Differential expression of HLA-DR, -DQ, and -DP antigens on malignant B cells

HLA class II antigens mediate interactions among cells involved in the immune response and play an important role in the process of self recognition. We made use of conventional alloantisera and six well-characterized monoclonal antibodies (MoAb) to study the HLA class II antigens on CALLA-positive malignant B cell populations and autologous normal B cell lines. Forty additional HLA class II-specific MoAb were also tested for their ability to bind to these cells. By using indirect immunofluorescence and immune precipitation assays, we find that malignant B cells often fail to express one or more of the three known types of HLA class II antigens. Cell lines with the following five phenotypes have been identified: HLA-DR+, -DQ+, -DP+; HLA-DR+, -DQ-, -DP+; HLA-DR-, -DQ+, -DP+; HLA-DR-, -DQ-, -DP+; and HLA-DR-, -DQ-, -DP-. These cell lines have been used to characterize the subregion specificity of MoAb that react with HLA class II antigens. This work confirms the existence of complicated patterns of serologic cross-reactivity between the three different types of HLA class II molecules. It also increases our understanding of the specificity of individual MoAb, thereby facilitating future investigation of the distribution and function of individual antigens. Our studies are consistent with the proposal that altered expression of HLA antigens on tumors might impair recognition of these cells by the immune system of the host, thereby contributing to the proliferation of a malignant clone.     

Patterns of constitutive and IFN-γ inducible expression of HLA class II molecules in human melanoma cell lines

Major histocompatibility complex (MHC) class II proteins (HLA-DR, HLA-DP and HLA-DQ) play a fundamental role in the regulation of the immune response. The level of expression of human leukocyte antigen (HLA) class II antigens is regulated by interferon-gamma (IFN-gamma) and depends on the status of class II trans-activator protein (CIITA), a co-activator of the MHC class II gene promoter. In this study, we measured levels of constitutive and IFN-gamma-induced expression of MHC class II molecules, analysed the expression of CIITA and investigated the association between MHC class II transactivator polymorphism and expression of different MHC class II molecules in a large panel of melanoma cell lines obtained from the European Searchable Tumour Cell Line Database. Many cell lines showed no constitutive expression of HLA-DP, HLA-DQ and HLA-DR and no IFN-gamma-induced increase in HLA class II surface expression. However, in some cases, IFN-gamma treatment led to enhanced surface expression of HLA-DP and HLA-DR. HLA-DQ was less frequently expressed under basal conditions and was less frequently induced by IFN-gamma. In these melanoma cell lines, constitutive surface expression of HLA-DR and HLA-DP was higher than that of HLA-DQ. In addition, high constitutive level of cell surface expression of HLA-DR was correlated with lower inducibility of this expression by IFN-gamma. Finally, substitution A-->G in the 5' flanking region of CIITA promoter type III was associated with higher expression of constitutive HLA-DR (p<0.005). This study yielded a panel of melanoma cell lines with different patterns of constitutive and IFN-gamma-induced expression of HLA class II that can be used in future studies of the mechanisms of regulation of HLA class II expression.     

Epstein-Barr virus entry utilizing HLA-DP or HLA-DQ as a coreceptor

Epstein-Barr virus (EBV) glycoprotein gp350/gp220 association with cellular CD21 facilitates virion attachment to B lymphocytes. Membrane fusion requires the additional interaction between virion gp42 and cellular HLA-DR. This binding is thought to catalyze membrane fusion through a further association with the gp85-gp25 (gH-gL) complex. Cell lines expressing CD21 but lacking expression of HLA class II molecules are resistant to infection by a recombinant EBV expressing enhanced green fluorescent protein. Surface expression of HLA-DR, HLA-DP, or HLA-DQ confers susceptibility to EBV infection on resistant cells that express CD21. Therefore, HLA-DP or HLA-DQ can substitute for HLA-DR and serve as a coreceptor in EBV entry.     

