Selective MHC expression in tumours modulates adaptive and innate antitumour responses

Progress towards developing vaccines that can stimulate an immune response against growing tumours has involved the identification of the protein antigens associated with a given tumour type. Epitope mapping of tumour antigens for HLA class I- and class II-restricted binding motifs followed by immunization with these peptides has induced protective immunity in murine models against cancers expressing the antigen. MHC class I molecules presenting the appropriate peptides are necessary to provide the specific signals for recognition and killing by cytotoxic T cells (CTL). The principle mechanism of tumour escape is the loss, downregulation or alteration of HLA profiles that may render the target cell resistant to CTL lysis, even if the cell expresses the appropriate tumour antigen. In human tumours HLA loss may be as high as 50%, inferring that a reduction in protein levels might offer a survival advantage to the tumour cells. Alternatively, MHC loss may render tumour cells susceptible to natural killer cell-mediated lysis because they are known to act as ligands for killer inhibitory receptors (KIRs). We review the molecular features of MHC class I and class II antigens and discuss how surface MHC expression may be regulated upon cellular transformation. In addition, selective loss of MHC molecules may alter target tumour cell susceptibility to lymphocyte killing. The development of clinical immunotherapy will need to consider not only the expression of relevant CTL target MHC proteins, but also HLA inhibitory to NK and T cells. Received: 20 March 1999 / Accepted: 3 May 1999     

High frequency of homozygosity of the HLA region in melanoma cell lines reveals a pattern compatible with extensive loss of heterozygosity

Malignant transformation of cells is frequently associated with abnormalities in human leukocyte antigen (HLA) expression. MHC class I loss or down-regulation in cancer cells is a major immune escape route used by a large variety of human tumours to evade antitumour immune responses mediated by cytotoxic T lymphocytes. The goal of our study was to explore HLA genotyping and phenotyping in a variety of melanoma tumour cell lines. A total of 91 melanoma cell lines were characterised for HLA class I and II genotype. In addition, 61 out of the 91 cell lines were also analysed for HLA class I and II cell surface molecule expression by flow cytometry. Unexpectedly, we found that 19.7% of the melanoma cell lines were homozygous for HLA class I genotypes, sometimes associated with HLA class II homozygosity (8.79%) and sometimes not (10.98%). The frequency of homozygosity was significantly higher compared with the control groups (1.6%). To identify the reasons underlying the high frequency of HLA homozygosity we searched for genomic deletions using eight pairs of highly polymorphic microsatellite markers covering the entire extended HLA complex on the short arm of chromosome 6. Our results were compatible with hemizygous deletions and suggest that loss of heterozygosity on chromosome arm 6p is a common feature in melanoma cell lines. In fact, although autologous normal DNA from the patients was not available and could not be tested, the retention in some cases of heterozygosity for a number of microsatellite markers would indicate a hemizygous deletion. In the rest of the cases, markers at 6p and 6q showed a single allele pattern indicating the probable loss of part or the whole of chromosome 6. These results led us to conclude that loss of heterozygosity in chromosome 6 is nonrandom and is possibly an immunologically relevant event in human malignant melanoma. Other well-established altered HLA class I phenotypes were also detected by flow cytometry that correspond to HLA class I total loss and HLA-ABC and/or specific HLA-B locus down-regulation.     

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.     

Multiple mechanisms generate HLA class I altered phenotypes in laryngeal carcinomas: high frequency of HLA haplotype loss associated with loss of heterozygosity in chromosome region 6p21

