Consistent cytotoxic-T-lymphocyte targeting of immunodominant regions in human immunodeficiency virus across multiple ethnicities.

Although there is increasing evidence that virus-specific cytotoxic-T-lymphocyte (CTL) responses play an important role in the control of human immunodeficiency virus (HIV) replication in vivo, only scarce CTL data are available for the ethnic populations currently most affected by the epidemic. In this study, we examined the CD8(+)-T-cell responses in African-American, Caucasian, Hispanic, and Caribbean populations in which clade B virus dominates and analyzed the potential factors influencing immune recognition. Total HIV-specific CD8(+)-T-cell responses were determined by enzyme-linked immunospot assays in 150 HIV-infected individuals by using a clade B consensus sequence peptide set spanning all HIV proteins. A total of 88% of the 410 tested peptides were recognized, and Nef- and Gag-specific responses dominated the total response for each ethnicity in terms of both breadth and magnitude. Three dominantly targeted regions within these proteins that were recognized by >90% of individuals in each ethnicity were identified. Overall, the total breadth and magnitude of CD8(+)-T-cell responses correlated with individuals' CD4 counts but not with viral loads. The frequency of recognition for each peptide was highly correlated with the relative conservation of the peptide sequence, the presence of predicted immunoproteasomal cleavage sites within the C-terminal half of the peptide, and a reduced frequency of amino acids that impair binding of optimal epitopes to the restricting class I molecules. The present study thus identifies factors that contribute to the immunogenicity of these highly targeted and relatively conserved sequences in HIV that may represent promising vaccine candidates for ethnically heterogeneous populations.

Increased frequency of HLA-DR2 in patients with paroxysmal nocturnal hemoglobinuria and the PNH/aplastic anemia syndrome.

Many autoimmune diseases are associated with HLA alleles, and such a relationship also has been reported for aplastic anemia (AA). AA and paroxysmal nocturnal hemoglobinuria (PNH) are related clinically, and glycophosphoinositol (GPI)-anchored protein (AP)-deficient cells can be found in many patients with AA. The hypothesis was considered that expansion of a PNH clone may be a marker of immune-mediated disease and its association with HLA alleles was examined. The study involved patients with a primary diagnosis of AA, patients with myelodysplastic syndrome (MDS), and patients with primary PNH. Tests of proportions were used to compare allelic frequencies. For patients with a PNH clone (defined by the presence of GPI-AP-deficient granulocytes), regardless of clinical manifestations, there was a higher than normal incidence of HLA-DR2 (58% versus 28%; z = 4.05). The increased presence of HLA-DR2 was found in all frankly hemolytic PNH and in PNH associated with bone marrow failure (AA/PNH and MDS/PNH). HLA-DR2 was more frequent in AA/PNH (56%) than in AA without a PNH clone (37%; z = 3.36). Analysis of a second cohort of patients with bone marrow failure treated with immunosuppression showed that HLA-DR2 was associated with a hematologic response (50% of responders versus 34% of nonresponders; z = 2.69). Both the presence of HLA-DR2 and the PNH clone were independent predictors of response but the size of PNH clone did not correlate with improvement in blood count. The results suggest that clonal expansion of GPI-AP-deficient cells is linked to HLA and likely related to an immune mechanism.

High throughput HLA sequence-based typing (SBT) utilizing the ABI Prism 3700 DNA Analyzer.

AIMS AND BACKGROUND: The genetic complexity of the human major histocompatibility complex (MHC) has required the development of various molecular typing methods. The purpose of this paper is to compare the results of two of these molecular methods: sequenced based typing (SBT) and polymerase chain reaction (PCR) using sequence specific primers (PCR-SSP). METHODS: The SBT method described utilizes an ABI Prism 3700 DNA Analyzer, which has been designed fro high throughput production of sequence data through highly automated operation with significant walk-away time. The ABI Prism 3700 DNA Analyzer is a 96-capillary electrophoresis instrument with the capability of running four 96-well plates black to back in a sixteen-hour period. Potentially, data from this machine can produce Class I sequences for A or B loci for 64 samples in this time frame. The SBT method encompassed exons 2, 3, and 4 with forward and reverse sequence orientation reactions using the PE Biosystems HLA-A and HLA-B Sequenced Based Typing Kits (PE Applied Biopsystems/Perkin-Elmer, Foster City, CA, USA). Most SBT methods previously employed only gather data from exons 2 and 3 which distinguishes most of the polymorphism necessary to identify the majority of alleles in the HLA region. However, in an effort to discern numerous null alleles in the HLA region, exon 4 data is also included. The PCR-SSP method utilized consists of one 96 well tray, with 95 primer mixes and one negative control, per sample designed to produce an intermediate/high resolution HLA-A, B typing. RESULTS: Data from one 96-well capillary run on the ABI Prism 3700 DNA Analyzer, which consists of results from 16 samples for HLA-A or HLA-B loci, was compared to data derived from sixteen HLA-A and HLA-B PCR-SSP typings. 75% of loci tested achieved a higher resolution HLA typing by the SBT method. DISCUSSION: The ability to provide allele level HLA typing results can have significant functional implications for the bone marrow transplant community and numerous vaccine studies.

