Surface Human Leukocyte Antigen Class Research Articles

294-OR: The Type 1 Diabetes Protective Variant of Tyrosine Kinase 2 (TYK2) Protects ß-Cells from Autoimmunity

Type 1 diabetes (T1D) is a complex autoimmune disease where genes and environmental factors impact pathogenesis. Analysis of the >150 loci identified by GWAS reveals the compelling theme that candidate genes cluster into pathways. A pathway connecting environment to T1D genetics is the anti-viral/Type 1 interferon (IFN1) pathway that is regulated by at least 7 T1D linked loci, including Tyrosine Kinase 2 (TYK2) which regulates IFN1 receptor signaling. We test the hypothesis that the T1D protective variant of TYK2 restrains signaling though the IFN1 receptor functions in β-cells reduce interactions with and killing by autoreactive, cytolytic CD8+ T lymphocytes (CTL) . These studies employed β-cells edited in the endogenous TYK2 locus with CRISPR/Cas9 to express the T1D protective TYK2110A or common allotype, TYK2110P. These cells were treated with Poly I:C (50ng/mL) , to see IFN1 response to an imitated viral infection. Outcomes were IFN1 production, gene expression, antigen presentation, and responses to CTL. When allotypic β-cells were treated with Poly I:C, both produced equivalent levels of IFN1. While β-cells with TYK2110P showed increased mechanisms of antigen presentation, this pathway was attenuated in TYK2110A β-cells (p<0.05) . These results were replicated by treating TYK2110P and TYK2110A IFN1 (100U/mL) , where we observed increased antigen presentation in TYK2110P β-cells but not TYK2110A β-cells (p<0.05) . Interestingly, prior to treatment cell surface human leukocyte antigen class I levels were reduced in TYK2110A β-cells (p<0.01) . Studies to test the interactions of autoreactive CTL with β-cells supported our hypothesis. TYK2110P β-cells were efficiently lysed by CTL and lysis was increased by pre-treatment of β-cells with IFN1 or PolyI:C. In stark contrast, TYK2110A β-cells exhibited low levels of CTL-lysis and pre-treatment with IFN1 or PolyI:C did not modify lysis. In conclusion, TYK2110A protects β-cells from autoimmune-mediated death. Disclosure A.Rodriguez tamayo: None. M.Huber: None. E.J.Butfiloski: None. C.E.Mathews: None. Funding National Institutes of Health PAI42288National Institutes of Health RR01DK127497

Abstract P071: Gemcitabine augments HLA class I expression in pancreatic cancer cells through alterations in transcript production and surface stability