Major histocompatibility complex (MHC) class I loss or downregulation in cancer cells is a major immune escape route used by a large variety of human tumors to evade anti-tumor immune responses mediated by cytotoxic T lymphocytes. Multiple mechanisms are responsible for such HLA class I alterations. However, the precise frequency of these molecular defects has not been clearly determined in tumors derived from specific tissues. To analyze such defects we aim to define the major HLA class I-altered phenotypes in different tumor types. In this paper we report on HLA class I expression in 70 laryngeal carcinomas. We used immunohistological techniques with a highly selective panel of anti-HLA monoclonal antibodies (mAb), and polymerase chain reaction (PCR) microsatellite amplification of previously selected microsatellite markers (STR) located in chromosome 6 and 15. DNA was obtained from microdissected tumor tissues and surrounding stroma to define the loss of heterozygosity (LOH) associated with chromosome 6p21. Our results showed that LOH in chromosome 6 produced HLA haplotype loss (phenotype II) in 36% of the tumors. In addition, HLA class I total loss (phenotype I) was found in 11%; HLA A or B locus downregulation (phenotype III) was detected in 20%; and HLA class I allelic loss (phenotype IV) in 10% of all cases. We sometimes observed two or more associated mechanisms in the same HLA-altered phenotype, such as LOH and HLA total loss in phenotype I. In only 23% of tumors it was not possible to identify any HLA class I alteration. We conclude that the combination of immunohistological techniques and molecular analysis of tumor DNA obtained from microdissected tumor tissues provides a means for the first time of determining the actual frequency of the major HLA class I-altered phenotypes in laryngeal carcinomas.     

LOH at 6p21.3 region and HLA class altered phenotypes in bladder carcinomas

Alterations in HLA class I antigen expression have been frequently described in different epithelial tumors and are thought to favor tumor immune escape from T lymphocyte recognition. Multiple molecular mechanisms are responsible for these altered HLA class I tumor phenotypes. Some are structural defects that produce unresponsiveness to treatment with interferons. Others include alterations in regulatory mechanisms that can be switched on by treatment of tumor cells with different cytokines. One important mechanism belonging to the first group is loss of heterozygosity (LOH) at chromosome region 6p21.3, which can lead to HLA haplotype loss. In this investigation, the frequency of LOH at 6p21 chromosome region was studied in 69 bladder carcinomas. Short tandem repeat analysis showed that 35% of cases had LOH in this chromosome region. By considering these results together with immunohistological findings previously published by our group, we identified a distribution pattern of HLA class I altered phenotypes in bladder cancer. The most frequently altered phenotype in bladder carcinomas was total loss of HLA class I expression (17 cases, 25%), followed by phenotype II associated with HLA haplotype loss (12 cases, 17.5%), and HLA allelic loss (ten cases, 14.5%). Nine cases (13%) were classified as having a compound phenotype, five cases (7%) as having HLA locus loss, and in 16 cases (23%) no alteration in HLA expression was detected. An important conclusion of this report is that a combination of different molecular and immunohistological techniques is required to precisely define which HLA alleles are lost during tumor progression and to characterize the underlying mechanisms of these losses. These studies should be performed when a cancer patient is to be included in an immunotherapy protocol that aims to stimulate different immune effector mechanisms.     

Regressing and progressing metastatic lesions: resistance to immunotherapy is predetermined by irreversible HLA class I antigen alterations

Despite the significant efforts to enhance immune reactivity against malignancies the clinical effect of anti-tumor vaccines and cancer immunotherapy is still below expectations. Understanding of the possible causes of such poor clinical outcome has become very important for improvement of the existing cancer treatment modalities. In particular, the critical role of HLA class I antigens in the success of T cell based immunotherapy has led to a growing interest in investigating the expression and function of these molecules in metastatic cancer progression and, especially in response to immunotherapy. In this report, we illustrate that two types of metastatic lesions are commonly generated in response to immunotherapy according to the pattern of HLA class I expression. We found that metastatic lesions, that progress after immunotherapy have low level of HLA class I antigens, while the regressing lesions demonstrate significant upregulation of these molecules. Presumably, immunotherapy changes tumor microenvironment and creates an additional immune selection pressure on tumor cells. As a result, two subtypes of metastatic lesions arise from pre-existing malignant cells: (a) regressors, with upregulated HLA class I expression after therapy, and (b) progressors with resistance to immunotherapy and with low level of HLA class I. Tumor cells with reversible defects (soft lesions) respond to therapy by upregulation of HLA class I expression and regress, while tumor cells with structural irreversible defects (hard lesions) demonstrate resistance to immunostimulation, fail to upregulate HLA class I antigens and eventually progress. These two types of metastases appear independently of type of the immunotherapy used, either non-specific immunomodulators (cytokines or BCG) or autologous tumor vaccination. Similarly, we also detected two types of metastatic colonies in a mouse fibrosarcoma model after in vitro treatment with IFN-gamma. One type of metastases characterized by upregulation of all MHC class I antigens and another type with partial IFN-gamma resistance, namely with lack of expression of L(d)-MHC class I molecule. Our observations may shed new light on the understanding of the mechanisms of tumor escape and might have implications for improvement of the efficacy of cancer immunotherapy.     