Generation of alloantigen-stimulated anti-human immunodeficiency virus activity is associated with HLA-A*02 expression.

Stimulation of peripheral blood mononuclear cells (PBMC) with allogeneic PBMC (ALLO) can result in activity that inhibits the replication of human immunodeficiency virus (HIV). The present study demonstrates that strong anti-HIV activity is dependent on expression of HLA-A*02 by the responding PBMC. Anti-HIV activity was equally effective against 2 primary isolates that use different coreceptors. Neither ALLO-stimulated cell proliferation nor cytokine and beta-chemokine production was associated with the expression of HLA-A*02. ALLO-stimulated production of strong anti-HIV activity required intact PBMC and was not inhibited by monoclonal antibodies directed against nonpolymorphic regions of human leukocyte antigens (HLAs). Anti-HIV activity was generated by ALLO-stimulated CD4(+) cells, CD8(+) T lymphocytes, and monocytes from HLA-A*02-positive patients. These findings provide the first evidence that the production of an HIV inhibitory factor or factors is associated with certain HLA genes and raise new possibilities concerning the role of the major histocompatibility complex in controlling viral infections via alloantigen stimulation.

Frequency of human leukocyte antigen-A 24 alleles in patients with melanoma determined by human leukocyte antigen-A sequence-based typing.

The analysis of immune responses of patients with melanoma has led to the identification of melanoma-associated antigens targeted by T cells. Cytotoxic T lymphocytes recognize peptides from melanoma-associated antigens presented on the cancer cell surface in the context of HLA class I molecules. Immunodominant melanoma-associated antigen epitopes are being evaluated for their ability to immunize patients with advanced melanoma. However, these vaccination efforts are limited by the extensive polymorphism of the HLA class I heavy chain, which occurs in functional domains of the molecule. Patients with melanoma with the HLA-A-24 phenotype were recruited for vaccination with the peptide AFLPWHRLF from the melanoma-associated antigen tyrosinase. This peptide is recognized in association with HLA-A*2402. The HLA-A24 family includes at least 15 alleles whose frequency and ability to present the same peptide are unknown. The distribution of HLA-A24 alleles was studied in a melanoma population for the practical purpose of identifying patients suitable for vaccination with HLA-A*2402 epitopes. An HLA-A locus-specific polymerase chain reaction method followed by sequencing was developed to determine the HLA-A alleles in genomic DNA. HLA-A 24 was also typed in healthy persons of various ethnic backgrounds to further explore the HLA-A24 family. In white persons, the HLA-A*2402 allele was most common (in 85% of white persons and in 97% of the patients with melanoma). Fewer persons carried the HLA-A*2403 allele (13% in all samples, 3% in melanoma patients). Finally, two new alleles, HLA-A*2422 and HLA-A*24 null, were identified. These results suggest that vaccination with HLA-A*2402-associated epitopes has the potential for broad use in this patient population.

Recognition of a shared human prostate cancer-associated antigen by nonclassical MHC-restricted CD8+ T cells.

