNA preparations obtained from metastatic nodes from immunodeficient mice showed that the mRNA expression level of these genes was highly positive. Interestingly, the MHC class I‐positive or negative phenotypes of the metastatic colonies correlated with in vivo immunogenicity. H‐2 positive metastasis grew more slowly than the H‐2 negative ones when injected intrafootpat in syngeneic immunocompetent animals and were finally rejected. These results provide evidence of the role of T cells in immune surveillance against tumors and identify a mechanism targeted by antitumor T lymphocytes to generate MHC class I‐negative tumor escape variants.

The escape of cancer from T lymphocytes: immunoselection of MHC class I loss variants harboring structural-irreversible “hard” lesions

The discovery of tumor antigens recognized by T lymphocytes has stimulated the development of a variety of cancer treatment protocols aimed at enhancing antitumor-specific T cell responses and tumor rejection. However, immunotherapy-mediated regression of established tumors and clearly positive clinical response to such treatment has not been achieved yet despite the induction of T cells directed against tumor antigens. The failure of the modern immunotherapy protocols can be explained by different tumor escape mechanisms that have been defined in various types of malignancy. The loss or downregulation of MHC class I antigens in tumor cells is one of the best analyzed mechanisms. In this review, we show experimental evidence obtained in our laboratory on human tumors and in a mouse cancer model suggesting that the molecular mechanism responsible for the MHC class I alteration in tumor cells might have a crucial impact on tumor recovery of normal H-2/HLA expression during the natural history of tumor development or after immunotherapy. When the preexisting molecular lesion underlying tumor MHC class I alteration is reversible (regulatory or soft), class I expression can be recovered leading to regression of tumor lesion. In contrast, if the HLA class I alteration is irreversible in nature (structural or hard), the lesion will progress killing the host. This is a new vision of the role of MHC class I alteration in tumors that can explain the failure of immunotherapy in a variety of different clinical protocols.

MHC class I molecules act as tumor suppressor genes regulating the cell cycle gene expression, invasion and intrinsic tumorigenicity of melanoma cells

The alteration of MHC class I (MHC-I) expression is a frequent event during cancer progression, allowing tumor cells to evade the immune system. We report that the loss of one major histocompatibility complex haplotype in human melanoma cells not only allowed them to evade immunosurveillance but also increased their intrinsic oncogenic potential. A second successive defect in MHC-I expression, MHC-I total downregulation, gave rise to melanoma cells that were more oncogenic per se in vivo and showed a higher proliferation rate and greater migratory and invasive potential in vitro. All these processes were reversed by restoring MHC-I expression via human leukocite antigen-A2 gene transfection. MHC-I cell surface expression was inversely correlated with intrinsic oncogenic potential. Modifications in the expression of various cell cycle genes were correlated with changes in MHC-I expression; the most important differences among the melanoma cell lines were in the transcriptional level of AP2-alpha, cyclin A1 and p21WAF1/CIP1. According to these results, altered MHC-I expression in malignant cells can directly increase their intrinsic oncogenic and invasive potential and modulate the expression of cell cycle genes. These findings suggest that human leukocite antigen class I molecules may act directly as tumor suppressor genes in melanoma.

The tumour suppressor Fhit positively regulates MHC class I expression on cancer cells

MHC class I (MHC‐I) molecules are ubiquitously expressed on the cells of an organism. Study of the regulation of these molecules in normal and disease conditions is important. In tumour cells, the expression of MHC‐I molecules is very frequently lost, allowing these cells to evade the immune response. Cancers of different histology have shown total loss of MHC‐I molecule expression, due to a coordinated transcriptional down‐regulation of various antigen‐processing machinery (APM) components and/or MHC‐I heavy chains. The mechanisms responsible for these alterations remain unclear. We determined the possible genes involved by comparing MHC‐I‐positive with MHC‐I‐negative murine metastases derived from the same fibrosarcoma tumour clone. MHC‐I‐negative metastases showed transcriptional down‐regulation of APM and MHC‐I heavy chains. The use of microarrays and subtraction cDNA libraries revealed four candidate genes responsible for this alteration, but two of them were ruled out by real‐time RT‐PCR analyses. The other two genes, AP‐2α and Fhit tumour suppressors, were studied by using siRNA to silence their expression in a MHC‐I‐positive metastatic cell line. AP‐2α inhibition did not modify transcriptional expression of APM components or MHC‐I heavy chains or surface expression of MHC‐I. In contrast, silencing of the Fhit gene produced the transcriptional down‐regulation of APM components and MHC‐I heavy chains and decreased MHC‐I surface expression. Moreover, transfection of Fhit in MHC‐I‐negative tumour cell lines restored MHC‐I cell surface expression. These data indicate that defects in Fhit expression may promote MHC‐I down‐regulation in cancer cells and allow escape from immunosurveillance#. Copyright

Alterations of HLA class I expression in human melanoma xenografts in immunodeficient mice occur frequently and are associated with higher tumorigenicity.

Animal models are widely used to study the biological behavior of human tumors in vivo. Murine immunodeficient models are used to test novel human anti-tumor therapies, and humanized mice are employed to study immunotherapeutic protocols. We find that human melanoma cell lines lose HLA class I surface expression after growth in immunodeficient mice and that this phenomenon occurs frequently and is reproducible. This HLA loss is due to a coordinated down-regulation of APM and HLA heavy chain expression at the transcriptional level. It is produced by epigenetic modifications and can be reversed by treatment with histone deacetylase inhibitors or IFN-gamma. These HLA alterations only appear during in vivo growth and not during successive in vitro passages. Interestingly, these new tumor variants with HLA class I loss show higher tumorigenicity per se and may represent a more advanced state of the original tumor. Lack of MHC class I expression on tumor cells represents a frequent escape mechanism from the immune response. Our results indicate that tumor variants with alterations in MHC can also appear in vivo after the immunoescape phase in the absence of anti-tumor immune response. Our findings suggest that any studied parameter, i.e., HLA expression, of malignant cells in xenograft models, has to be evaluated before and after growth in immunodeficient mice, in order to design more appropriate immunotherapy and chemotherapy treatments against tumor cells growing in vivo.

T lymphocytes restrain spontaneous metastases in permanent dormancy

Tumor dormancy is a clinical phenomenon related to immune equilibrium during cancer immunoediting. The mechanisms involved in dormant metastases are poorly understood due to the lack of preclinical models. Here, we present a nontransgenic mouse model in which spontaneous metastases remain in permanent immunomediated dormancy with no additional antitumor treatment. After the injection of a GR9-B11 mouse fibrosarcoma clone into syngeneic BALB/c mice, all animals remained free of spontaneous metastases at the experimental endpoints (3-8 months) but also as long as 24 months after tumor cell injection. Strikingly, when tumor-bearing mice were immunodepleted of T lymphocytes or asialo GM1-positive cells, the restraint on dormant disseminated metastatic cells was relieved and lung metastases progressed. Immunostimulation was documented at both local and systemic levels, with results supporting the evidence that the immune system was able to restrain spontaneous metastases in permanent dormancy. Notably, the GR9-B11 tumor clone did not express MHC class I molecules on the cell surface, yet all metastases in immunodepleted mice were MHC class I-positive. This model system may be valuable for more in-depth analyses of metastatic dormancy, offering new opportunities for immunotherapeutic management of metastatic disease.