c-myc down-regulates class I HLA expression in human melanomas

Expression of class I HLA antigen has been shown to be reduced in a number of human tumours. Here we show that in a panel of 11 melanoma cell lines with variable class I HLA expression an inverse correlation exists between the mRNA levels of c-myc and class I HLA. This suggests that high expression of the c-myc oncogene might inhibit the class I HLA expression. To test this hypothesis a melanoma cell line with a low c-myc and high class I HLA mRNA expression was transfected with a c-myc expression vector. All clones expressing the transfected c-myc gene show reduced class I HLA mRNA and beta 2-microglobulin mRNA expression. Reduced class I HLA mRNA levels result in a lowered class I protein expression on the cell surface. Treatment with gamma-interferon fully restores the class I HLA and beta 2-microglobulin expression in these cells. This effect is preceded by a transient decrease of the c-myc mRNA level. These results show that the class I HLA expression is modulated by the level of c-myc expression, thus opening up the possibility that high expression of this oncogene influences the interaction of melanoma cells with the immune system.     

Altered expression of endoplasmic reticulum aminopeptidases ERAP1 and ERAP2 in transformed non-lymphoid human tissues

The endoplasmic reticulum (ER) aminopeptidases ERAP1 and ERAP2 contribute to generate HLA class I binding peptides. Recently, we have shown that the expression of these enzymes is high and coordinated (with each other and with HLA class I molecules) in immortalized B cells, but variable and imbalanced in human tumour cell lines of various non-lymphoid lineages. Herein, this issue was investigated in vivo by testing ERAP1 and ERAP2 expression in normal non-lymphoid tissues and their malignant counterparts. ERAP1 and ERAP2 were detected exclusively in the epithelial cells of over half of the tested normal tissues. Four ERAP1/ERAP2 phenotypes (+/+, -/-, +/- and -/+) were detected, and the presence of either or both enzymes was not necessarily associated with HLA class I expression. In more than 160 neoplastic lesions, the expression of either or both aminopeptidases was retained, lost (most frequently, particularly ERAP1) or acquired as compared to the normal counterparts, depending on the tumour histotype. The double-negative (-/-) phenotype was the most frequent, and significantly (P = 0.013) associated with a lack of detectable HLA class I antigens. In selected neoplastic lesions, ERAP1 and ERAP2 were also tested for their enzymatic (peptide-trimming) activities. Expression and function were found to correlate, indicating that immunohistochemistry detects active enzymes in vivo. Thus, dissociation in the expression of ERAP1, ERAP2 and HLA class I may already be present in some normal tissues, but malignant transformation causes additional losses, gains and imbalances in specific tumour histotypes, and these alter the peptide-trimming ability of tumour cells in vivo.     

Characterization of HLA class I altered phenotypes in a panel of human melanoma cell lines

Background Altered HLA class I cell surface expression is one of the major mechanisms by which tumor cells escape from T lymphocytes. Immunohistochemistry-defined phenotypes of lost HLA class I expression have been described in human solid tumors, nut less information is available on melanoma cell lines. Objectives To describe the frequency and distribution of different types of HLA class I antigen alterations in 91 melanoma cell lines from the European Searchable Tumour Cell and Databank (ESTDAB). Methods The HLA class I expression was assessed by flow cytometry and HLA genotyping. Results We found various types of HLA class I cell surface alterations in about 67% of the melanoma cell lines. These alterations range from total to selective HLA class I loss due to loss of heterozygosity (LOH), haplotype loss, β2-microglobulin gene mutation, and/or total or selective down-regulation of HLA class I molecules. The most frequently observed phenotype is down-regulation of HLA-B locus that was reversible after treatment with IFN -γ. Conclusions In general, HLA class I alterations in the majority of the cells analyzed were of regulatory nature and could be restored by IFN-γ. Analysis of the frequency of distinct HLA class I altered phenotypes in these melanoma cell lines revealed specific differences compared to other types of tumors. Rosa Méndez and Teresa Rodríguez have equally contributed to this work and both should be considered as first authors.     