To identify prostate cancer-associated Ags, tumor-reactive T lymphocytes were generated using iterative stimulations of PBMC from a prostate cancer patient with an autologous IFN-gamma-treated carcinoma cell line in the presence of IL-2. A CD8+ T cell line and TCR alphabeta+ T cell clone were isolated that secreted IFN-gamma and TNF-alpha in response to autologous prostate cancer cells but not to autologous fibroblasts or lymphoblastoid cells. However, these T cells recognized several normal and malignant prostate epithelial cell lines without evidence of shared classical HLA molecules. The T cell line and clone also recognized colon cancers, but not melanomas, sarcomas, or lymphomas, suggesting recognition of a shared epithelium-associated Ag presented by nonclassical MHC or MHC-like molecules. Although Ag recognition by T cells was inhibited by mAb against CD8 and the TCR complex (anti-TCR alphabeta, CD3, Vbeta12), it was not inhibited by mAb directed against MHC class Ia or MHC class II molecules. Neither target expression of CD1 molecules nor HLA-G correlated with T cell recognition, but beta2-microglobulin expression was essential. Ag expression was diminished by brefeldin A, lactacystin, and cycloheximide, but not by chloroquine, consistent with an endogenous/cytosolic Ag processed through the classical class I pathway. These results suggest that prostate cancer and colon cancer cells can process and present a shared peptidic Ag to TCR alphabeta+ T cells via a nonclassical MHC I-like molecule yet to be defined.

High-resolution HLA-A*0201 subtyping using directed heteroduplex analysis.

HLA-A02* has become an important target for cytotoxic T lymphocyte-based immunotherapy reflecting the high prevalence of this allele in patient populations. There are at least 26 different A*02 alleles, and their subtype specificity has significant functional implications for T-cell-mediated recognition of immunologic targets. We have developed a novel method for HLA-A*02 allelic screening using directed heteroduplex analysis (DHDA). DNA samples from Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines (EBV-B) representing 10 different HLA-A*02 alleles (0201, 0202, 0204, 0205, 0206, 0208, 0210, 0211, 0216, 0217) were prepared. In addition, DNA was prepared from 81 individuals representing a wide variety of A*02 subtypes previously determined by sequence specific primer (SSP) polymerase chain reaction (PCR) including individuals heterozygous for two A*02 specificities. Probes and samples were generated by PCR amplification using HLA-A*02 specific primers encompassing exons 2 and 3, where most of the functionally significant allelic polymorphism is clustered. DHDA was performed by generating heteroduplex molecules composed of a fluorescein-labeled allelic probe sequence and an unlabeled allelic PCR product. Gel retardation was consistent for allele-probe combinations. We were able to identify several A*02 alleles prepared from EBV-B cell lines that, when used as probes, had very impressive specificity and sensitivity. Combinations of two probes were identified (0205 + 0211 and 0208 + 0211) that allowed differentiation of A*0201 alleles from all other A*02 alleles tested. All samples typed by probe combinations had DHDA typing and SSP typing confirmed by DNA sequencing. This study expands the molecular typing repertoire available to the modern HLA laboratory, and shows that DHDA has significant promise as a reliable screening method for HLA A*02 subtyping.

Association of major histocompatibility complex determinants with the development of symptomatic and asymptomatic genital herpes simplex virus type 2 infections.

The clinical spectrum of herpes simplex virus (HSV) infections, ranging from asymptomatic to frequently distressing outbreaks, suggests that there may be immunologic determinants of disease severity that are associated with human leukocyte antigen (HLA) expression. A controlled, prospective study identified several major histocompatibility complex (MHC) class I and II antigens whose frequencies are associated with HSV-2 infection or with frequent symptomatic genital recurrences. Previous studies were hampered by the inability to serologically identify patients with asymptomatic HSV-2 infection. Clinical evaluation and Western blot assay were used to identify 3 subject cohorts: 1 with no prior HSV infections, 1 with HSV-2 antibodies but no recognized symptoms, and 1 with HSV-2 antibodies and frequent genital recurrences. Statistical comparisons of HLA frequencies among these cohorts showed associations of HLA-B27 and -Cw2 with symptomatic disease. Also, HLA-Cw4 was significantly associated with HSV-2 infection. These associations indicate that immunologic factors linked to the MHC influence the risk of HSV-2 infection and disease expression.

HLA antigens in childhood onset schizophrenia.

