Cancer cells escape T-cell-mediated destruction by losing human leukocyte antigen (HLA) class I expression via various mechanisms, including loss of beta2-microglobulin (b2m). Our study illustrates the immune escape of HLA class I-negative tumor cells and chronological sequence of appearance of tumor b2m gene mutation in successive lesions obtained from a patient with metastatic melanoma. We observed a gradual decrease in HLA expression in consecutive lesions with few HLA-negative nodules in the primary tumor and the emergence of a totally negative lesion at later stages of the disease. We detected loss of b2m in b2m-negative nests of the primary tumor caused by a combination of two alterations: (i) a mutation (G to T substitution) in codon 67 in exon 2 of b2m gene, producing a stop codon and (ii) loss of the second gene copy by loss of heterozygosity (LOH) in chromosome 15. The same b2m mutation was found in a homogeneously b2m-negative metastasis 10 months later and in a cell line established from a biopsy of a postvaccination lymph node. Microsatellite analysis revealed the presence of LOH in chromosomes 6 and 15 in tumor samples, showing an accumulation of chromosomal loss at specific short tandem repeats in successive metastases during disease progression. HLA loss correlated with decreased tumor CD81 T-cell infiltration. Early incidence of b2m defects can cause an immune selection and expansion of highly aggressive melanoma clones with irreversible genetic defects causing total loss of HLA class I expression and should be taken into consideration as a therapeutic target in the development of cancer immunotherapy protocols.

We wanted to analyze whether tumor HLA class I (HLA-I) expression influences the pattern of the immune cell infiltration and stromal cell reaction in the tumor microenvironment. Tumor tissues obtained from 57 patients diagnosed with lung carcinomas were analyzed for HLA expression and leukocyte infiltration. 28 patients out of the 57 were completely negative for HLA-I expression (49.1%) or showed a selective HLA-A locus downregulation (three patients, 5.2%). In 26 out of 57 tumors (47.8%) we detected a positive HLA-I expression but with a percentage of HLA-I negative cells between 10 and 25%. The HLA-I negative phenotype was produced by a combination of HLA haplotype loss and a transcriptional downregulation of β2-microglobulin (β2-m) and LMP2 and LMP7 antigen presentation machinery genes. The analysis and localization of different immune cell populations revealed the presence of two major and reproducible patterns. One pattern, which we designated "immune-permissive tumor microenvironment (TME)," was characterized by positive tumor HLA-I expression, intratumoral infiltration with cytotoxic T-CD8+ cells, M1-inflammatory type macrophages, and a diffuse pattern of FAP+ cancer-associated fibroblasts. In contrast, another pattern defined as "non-immune-permissive TME" was found in HLA-I negative tumors with strong stromal-matrix interaction, T-CD8+ cells surrounding tumor nests, a dense layer of FAP+ fibroblasts and M2/repair-type macrophages. In conclusion, this study revealed marked differences between HLA class I-positive and negative tumors related to tissue structure, the composition of leukocyte infiltration and stromal response in the tumor microenvironment.

The dynamic interaction between the host immune system and growing cancer has been of central interest to the field of tumor immunology over the past years. Recognition of tumor-associated antigens (TAA) by self-HLA (human leukocyte antigen) class I-restricted CD8+ T cells is a main feature in the detection and destruction of malignant cells. The discovery and molecular characterization of TAA has changed the field of cancer treatment and introduced a new era of cancer immunotherapy aimed at increasing tumor immunogenicity and T-cell-mediated anti-tumor immunity. Unfortunately, while these new protocols of cancer immunotherapy are mediating induction of tumor-specific T lymphocytes in patients with certain malignancies, they have not yet delivered substantial clinical benefits, such as induction of tumor regression or increased disease-free survival. It has become apparent that lack of tumor rejection is the result of immune selection and escape by tumor cells that develop low immunogenic phenotypes. Substantial experimental data support the existence of a variety of different mechanisms involved in the tumor escape phase, including loss or downregulation of HLA class I antigens. These alterations could be caused by regulatory ('soft') or by structural/irreversible ('hard') defects. On the basis of the evidence obtained from experimental mouse cancer models and metastatic human tumors, the structural defects underlying HLA class I loss may have profound implications on T-cell-mediated tumor rejection and ultimately on the outcome of cancer immunotherapy. Strategies to overcome this obstacle, including gene therapy to recover normal expression of HLA class I genes, require consideration. In this review, we outline the importance of monitoring and correction of HLA class I alterations during cancer development and immunotherapy.

Optimal tumor cell surface expression of human leukocyte antigen (HLA) class I molecules is essential for the presentation of tumor-associated peptides to T-lymphocytes. However, a hallmark of many types of tumor is the loss or downregulation of HLA class I expression associated with ineffective tumor antigen presentation to T cells. Frequently, HLA loss can be caused by structural alterations in genes coding for HLA class I complex, including the light chain of the complex, β2-microglobulin (β2m). Its best-characterized function is to interact with HLA heavy chain and stabilize the complex leading to a formation of antigen-binding cleft recognized by T-cell receptor on CD8+ T cells. Our previous study demonstrated that alterations in the β2m gene are frequently associated with cancer immune escape leading to metastatic progression and resistance to immunotherapy. These types of defects require genetic transfer strategies to recover normal expression of HLA genes. Here we characterize a replication-deficient adenoviral vector carrying human β2m gene, which is efficient in recovering proper tumor cell surface HLA class I expression in β2m-negative tumor cells without compromising the antigen presentation machinery. Tumor cells transduced with β2m induced strong activation of T cells in a peptide-specific HLA-restricted manner. Gene therapy using recombinant adenoviral vectors encoding HLA genes increases tumor antigen presentation and represents a powerful tool for modulation of tumor cell immunogenicity by restoration of missing or altered HLA genes. It should be considered as part of cancer treatment in combination with immunotherapy.