Abstract Background: Pancreatic adenocarcinoma, or PDAC, is the fourth leading cause of cancer-related deaths in the United States. Gemcitabine, a nucleoside analog, is a primary standard of care in pancreatic cancer. In addition to its normative cytotoxic function, evidence suggests that this chemotherapy drug also harnesses immunomodulatory capabilities in the form of increasing human leukocyte antigen (HLA) class I expression in lung, breast, colon, and cholangiocarcinoma cells. HLA class I is a complex (alpha heavy chain and beta 2-microglobulin light chain) located at the surface of nearly all cells where it presents peptides, including cancer-associated peptides, to cytotoxic T cells. Recognition of these peptides as atypical leads to T-cell mediated lysis of the presenting cell. Subsequently, understanding the ability of gemcitabine and the mechanisms by which it influences the HLA class I complex are of great importance. Methods: To investigate the effect of gemcitabine treatment on HLA class I expression in pancreatic cancer, alterations in HLA class I protein levels were monitored via western blot analysis, flow cytometry, and quantitative polymerase chain reaction (qPCR) in three pancreatic cancer cell lines. Changes in surface stability of the HLA class I complex were evaluated through brefeldin A (BFA) assays at the 72-hour time point. For western blot and flow cytometry experiments, impacts on the alpha heavy chain were further assessed for all three types of alpha heavy chains (HLA-A, HLA-B/C) at 72 and 96 hours. In qPCR experiments, alterations were analyzed for the HLA-A, HLA-B, and beta 2-microglobulin transcripts after a 48-hour gemcitabine exposure period. Results: Administration of gemcitabine to pancreatic cancer cell lines (S2-013, Capan-1, PANC-1) increased total protein levels of both HLA class I constituents (alpha heavy chain and beta-2-microglobulin light chain). All PDAC cell lines evaluated demonstrated enhanced surface expression of HLA-A2 and HLA-B/C with maximal increases of 3 and 2.5-fold respectively, as indicated by flow cytometry. Our qPCR analysis of the Capan-1 and S2-013 cell lines revealed increases in the levels of HLA-A, HLA-B, and beta 2-microglobulin transcripts (3-12-fold). BFA assays suggested that gemcitabine treatment also enhances stability of the surface HLA class I in the S2-013 and PANC-1 cell lines. Conclusion: In summary, gemcitabine exhibits an immunomodulatory ability to stimulate expression of HLA class I in pancreatic cancer cells. We have demonstrated that this increase in HLA class I is seen at the mRNA, total protein, surface, and stability level. Characterizing the ability of gemcitabine to influence the HLA class I complex and defining the mechanisms by which it does so will increase the potential for identification of suitable immunotherapies, including novel peptide-based cancer vaccines with enhanced treatment efficacy for PDAC patients. Citation Format: Alaina C. Larson, Shelby M. Knoche, Joyce C. Solheim. Gemcitabine augments HLA class I expression in pancreatic cancer cells through alterations in transcript production and surface stability [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P071.

Distinct Molecular Mechanisms of Altered HLA Class II Expression in Malignant Melanoma.

Simple SummaryThere exists limited knowledge about the underlying molecular processes controlling the expression of HLA class II APM components and their prognostic significance in melanoma. Therefore, this study analyzed the basal and regulated expression of HLA class II antigens and components in melanoma cell lines and patients’ lesions in conjunction to T-cell infiltration. The heterogeneous constitutive HLA class II APM expression was caused by distinct molecular mechanisms and was partially linked to immune cell infiltration and clinical parameters. These results contribute not only to a better understanding of the regulation of HLA class II expression in melanoma, but might have an impact on the design of novel (immuno)therapies for the treatment of this disease.Background: The human leukocyte antigen (HLA) class II molecules are constitutively expressed in some melanoma, but the underlying molecular mechanisms have not yet been characterized. Methods: The expression of HLA class II antigen processing machinery (APM) components was determined in melanoma samples by qPCR, Western blot, flow cytometry and immunohistochemistry. Immunohistochemical and TCGA datasets were used for correlation of HLA class II expression to tumor grading, T-cell infiltration and patients’ survival. Results: The heterogeneous HLA class II expression in melanoma samples allowed us to characterize four distinct phenotypes. Phenotype I totally lacks constitutive HLA class II surface expression, which is inducible by interferon-gamma (IFN-γ); phenotype II expresses low basal surface HLA class II that is further upregulated by IFN-γ; phenotype III lacks constitutive and IFN-γ controlled HLA class II expression, but could be induced by epigenetic drugs; and in phenotype IV, lack of HLA class II expression is not recovered by any drug tested. High levels of HLA class II APM component expression were associated with an increased intra-tumoral CD4+ T-cell density and increased patients’ survival. Conclusions: The heterogeneous basal expression of HLA class II antigens and/or APM components in melanoma cells is caused by distinct molecular mechanisms and has clinical relevance.