Evidence of immune system abnormalities in adult schizophrenia has prompted examination of the human leukocyte antigen (HLA) system. Childhood onset schizophrenia offers a unique opportunity to test neurodevelopmental hypotheses of schizophrenia, including those which implicate components of the immune system. In the present study, class I and II HLA antigens were typed using sequence-specific primers and the polymerase chain reaction in 28 childhood onset schizophrenics and 51 ethnically matched healthy subjects. Groups were compared for frequencies of HLA antigens reported to be associated with schizophrenia and/or autoimmune disorders. We hypothesized that antigen frequencies would differ between schizophrenic and healthy children, suggesting that some dimension of the neurodevelopmental disturbance experienced by these children may be mediated by subtle abnormalities of immune function. There were no significant differences between schizophrenic and healthy subjects in the frequency of any antigen tested. These findings do not support HLA-associated pathology in childhood onset schizophrenia.

HLA allele distribution distinguishes sporadic inclusion body myositis from hereditary inclusion body myopathies.

We studied the HLA class II associations in patients with sporadic inclusion body myositis (s-IBM) and hereditary inclusion body myopathies (h-IBM) and attempted to distinguish these myopathies on the basis of HLA allele assignments. Forty-five patients, 30 with s-IBM and 15 with h-IBM, underwent HLA class II allele-specific typing using polymerase chain reaction sequence-specific primers for 71 alleles contained in the DRbeta1, DRbeta3-5, and DQbeta1 loci. In s-IBM, we found a high (up to 77%) frequency of DRbeta1*0301, DRbeta3*0101 (or DRbeta3*0202) and DQbeta1*0201 alleles. No significant association with alleles in the DR and DQ haplotypes was found among the 15 h-IBM patients. The strong association of prominent alleles with s-IBM, but not h-IBM, suggests that s-IBM is a distinct disorder with an immunogenetic background that differs from h-IBM.

Differences in frequency distribution of HLA-A2 subtypes between North American and Italian white melanoma patients: relevance for epitope specific vaccination protocols.

Cytotoxic T lymphocytes (CTL) associated in vivo with tumor regression recognize the product of nonmutated genes expressed by most melanoma cells as peptides bound to human leukocyte antigen (HLA) molecules. Multiple HLA-A*0201 restricted peptides derived from melanoma associated antigens (MAA) have been described, and peptide-based vaccination protocols against melanoma are being developed worldwide for the treatment of HLA-A2 melanoma patients based on the assumption that most serologically typed HLA-A2+ individuals will be suitable for such vaccinations. Serologic typing of HLA-A2, however, encompasses a family of at least 17 related alleles recognized by molecular typing techniques and differing at one or more functional residues of the HLA class I molecule. We have recently shown that naturally occurring single-residue variants of HLA-A*0201 are responsible for significant differences in CTL response to MAA-peptide stimulation. Existing data for HLA-A*02 subtype frequencies among whites (who are most affected by melanoma) derive from analyses of Northern European and North American populations that are of similar heritage and predict an exceedingly rare (< 5%) frequency of non-HLA-A*0201 alleles. Melanoma however, affects other white populations in which the prevalence of HLA-A*02 alleles could be more variable. This study was done to identify HLA-A*02 subtypes and their prevalence in two ancestrally different white melanoma populations. HLA-A*02 subtype frequencies were compared by polymerase chain reaction between serologically HLA-A2+ melanoma patients referred for treatment to the Istituto Nazionale Tumori of Milan (n = 93), Italy or the National Cancer Institute, Bethesda, MD, U.S.A. (n = 100). This analysis demonstrated differences in subtype specificity and distribution between the two populations, with a significantly higher percentage of non HLA-A*0201 subtypes in the Italian population. Only 2% of serologically HLA-A2+ Northern American white melanoma patients did not express HLA-A*0201. In contrast, 15% of HLA-A2+ Italian patients were not HLA-A*0201 (p2 value = 0.001). As allele-specific/peptide-based vaccination protocols are presently pursued at several institutions, a proportion of patients might be inappropriately enrolled basing their eligibility on serologically defined HLA-typing.

DR2/DQw1 inheritance and haplotype sharing in affected siblings from multiple sclerosis families.

Although the human leukocyte antigen DR2/DQw1 allele has been associated with multiple sclerosis, studies of DR2/DQw1 inheritance in multiple sclerosis multiplex families have yielded conflicting results. We examined this question in "high-incidence" families, defined as families with more than 50% of siblings affected. DR2/DQw1 allele frequencies were significantly increased, particularly in mothers and affected siblings (p < 0.0001). The transmission of DR2/DQw1 from both parents was more frequent in affected offspring (p = 0.005). While evidence for segregation of disease with a particular parental allele was lacking in most families, the frequency of haplotype sharing was higher in affected sib pairs (p < 0.01).