Immune selective pressure and HLA class I antigen defects in malignant lesions

The revived cancer immune surveillance theory has emphasized the active role the immune system plays in eliminating tumor cells and in facilitating the emergence of their immunoresistant variants. MHC class I molecule abnormalities represent, at least in part, the molecular phenotype of these escape variants, given the crucial role of MHC class I molecules in eliciting tumor antigen-specific T cell responses, the high frequency of HLA class I antigen abnormalities in malignant lesions and their association with a poor clinical course of the disease. Here, we present evidence for this possibility and review the potential mechanisms by which T cell selective pressure participates in the generation of tumor cells with MHC class I molecule defects. Furthermore, we discuss the strategies to counteract tumor cell immune evasion.     

Recombinant IgE antibodies for passive immunotherapy of solid tumours: from concept towards clinical application

Therapeutic antibodies have revolutionised treatment of some cancers and improved prognosis for many patients. Over half of those available are approved for haematological malignancies, but efficacious antibodies for solid tumours are still urgently needed. Clinically available antibodies belong to the IgG class, the most prevalent antibody class in human blood, while other classes have not been extensively considered. We hypothesised that the unique properties of IgE, a class of tissue-resident antibodies commonly associated with allergies, which can trigger powerful immune responses through strong affinity for their particular receptors on effector cells, could be employed for passive immunotherapy of solid tumours such as ovarian and breast carcinomas. Our laboratory has examined this concept by evaluating two chimaeric antibodies of the same specificity (MOv18) but different isotype, an IgG1 and an IgE against the tumour antigen folate receptor α (FRα). The latter demonstrates the potency of IgE to mount superior immune responses against tumours in disease-relevant models. We identified Fcε receptor-expressing cells, monocytes/macrophages and eosinophils, activated by MOv18 IgE to kill tumour cells by mechanisms such as ADCC and ADCP. We also applied this notion to a marketed therapeutic, the humanised IgG1 antibody trastuzumab and engineered an IgE counterpart, which retained the functions of trastuzumab in restricting proliferation of HER2/neu-expressing tumour cells but also activated effector cells to kill tumour cells by different mechanisms. On-going efficacy, safety evaluations and future first-in-man clinical studies of IgE therapeutics constitute key metrics for this concept, providing new scope for antibody immunotherapies for solid tumours.     

Signal interaction between the tumour and inflammatory cells in patients with gastrointestinal cancer: Implications for treatment

Over the last 15 years there has been a change in how we understand the impact of the interaction between the tumour and the host on cancer outcomes. From the simplistic view that the make-up of tumours cells largely determines their aggressiveness to a more complex view that the interaction between the products of tumour and host cell signal transduction pathways is crucial in determining whether the tumour cell is eliminated or survives in the host. Of the host cells, those with an immune/inflammatory function are most well documented to inhibit or promote tumour cell proliferation and dissemination.It is only in the last few years that there has been greater recognition of the impact of intracellular, cellular and systemic immune/inflammatory phenotypes on patient outcomes independent of current tumour staging and that these phenotypes are useful in informing oncological research and practice. In the present review we will examine the importance of inflammatory phenotypes at the intra-cellular, cellular and systemic levels on outcomes in patients with gastrointestinal cancer with focus on colorectal cancer. Based on these phenotypes we will examine and discuss the prospects for therapeutic intervention.     