Generation of Retinal Pigment Epithelial Cells Derived from Human Embryonic Stem Cells Lacking Human Leukocyte Antigen Class I and II

SummaryHuman embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells could serve as a replacement therapy in advanced stages of age-related macular degeneration. However, allogenic hESC-RPE transplants trigger immune rejection, supporting a strategy to evade their immune recognition. We established single-knockout beta-2 microglobulin (SKO-B2M), class II major histocompatibility complex transactivator (SKO-CIITA) and double-knockout (DKO) hESC lines that were further differentiated into corresponding hESC-RPE lines lacking either surface human leukocyte antigen class I (HLA-I) or HLA-II, or both. Activation of CD4+ and CD8+ T-cells was markedly lower by hESC-RPE DKO cells, while natural killer cell cytotoxic response was not increased. After transplantation of SKO-B2M, SKO-CIITA, or DKO hESC-RPEs in a preclinical rabbit model, donor cell rejection was reduced and delayed. In conclusion, we have developed cell lines that lack both HLA-I and -II antigens, which evoke reduced T-cell responses in vitro together with reduced rejection in a large-eyed animal model.

Apparent Lack of BRAFV600E Derived HLA Class I Presented Neoantigens Hampers Neoplastic Cell Targeting by CD8+ T Cells in Langerhans Cell Histiocytosis.

Langerhans Cell Histiocytosis (LCH) is a neoplastic disorder of hematopoietic origin characterized by inflammatory lesions containing clonal histiocytes (LCH-cells) intermixed with various immune cells, including T cells. In 50–60% of LCH-patients, the somatic BRAFV600E driver mutation, which is common in many cancers, is detected in these LCH-cells in an otherwise quiet genomic landscape. Non-synonymous mutations like BRAFV600E can be a source of neoantigens capable of eliciting effective antitumor CD8+ T cell responses. This requires neopeptides to be stably presented by Human Leukocyte Antigen (HLA) class I molecules and sufficient numbers of CD8+ T cells at tumor sites. Here, we demonstrate substantial heterogeneity in CD8+ T cell density in n = 101 LCH-lesions, with BRAFV600E mutated lesions displaying significantly lower CD8+ T cell:CD1a+ LCH-cell ratios (p = 0.01) than BRAF wildtype lesions. Because LCH-lesional CD8+ T cell density had no significant impact on event-free survival, we investigated whether the intracellularly expressed BRAFV600E protein is degraded into neopeptides that are naturally processed and presented by cell surface HLA class I molecules. Epitope prediction tools revealed a single HLA class I binding BRAFV600E derived neopeptide (KIGDFGLATEK), which indeed displayed strong to intermediate binding capacity to HLA-A*03:01 and HLA-A*11:01 in an in vitro peptide-HLA binding assay. Mass spectrometry-based targeted peptidomics was used to investigate the presence of this neopeptide in HLA class I presented peptides isolated from several BRAFV600E expressing cell lines with various HLA genotypes. While the HLA-A*02:01 binding BRAF wildtype peptide KIGDFGLATV was traced in peptides isolated from all five cell lines expressing this HLA subtype, KIGDFGLATEK was not detected in the HLA class I peptidomes of two distinct BRAFV600E transduced cell lines with confirmed expression of HLA-A*03:01 or HLA-A*11:01. These data indicate that the in silico predicted HLA class I binding and proteasome-generated neopeptides derived from the BRAFV600E protein are not presented by HLA class I molecules. Given that the BRAFV600E mutation is highly prevalent in chemotherapy refractory LCH-patients who may qualify for immunotherapy, this study therefore questions the efficacy of immune checkpoint inhibitor therapy in LCH.

HLA dependent immune escape mechanisms in B-cell lymphomas: Implications for immune checkpoint inhibitor therapy?