Expression of HLA class I molecules and the transporter associated with antigen processing in hepatocellular carcinoma.

The expression of the HLA class I molecules on the cell surface was investigated in hepatocellular carcinoma (HCC) cell lines using complement-mediated cytotoxicity (CMC) and flow cytometric analysis. Although HLA-A antigens were detected by CMC in all cell lines tested, HLA-B and -C antigens were not detectable in six of seven HCC cell lines. These results were also confirmed by flow cytometric analysis focusing on HLA-Bw4 and Bw6 public antigens. Furthermore, complementary DNA (cDNA) from each cell line was tested for the expression of HLA-A, -B, -C and the transporter associated with antigen processing genes (TAP1 and TAP2). Two cell lines showed a reduced level of one or both of the TAP messenger RNAs (mRNAs), and one of these showed a reduction of HLA-B and -C gene expression as well, but the others had detectable mRNA levels. These results demonstrate that hepatocellular carcinoma cell lines tested in the current study lose or decrease the expression of HLA-B and -C alleles on the cell surface, even though mRNA encoding these alleles is present, suggesting that the loss of the HLA molecules might be caused by posttranscriptional events or failure to transport and load peptides necessary for HLA expression. The selective loss of HLA-B and -C, but not -A, molecules (which also excludes a beta 2-microglobulin defect) is intriguing, and may be attributable to the ability of some of the HLA-A molecules to load signal peptides not requiring TAP transport, or to natural selection of HLA-B or -C locus-specific immune surveillance.

Analysis of expression of the melanoma-associated antigens MART-1 and gp100 in metastatic melanoma cell lines and in in situ lesions.

MART-1 and gp100 melanoma associated antigens (MAA) are expressed by cells of the melanocytic lineage and are recognized by the majority of HLA-A2 restricted tumor-infiltrating lymphocytes. Heterogeneity of expression of MAA in tumor deposits may affect the natural history or response to therapy of patients with melanoma. In this study, we evaluated the expression of these MAA with a new monoclonal antibody (mAb) directed against MART-1 (M2-7C10) and the commercially available HMB45 mAb directed against gp100. Expression was tested in vitro by intracellular fluorescence analysis and in vivo by immunophenotyping of tissue specimens. Nine melanoma cell lines and 25 tissue specimens from metastatic melanoma were analyzed. One cell line did not express MART-1 or gp100. The expression of both antigens was more heterogeneous and significantly reduced (p < 0.01) in melanoma cell lines compared with melanocytes, suggesting progressive loss of expression of MAA by neoplastic cells. None of the nonmelanoma cancer lines tested stained for MART-1 or gp100. Analysis of melanoma lesions by immunohistochemistry showed significant heterogeneity of expression of both MART-1 and gp100 MAA either as a percentage of cells expressing MAA or as intensity of expression. Ten of 25 frozen sections expressed MART-1 in < 50% of the cells. In 6 of 25 lesions, immunoreactivity for MART-1 was totally absent. Fine needle aspiration of metastatic lesions seemed to yield information accurately about amount and heterogeneity of expression of MAA in tumor lesions in vivo. Heterogeneity of expression of MAA may be one of several mechanisms leading to tumor escape from immune recognition, and pretreatment evaluation of tumor lesion for expression of these antigens may help in selecting patients best suited to antigen-specific vaccine therapies.

Novel HLA-B alleles, B*8201, B*3515 and B*5106, add to the complexity of serologic identification of HLA types.

Three class I alleles, B*8201, B*3515 and B*5106, have been described using DNA and cDNA sequencing. The B*8201 allele is most structurally related to B*5602, differing from it by 14 nucleotide substitutions resulting in 5 amino acid differences. The other two alleles, B*3515 and B*5106, differ from their most closely related HLA-B alleles by 2-3 nucleotide substitutions resulting in 1-2 amino acid substitutions, respectively. The majority of nucleotide substitutions marking these new alleles are observed in other HLA-B alleles suggesting that gene conversion and/or reciprocal recombination have created this diversity. All of the amino acid substitutions are predicted to alter the antigen binding site of the HLA-B molecule. The newly defined HLA-B allelic products were originally defined by their unusual serologic reactivity patterns. The B*8201 allelic product is serologically typed as a B "blank" or as a variant of B22 or B45. These patterns and the serologic reactivity of the other newly described allelic products are consistent with the protein sequence homology among specific HLA-B molecules. While serology remains a powerful tool for detecting HLA diversity, alleles generated by events resulting in the sharing of HLA sequence polymorphisms among alleles at a locus will continue to create complexity in the interpretation of typing results.