Distribution of HLA class I altered phenotypes in colorectal carcinomas: high frequency of HLA haplotype loss associated with loss of heterozygosity in chromosome region 6p21

HLA class I loss or down-regulation is a widespread mechanism used by tumor cells to avoid tumor recognition by cytotoxic T lymphocytes, and thus favor tumor immune escape. Multiple mechanisms are responsible for these HLA class I alterations. In different epithelial tumors, loss of heterozygosity (LOH) at chromosome region 6p21.3, leading to HLA haplotype loss, occurs in 6–50% of all cases depending on the tumor entity. In this paper we report the frequency of LOH at 6p21 in 95 colorectal carcinomas (CRC) previously analyzed for altered HLA class I expression with immunohistological techniques. We used PCR microsatellite amplification of selected STR markers located on Chromosome 6 to identify LOH with DNA from microdissected tumor tissues and the surrounding stroma. Sequence-specific oligonucleotide analysis was performed in microdissected stroma and tumor cells for HLA typing, and to detect HLA haplotype loss. A high frequency (40%) of HLA haplotype loss was found in CRC. Eight tumors showed microsatellite instability. We sometimes observed two or more mechanisms responsible for HLA alteration within the same HLA-altered phenotype, such as LOH and HLA class I total loss. In 25 tumors (26%) no HLA class I alteration could be identified. These data are potentially relevant for CRC patients undergoing T-cell-based immunotherapy.     

HLA class I expression in metastatic melanoma correlates with tumor development during autologous vaccination

Our knowledge of the mechanisms underlying tumor-specific immune response and tumor escape has considerably increased. HLA class I antigen defects remain an important tumor escape mechanism since they influence the interactions between tumor cells and specific T and NK cells in the course of malignant disease. We have studied here HLA class I expression in six subcutaneous metastases obtained from a melanoma patient immunized with an autologous melanoma cell vaccine (M-VAX). We report in this paper that HLA class I antigen expression on these metastatic lesions strongly correlated with the course of the disease. The three metastases that were partially regressing at the time of their excision showed a strong HLA class I expression, whereas the progressing ones showed a very weak or negative staining with most of the anti-HLA class I mAbs used. Real-time quantitative PCR of the samples obtained from microdissected tumor tissue revealed a significant difference in the mRNA levels of HLA-ABC heavy chain and beta2m between the two types of metastases, i.e., lower levels in progressing metastases and high levels in regressing ones, confirming the immunohistological findings. This is, to our knowledge, the first report where the clinical outcome of different HLA class I positive and negative melanoma metastases can be clearly correlated with the regression and progression of the disease, respectively.     

Cancerous HLA class I expression and regulatory T cell infiltration in gastric cancer

Background

Since antitumor immune reactions between tumors and intratumoral immunocytes have been verified in several human tumors, immunological therapeutic strategies must be considered to obtain the proper efficacy of tumor shrinkage under these conditions. Human leukocyte antigen (HLA) class I expression in cancer cells and degree of infiltration of regulatory T cells (Tregs) in the stroma have been regarded as important markers of antitumor immune reactions in the context of independent immunological mechanisms. In the current study, we investigated HLA class I expression and Treg cells infiltration in gastric cancer and discussed the clinical implications of this combinatory analysis in gastric cancer.

Patients and methods

A total of 141 gastric cancer patients who received R0 gastrectomy at Kagoshima University Hospital were studied. Immunohistochemically, in 141 gastric cancer patients, HLA class I expression and Treg cell infiltration in cancerous tissue were evaluated using HLA class I (EMR8-5) and forkhead box p3 (FOXP3) monoclonal antibodies. The correlation between clinical factors and tumor-infiltrating Treg cells was analyzed.

Results

HLA class I expression was positively associated with depth of tumor invasion (P?r?=?0.04). A better postoperative outcome was associated with fewer numbers of Treg infiltration (P?=?0.034). A combination of HLA and Treg analysis may lead to a more accurate prediction of postoperative outcome (P?=?0.02).

Conclusions

Two different antitumor immunological markers, Treg infiltration and HLA class I expression, affected clinicopathological factors in gastric cancer by different mechanisms. Thus, an immunological combination of HLA class I expression and Treg cell infiltration may more accurately predict postoperative outcome. Immunological balance needs to be restored after evaluation of each immunological deficit in gastric cancer.     