ABSTRACTAntigen presentation by tumor cells in the context of Human Leukocyte Antigen (HLA) is generally considered to be a prerequisite for effective immune checkpoint inhibitor therapy. We evaluated cell surface HLA class I, HLA class II and cytoplasmic HLA-DM staining by immunohistochemistry (IHC) in 389 classical Hodgkin lymphomas (cHL), 22 nodular lymphocyte predominant Hodgkin lymphomas (NLPHL), 137 diffuse large B-cell lymphomas (DLBCL), 39 primary central nervous system lymphomas (PCNSL) and 19 testicular lymphomas. We describe a novel mechanism of immune escape in which loss of HLA-DM expression results in aberrant membranous invariant chain peptide (CLIP) expression in HLA class II cell surface positive lymphoma cells, preventing presentation of antigenic peptides. In HLA class II positive cases, HLA-DM expression was lost in 49% of cHL, 0% of NLPHL, 14% of DLBCL, 3% of PCNSL and 0% of testicular lymphomas. Considering HLA class I, HLA class II and HLA-DM together, 88% of cHL, 10% of NLPHL, 62% of DLBCL, 77% of PCNSL and 87% of testicular lymphoma cases had abnormal HLA expression patterns. In conclusion, an HLA expression pattern incompatible with normal antigen presentation is common in cHL, DLBCL, PCNSL and testicular lymphoma. Retention of CLIP in HLA class II caused by loss of HLA-DM is a novel immune escape mechanism, especially prevalent in cHL. Aberrant HLA expression should be taken into account when evaluating efficacy of checkpoint inhibitors in B-cell lymphomas.

Adenovirus expressing β2-microglobulin recovers HLA class I expression and antitumor immunity by increasing T-cell recognition.

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.

Homozygosity for killer immunoglobin-like receptor haplotype A predicts complete molecular response to treatment with tyrosine kinase inhibitors in chronic myeloid leukemia patients

Several recent reports suggest a possible role for killer immunoglobulin-like receptors (KIR) in the onset of chronic myeloid leukemia (CML) and response to therapy with tyrosine kinase inhibitors (TKIs). To explore this hypothesis, we studied KIRs and their human leukocyte antigen class I ligands in 59 consecutive patients with chronic-phase CML (mean age, 53 years; range, 23-81 years) and a group of 121 healthy control participants belonging to the same ethnic group as the patients. The 2-year cumulative incidence of complete molecular response, obtained after a median of 27 months (range, 4-52 months), was 51.2%. An increased frequency of the activating receptor KIR2DS1 (pm = 0.05) and a reduced frequency of the KIR-ligand combination KIR2DS2/2DL2 absent/C1 present (pm = 0.001) were significantly associated with CML. Moreover, KIR repertoires in patients appeared to influence response to TKI therapy. Homozygosity for KIR haplotype A (pm = 0.01), a decreased frequency of the inhibitory KIR gene KIR2DL2 (pm = 0.02), and low numbers of inhibitory KIR genes (pm = 0.05) were all significantly associated with achievement of complete molecular remission. These data suggest that a decrease in properly stimulated and activated NK cells might contribute to the occurrence of CML and indicate homozygosity for KIR haplotype A as a promising immunogenetic marker of complete molecular response that could help clinicians decide whether to withdraw treatment in patients with CML.

Two Newly Diagnosed HLA Class II-Deficient Patients Identified by Rapid Vector-Based Complementation Analysis Reveal Discoordinate Invariant Chain Expression Levels

Background: Primary immunodeficiencies represent the ‘molecular Achilles’ heels’ of human immunity. Detailed analyses of primary immunodeficiencies extend our knowledge of pivotal immunological processes, lead to novel diagnostic algorithms and shorten the time to diagnosis. Methods: Clinical/immunological phenotypes of 2 unrelated patients from Austria with combined immunodeficiency were determined. Leukocyte subpopulations of these patients, their parents and healthy controls were analyzed by flow cytometry. Patient-derived Epstein-Barr virus (EBV)-transformed B cell lines were established and complemented by candidate cDNAs. Suspected mutations were confirmed by DNA sequencing. Results: Phenotyping revealed a lack of constitutive human leukocyte antigen (HLA) class II expression on antigen-presenting cells of both patients, compatible with MHC class II deficiency. Rapid vector-based complementation analysis of the patients’ B cells identified HLA class II transactivator (CIITA) deficiency in patient VIP1 and regulatory factor X (RFX)AP deficiency in patient VIP2. CIITA deficiency was caused by a homozygous p.Glu381X mutation. RFXAP deficiency resulted from a homozygous p.Ser123ThrfsX15 mutation, not described in the Middle European population so far. Of note, HLA class II-associated invariant chain (Ii) expression levels were significantly reduced in VIP1 and 3 additional EBV-transformed B cell lines of CIITA-deficient patients but normal in EBV-transformed B cells from VIP2. In addition, peripheral blood B cells from the parents of VIP1 showed significantly reduced HLA-DR and -DP expression levels compared to healthy controls. Conclusions: Analysis of patients’ intracellular Ii and their parents’ surface HLA class II expression levels might help to identify CIITA-deficient patients already during initial phenotyping.