Combination therapy with interferon-gamma and interleukin-2 for the treatment of metastatic melanoma.

The toxicity and clinical response to treatment with the combination of interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) in patients with metastatic melanoma was evaluated. From May 1993 through February 1994, 20 patients were treated with 24 courses of IFN-gamma with or without IL-2. A 7-day course of subcutaneous IFN-gamma alone was administered to cohorts of two or three patients each at doses of 0.1, 0.2, or 0.3 mg/m2. Thirteen patients received escalating doses of IFN-gamma between 0.2 and 0.5 mg/m2 followed by the intravenous (i.v.) administration of IL-2 (720,000 IU/kg) given three times a day. A treatment course consisted of two cycles (maximum of 15 doses of IL-2 per cycle) separated by a 10-day interval. Five additional patients were treated with five courses of IFN-gamma, IL-2, and tumor-infiltrating lymphocytes (TILs). All patients treated had the diagnosis of metastatic melanoma. The maximal tolerated dose of subcutaneous IFN-gamma was established at 0.3 mg/m2 with dose-limiting hepatotoxicity. Immunohistochemistry analyses showed detectable upregulation of MHC class I alleles in one (8%) of 12 patients. Two of 20 patients who received the combination of IFN-gamma and IL-2 had responses, one partial and one complete response. The duration of response was 7 months for the partial response and 12 months for the complete response. IFN-gamma was tolerated with minimal side effects of nausea, vomiting, malaise, and decreased hematopoiesis. No increased toxicities were found with the combination treatment, as compared with IL-2 alone. One death occurred on the third day of treatment with IFN-gamma alone from hemorrhage into brain metastases. There were no responders in the five patients who received the combination treatment of TIL, IL-2, and IFN-gamma. From these findings, we conclude that further studies looking at this combination treatment are not warranted.

HLA associations in the antitumor response against malignant melanoma.

In this study we analyzed the human leukocyte antigen (HLA) pattern of North American Caucasian patients with metastatic melanoma as compared with the North American Caucasian (NAC) population. We also investigated whether the HLA type of melanoma patients had an effect on their tolerance and response to interleukin-2 (IL-2)-based therapy. Four hundred twelve serologic phenotypes of Caucasian melanoma patients referred to the National Cancer Institute, National Institutes of Health, from February 1989 through December 1993 were collected by typing the patient's peripheral blood lymphocytes. Furthermore, 74 melanoma patients were typed for HLA class II by high-resolution sequence specific primer-polymerase chain reaction. Response rate and treatment-related toxicity in those patients receiving IL-2-based treatment (N = 272) were compared with HLA serologic types. The frequency of four HLA-B alleles was significantly different in the melanoma compared with the NAC population: of these, HLA-B5, -B8, and -B15 had a frequency falling between the NAC and the Northern European population. No other significant differences between melanoma patients and NAC population were noted for other HLA loci. A correlation was noted between HLA-DR3 and -DR4 alleles and decreased tolerance to IL-2, whereas homozygosity for HLA-DR decreased the chance of response. There were no significant associations between HLA type and response. It is unlikely that the associations noted between some HLA-B alleles and melanoma bear significantly on the etiology of the disease. The differences seen between American melanoma patients and the NAC population are probably best explained by geographical ancestry. The association between HLA-DR and tolerance to IL-2 therapy noted in this study may offer insight toward the understanding of mechanisms regulating the cascade of events after the systemic administration of IL-2.

Quantitative correlation between HLA class I allele expression and recognition of melanoma cells by antigen-specific cytotoxic T lymphocytes.