TCR mimic monoclonal antibodies induce apoptosis of tumor cells via immune effector-independent mechanisms

mAbs that recognize peptides presented on the cell surface by MHC class I molecules are potential therapeutic agents for cancer therapy. We have previously demonstrated that these Abs, which we termed TCR mimic mAbs (TCRm), reduce tumor growth in models of breast carcinoma. However, mechanisms of TCRm-mediated tumor growth reduction remain largely unknown. In this study, we report that these Abs, in contrast to several mAbs used currently in the clinic, destroy tumor cells independently of immune effector mechanisms such as Ab-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). We found that TCRm-mediated apoptosis of tumor cells was associated with selective and specific binding of these Abs to peptide/HLA class I complexes, which triggered the activation of JNK and intrinsic caspase pathways. This signaling was accompanied by the release of mitochondrial cytochrome c and apoptosis-inducing factor. TCRm-induced apoptosis in tumor cells was completely inhibited by soluble MHC tetramers loaded with relevant peptide as well as with inhibitors for JNK and caspases. Furthermore, mAbs targeting MHC class I, independent of the peptide bound by HLA, did not stimulate apoptosis, suggesting that the Ab-binding site on the MHC/peptide complex determines cytotoxicity. This study suggests the existence of mechanisms, in addition to ADCC and CDC, through which these therapeutic Abs destroy tumor cells. These mechanisms would appear to be of particular importance in severely immunocompromised patients with advanced neoplastic disease, since immune cell-mediated killing of tumor cells through ADCC and CDC is substantially limited in these individuals.     

HLA expression in cancer: implications for T cell-based immunotherapy

HLA class I expression is altered in a significant fraction of the tumor types reviewed here, reflecting either immune pressure or, simply, the accumulation of pathological changes and alterations. However, in all tumor types analyzed, a majority of the tumors express HLA class I. with a general tendency for the more severe alterations to be found in later-stage and less differentiated tumors. These results are encouraging for the development of specific immunotherapies, especially considering that (1) the relatively low sensitivity of immunohistochemical techniques might underestimate HLA expression in tumors, (2) class I expression can be induced in tumor cells as a result of local inflammation and lymphokine release, and (3) class I-negative cells would be predicted to be sensitive to Iysis by natural killer cells.     

Lymphocyte activation in response to melanoma: interaction of NK-associated receptors and their ligands

In recent years, studies on the molecular and cellular mechanisms of immune responses against melanoma have contributed to a better understanding of how these tumours can be recognised by cytotoxic cells and the mechanisms they have developed to escape from innate and adaptive immunity. Lysis of melanoma cells by natural killer (NK) cells and cytolytic T cells is the result of a fine balance between signals transmitted by activating and inhibitory receptors. In addition to the T cell receptor, these were initially described as NK cell-associated receptors (NKRs) and were later also found on subsets of T lymphocytes, particularly effector-memory and terminally differentiated CD8 T cells. An increase of NKR(+)CD8(+) T cells has been found in melanoma patients, correlating with the expansion of differentiated effector CD8(+)CD28(null) CD27(null) T cells. NKRs can regulate the lysis of target cells expressing appropriate ligands. Activating receptors recognise ligands on tumours whereas inhibitory receptors are specific for MHC class I antigens and sense missing self. Altered expression of MHC class I antigens is frequently found on melanoma cells, preventing recognition by specific cytolytic T cells but favouring NK cell recognition. Changes in the expression of NKR-ligands in melanoma contribute in explaining the differences in the capacity of cytotoxic immune cells to control melanoma growth and dissemination.     

Application of isoelectric focusing for studies of major histocompatibility complex class I antigen expression on human carcinomas and sarcomas

Summary By one-dimension isoelectric focusing we analysed the major histocompatibility complex class I antigen expression on human tumours. Blood lymphocytes of the patients, processed in parallel, served as a basis for comparison. The prerequisite for the analysis is the preparation of metabolically active tumour cell suspensions devoid of significant leucocyte contamination. The method was found to be suitable for study of the expression of HLA alleles on ex vivo tumour cells and allowed the detection of changes imposed by in vitro treatment with interferon and tumour necrosis factor .