Effect of invariant chain on major histocompatibility complex class I molecule expression and stability on human breast tumor cell lines

Invariant chain (Ii) binds to the human leukocyte antigen (HLA) class II molecule and assists it in the process of peptide acquisition. In addition, Ii binds to the HLA class I molecule, although there has been little study of its effects on the HLA class I molecule. In addition to its normal expression on antigen-presenting cells, Ii expression is up regulated in a variety of tumors. By flow cytometric analysis, we found that expression of Ii resulted in an increase in the number of cell surface HLA class I molecules and in the proportion of unstable HLA class I molecules at the surface of breast tumor cell lines. These data suggest that the expression of Ii by tumor cells may quantitatively and qualitatively alter the presentation of antigens on those cells.

Derivation and immunological characterization of mesenchymal stromal cells from human embryonic stem cells

We have previously shown the simultaneous generation of CD73(+) mesenchymal stromal cells (MSCs) along with CD34(+) hematopoietic cells from human embryonic stem cells (ESCs) when they are cocultured with OP9 murine stromal cells. We investigated whether MSCs can be derived from human ESCs without coculturing with OP9 cells, and if such cells exhibit immunological properties similar to MSCs derived from adult human bone marrow (BM). Our starting populations were undifferentiated human ESCs cultured on Matrigel-coated plates without feeder cells. The differentiated fibroblast-looking cells were tested for expression of MSC markers and their potential for multilineage differentiation. We investigated surface expression of human leukocyte antigen (HLA) molecules on these MSCs before and after treatment with interferon-gamma (IFN-gamma). We also tested the proliferative response of T-lymphocytes toward MSCs and the effects of MSCs in mixed lymphocyte reaction (MLR) assays. We derived populations of MSCs from human ESCs with morphology, cell surface marker characteristics, and differentiation potential similar to adult BM-derived MSCs. Similar to BM-derived MSCs, human ESC-derived MSCs express cell surface HLA class I (HLA-ABC) but not HLA class II (HLA-DR) molecules. However, stimulation with IFN-gamma induced the expression of HLD-DR molecules. Human ESC-derived MSCs did not induce proliferation of T-lymphocytes when cocultured with peripheral blood mononuclear cells. Furthermore, ESC-derived MSCs suppressed proliferation of responder T-lymphocytes in MLR assays. MSCs can be derived from human ESCs without feeder cells. These human ESC-derived MSCs have cell surface markers, differentiation potentials, and immunological properties in vitro that are similar to adult BM-derived MSCs.

Concordant expression of proto‐oncogene promyelocytic leukemia and major histocompatibility antigen HLA class I in human hepatocellular carcinoma

Many malignant cancer cells downregulate human leukocyte antigen (HLA) class I antigen expression to evade T cell recognition. However, hepatocellular carcinoma (HCC) is exceptional to the general findings in cancer cells, and the mechanisms for its upregulation remain unclear. It has been reported that promyelocytic leukemia (PML) proto-oncogene controls the transcription of multiple class I antigen presentation genes in murine cancer cells. To find out the functional role of PML gene on the increased HLA class I antigen expression in HCC cells, we analyzed the expression of proto-oncogene PML and multiple class I antigen presentation genes in HCC specimens obtained in China. The results showed concordant changes of proto-oncogene PML and cell surface HLA-A expression in 44 paraffin-embedded HCC tissues. Furthermore, co-upregulated expression of PML genes and class I antigen presentation genes could be detected in 9 of 15 fresh HCC tissues by reverse transcription polymerase chain reaction (RT-PCR). In addition, studies using HCC cell lines showed that increased expression of HLA class I molecules paralleled with PML upregulation were detected in QGY-7701 HCC cell line with RT-PCR, western blot, and flow cytometry, and that the overexpression of exogenous PML in a low-expression class I cell line BEL-7405 could induce the expression of multiple class I antigen-presenting molecule genes and slightly but significantly increase the expression of cell surface HLA class I molecules. In conclusion, the expression of proto-oncogene PML and HLA class I molecules were concordantly upregulated and the expression of PML gene might be one of the mechanisms that leads to the increased expression of class I antigen in HCC.