MHC class I antigen expression is necessary for CD8+ T-cell-mediated recognition of tumors. Recently, several mechanisms leading to loss or decreased expression of MHC antigens on the tumor cell surface have been described that may account for tumor escape from immune recognition. It is yet unknown whether tumor recognition by CTL occurs at a threshold amount of MHC molecules or correlates with the level of HLA-allele expression. In this study, a model was developed in which clones derived from the 624-MEL melanoma cell line and expressing varying amounts of HLA-A2 molecules were lysed in a standard 51Cr release assay by an HLA-A2-restricted CTL clone (A42) or a bulk culture of tumor-infiltrating lymphocytes. The A42 clone and the tumor-infiltrating lymphocyte culture were characterized previously as specifically recognizing the melanoma antigen MART-1(27-35) peptide. A marked heterogeneity in the susceptibility to lysis by A42 was observed in tumor clones and was not due to heterogeneous expression of MART-1 by the clones or loss of accessory molecules involved in the lymphocyte-target interaction. Lysis by A42 and by the tumor-infiltrating lymphocyte culture significantly correlated with the level of HLA-A2 expression, evaluated as mean channel number of fluorescence by flow cytometry (P < 0.001). Transfection of an HLA-A2-negative clone (624.28) with the HLA-A2.1 gene produced a panel of clones expressing different levels of HLA-A2, the lysis of which was highly correlated with the expression of HLA-A2 (P < 0.001). The addition of exogenous MART-1(27-35) peptide enhanced lysis of clones expressing intermediate amounts of HLA-A2 but did not affect clones with high expression. These data suggest that the number of HLA molecules present on the surface of tumor cells can quantitatively affect their lysis by CTL in situations with borderline amounts of peptide and/or MHC.

Loss of HLA haplotype and B locus down-regulation in melanoma cell lines.

The expression of HLA class I molecules on tumor cells is vital for CD8+T cell recognition of tumor Ags. Loss of HLA class I Ag expression as a result of defective beta 2-microglobulin genes has been described in melanoma cells. To further evaluate mechanisms of tumor escape, HLA class I Ag expression was compared in 24 metastatic melanoma cell lines and 20 melanocyte strains by FACS analysis with use of allele-specific mAbs. Total loss of HLA class I Ag expression was not noted; instead, two relatively common phenomena were identified: 1) A variable degree of expression of HLA-B Ags by melanoma cell lines and melanocytes; however, HLA-A Ags were consistently expressed in all cell types. Furthermore, HLA-B locus Ag expression was detected in vivo in only one of six frozen section specimens obtained from six patients having metastatic melanoma. 2) Loss of allelic expression was noted in two of 14 HLA-A2 (14%) and one of three HLA-A29 (33%) melanoma cell lines and included a full haplotype, which suggests loss of a genomic fragment. Allele-specific PCR amplification demonstrated deletion of genes in linkage disequilibrium within the MHC class II, III, and I regions. Aberrations of HLA class I expression in tumor lines should be considered when assessing MHC-restricted phenomena in in vitro models.

Locus-specific analysis of human leukocyte antigen class I expression in melanoma cell lines.

Surface expression of human leukocyte antigen (HLA) class I antigens on melanoma lines was evaluated by locus-specific monoclonal antibodies (mAbs) with three different techniques: Fluorescence-activated cell sorting (FACS), immunohistochemistry with cytospin preparation (ICP), and complement-mediated cytotoxicity (CMC). Eleven HLA class I-expressing cell lines developed from metastases were used. Specific expression of HLA loci was examined under routine culture conditions and after 48-h incubation in interferon-gamma (IFN-gamma; 500 U/ml). Loss of allelic expression was seen in one line (586-MEL): Products of genes coding for HLA-A29 and -B44, in strong linkage disequilibrium, were not detectable. HLA-A antigens were consistently detected by all methodologies and minimally affected by pretreatment with IFN-gamma. HLA-B antigens were detectable in 8 of 11 lines by ICP and 3 of 11 lines by CMC. By FACS the supratypic specificity HLA-Bw6 was expressed at low levels in most lines (mean fluorescence 47.2 +/- 13.4 and rose to 259.8 +/- 45.9 after incubation with IFN-gamma; p < 0.001). HLA-Cw antigen detection by CMC correlated with HLA-B (p < 0.01), suggesting that down-regulation and sensitivity to IFN-gamma are shared by the two loci. This low expression of the HLA-B antigens may play a role in the evasion of the host immune response and its up-regulation may be useful in allowing tumor antigen recognition.