Low‐molecular‐weight protein (LMP)2/LMP7 abnormality underlies the downregulation of human leukocyte antigen class I antigen in a hepatocellular carcinoma cell line

Tumor cells may alter the expression of numerous components involved in antigen-processing machinery to decrease human leukocyte antigen (HLA) class I expression, allowing the tumor cells to escape immune surveillance. The purpose of the present study was to investigate the involvement of these components in the downregulation of HLA class I expression in human hepatocellular carcinoma cell line BEL7,404. Expression of HLA-I and antigen presentation-related genes were analyzed by flow cytometry and polymerase chain reaction. The HLA class I-deficient BEL7,404 cell was transfected with the low-molecular-weight protein (LMP) 2 and LMP7 gene and were analyzed by flow cytometry for restoration of surface HLA class I expression. The BEL7,404 cells downregulated the expression of HLA class I antigen and lacked expression of LMP2 and LMP7. Interferon (IFN)-gamma treatment increased the expression of LMP2 but not LMP7. The LMP2-transfected BEL7,404 cells or LMP2 and LMP7-cotransfected cells restored surface HLA class I expression while LMP7-transfected cells did not. However, in IFN-gamma-treated BEL7,404 cells, transfection with the LMP7 gene induced more HLA class I expression than mock transfection. The LMP2 gene was required for the expression of HLA class I molecules in BEL7,404. The LMP7 was not the major reason for loss of HLA class I in BEL7,404 cells, although the supply of exogenous LMP7 could increase surface expression of HLA class I antigen.

Defective Human Leukocyte Antigen Class I-associated Antigen Presentation Caused by a Novel β2-Microglobulin Loss-of-function in Melanoma Cells

The major histocompatibility complex class I molecules consist of three subunits, the 45-kDa heavy chain, the 12-kDa beta(2)-microglobulin (beta(2)m), and an approximately 8-9-residue antigenic peptide. Without beta(2)m, the major histocompatibility complex class I molecules cannot assemble, thereby abolishing their transport to the cell membrane and the subsequent recognition by antigen-specific T cells. Here we report a case of defective antigen presentation caused by the expression of a beta(2)m with a Cys-to-Trp substitution at position 25 (beta(2)m(C25W)). This substitution causes misfolding and degradation of beta(2)m(C25W) but does not result in complete lack of human leukocyte antigen (HLA) class I molecule expression on the surface of melanoma VMM5B cells. Despite HLA class I expression, VMM5B cells are not recognized by HLA class I-restricted, melanoma antigen-specific cytotoxic T lymphocytes even following loading with exogenous peptides or transduction with melanoma antigen-expressing viruses. Lysis of VMM5B cells is restored only following reconstitution with exogenous or endogenous wild-type beta(2)m protein. Together, our results indicate impairment of antigenic peptide presentation because of a dysfunctional beta(2)m and provide a mechanism for the lack of close association between HLA class I expression and susceptibility of tumor cells to cytotoxic T lymphocytes-mediated lysis in malignant diseases.

An immunohistochemical study of altered immunomodulatory molecule expression in head and neck squamous cell carcinoma.

For the presentation of peptide antigens to cytotoxic CD8+ T lymphocytes of the immune system, the expression of human leukocyte antigen (HLA) class I molecules on the cell surface is necessary. There is increasing evidence that surface HLA class I antigen expression is altered in a variety of human tumours by either loss or down-regulation of these molecules, which may be a strategy for evasion of immunosurveillance by malignant cells. This study has examined the expression of HLA class I molecules in head and neck squamous cell carcinoma (HNSCC) specimens by immunohistochemistry, using a wide panel of antibodies directed against allele-specific as well as monomorphic determinants of these molecules. The expression of TAP proteins, HLA-DR and the co-stimulatory molecule ICAM-1 were also studied. In addition, the expression of the tumour-associated antigens (TAA) p53 and MAGE genes was determined. Aberrant allelic expression of HLA class I antigens was detected in 17 out of 34 (50%) of the specimens stained, whereas HLA class I expression determined by W6/32 staining was found to be heterogeneous in only 2 out of 34 (6%) cases. Decreased expression of ICAM-1 was observed in 12 out of 34 (35%) tumour specimens and de novo expression of HLA-DR (HLA class II) by carcinoma cells in 13 out of 34 (38%) cases. Aberrant expression of HLA class I antigens was frequently observed in cases in which MAGE genes and p53 overexpression were detected. The altered expression of these immunomodulatory molecules in HNSCC may affect prognosis and has important implications for peptide-based immunotherapy strategies for these patients.

Human cytomegalovirus does not induce human leukocyte antigen class II expression on arterial endothelial cells.

Human cytomegalovirus (CMV) has been associated with allograft rejection and, in particular, with transplant-associated arteriosclerosis. However, the role CMV plays in the development of transplant-associated arteriosclerosis remains unclear. CMV can infect the endothelium, the interface between allograft tissue and the host immune cells, but the direct induction of endothelial human leukocyte antigen (HLA) class II by CMV remains controversial. Our previous studies with venous endothelial cells (EC) have shown that CMV does not directly induce this antigen on infected EC and, furthermore, renders these cells refractory to interferon (IFN)-gamma induction. However, questions have arisen regarding the relevance of these findings to arterial endothelia. Thus, we have extended these studies to determine whether similar interactions occur in arterial EC. EC derived from human coronary artery, aorta, and umbilical artery were assayed by immunofluorescence flow cytometry and dual immunohistochemical staining following IFN-gamma treatment and/or inoculation with CMV. Data generated by these experiments demonstrate that regardless of vascular origin: (1) CMV does not directly induce endothelial surface or cytoplasmic HLA class II, and (2) although uninfected arterial EC are HLA class II inducible by IFN-gamma, infected cells are completely refractory to this effect. These results suggest that CMV-mediated inhibition of HLA class II expression is a phenomenon shared by human arterial and venous endothelia of both fetal and adult origin.

Role of major histocompatibility complex class I expression and natural killer-like T cells in the genetic control of endometriosis

To evaluate whether the expression of human leukocyte antigen (HLA) class I on eutopic and ectopic endometrial cells modify the susceptibility to lysis mediated by lymphocytes. Evaluation of T lymphocyte cytotoxic activity and HLA class I expression on endometrial cells. Subjects were recruited at laparoscopy. Patients with endometriosis (n = 7). Healthy women as controls (n = 10). Human leukocyte antigen class I molecule analysis of endometrial cells was carried out by immunofluorescence and flow cytometry. Phenotyping of T lymphocytes was performed to analyze T-cell subsets. Cytotoxicity was performed to determine cytolytic activity against endometrial cells. In vitro culture of endometrial cells down-regulates the expression of HLA class I molecules and enhances the susceptibility to lysis mediated by natural killer (NK)-like T lymphocytes. Cytolytic T-cell clones, expressing the CD94 antigen, are inhibited by the HLA-B7 allele on endometrial cells. Ectopic endometrial cells modulate the expression of HLA class I molecules. The resistance to lysis of endometrial cells is related to expression of surface HLA class I molecules, which send a negative signal for lysis mediated by NK-like T lymphocytes. The HLA-B7 allele inhibits the cytotoxic activity, suggesting that the growth of ectopic endometrial cells might be under a genetic control.