HLA-DP, HLA-DQ, and HLA-DR have different requirements for invariant chain and HLA-DM

The MHC is central to the adaptive immune response. The human MHC class II is encoded by three different isotypes, HLA-DR, -DQ, and -DP, each being highly polymorphic. In contrast to HLA-DR, the intracellular assembly and trafficking of HLA-DP molecules have not been studied extensively. However, different HLA-DP variants can be either protective or risk factors for infectious diseases (e.g. hepatitis B), immune dysfunction (e.g. berylliosis), and autoimmunity (e.g. myasthenia gravis). Here, we establish a system to analyze the chaperone requirements for HLA-DP and to compare the assembly and trafficking of HLA-DP, -DQ, and -DR directly. Unlike HLA-DR1, HLA-DQ5 and HLA-DP4 can form SDS-stable dimers supported by invariant chain (Ii) in the absence of HLA-DM. Uniquely, HLA-DP also forms dimers in the presence of HLA-DM alone. In model antigen-presenting cells, SDS-stable HLA-DP complexes are resistant to treatments that prevent formation of SDS-stable HLA-DR complexes. The unexpected properties of HLA-DP molecules may help explain why they bind to a more restricted range of peptides than other human MHC class II proteins and frequently present viral peptides.     

Separate ontogeny of two macrophage-like accessory cell populations in the human fetus

Human macrophage-like accessory cells were analyzed as they emerge in the absence of extrinsic antigens during fetal development. Monoclonal antibodies to monocytes/macrophages were used in combination with antibodies to HLA class II molecules. In the yolk sac and mesenchyme sampled at wk 4 to 6 of fertilization age, cells with dendritic morphology formed two populations distinguishable by phenotypic criteria: type i (majority) carried both macrophage-associated (RFD7+) and monocyte-associated markers (UCHM1+) but no detectable HLA-DR antigen, and type ii (minority) constitutively expressed class II (HLA-DR and -DP) but no RFD7 and UCHM1. The emergence of this heterogeneity preceded the formation of both thymus and bone marrow. During additional development, type i and type ii cells seeded to different microenvironments and underwent some additional phenotypic changes. Cells of type i, the RFD7+ population with high lysosomal (acid phosphatase) activity, were seen in the thymic cortex, marginal zone of lymph nodes, splenic red pulp, and in the midst of erythropoietic activity within the bone marrow. These cells were UCHM1- and class II-. Cells of type ii formed the population of HLA-DR+, RFD7- interdigitating cells, early inhabitants of T cell areas in the developing thymic medulla, lymph nodes, spleen, and tonsil. The Type ii cells that had already settled in their nichès expressed not only HLA-DR and -DP but also HLA-DQ, and another class II antigen identified by the antibody RFD1, which shows the restricted tissue distribution of HLA-DQ, but is governed by genes that are outside of and telomeric to the HLA-DQ region (or HLA-DR). Finally, subpopulations of macrophages (RFD7+, acid phosphatase-positive) in the fetal gastrointestinal and hepatic systems were HLA-DR+; the latter appear to include precursors of Kupffer cells in the developing liver.     

Expression of MHC class II epitopes on human T lymphocyte clones

Twenty-four CD4+ alloreactive helper T cell clones and eight CD8+ cytotoxic T cell clones from five different donors, all of which were dependent on alloantigen and IL 2 for continued growth, were analyzed by FACS for cell surface expression of HLA-DR, -DQ, and -DP epitopes using monoclonal antibodies against monomorphic and polymorphic determinants. Clones were tested early (less than 30 population doublings) and late (greater than 45 population doublings) in their life-spans and at various times (3-5 days) after antigenic restimulation. All clones expressed high levels of HLA-DR at all times, and lower but significant levels of both HLA-DQ and -DP. In contrast, B lymphoblastoid cell lines expressed equivalent amounts of HLA-DR and -DQ, but less HLA-DP. There was no evidence of differential regulation or expression of the three major MHC class II isotypes on different T cell subsets, and neither did antibodies specific for polymorphic epitopes fail to react on clones from donors carrying the appropriate alleles.     

Differential expression of HLA class II antigens in fetal human spleen: relationship of HLA-DP, DQ, and DR to immunoglobulin expression

Frozen sections of human fetal spleen from 12 to 20 wk gestation were examined by using polyclonal antibodies to Ig isotypes, monoclonal antibodies to HLA class II subregion locus products, B and T cells, and follicular dendritic cells. Scattered lymphoid cells in spleen sections from fetuses of 12 to 13 wk gestational age expressed IgM but not IgD. The appearance of lymphoid cells expressing IgD occurred at 14 to 15 wk before the formation of loose clusters of B cells at 16 wk. IgD expression was associated mainly with cells in these clusters, which by 17 wk had become definite follicles. Follicular dendritic cells were not detectable until 20 wk. OKT3-positive T cells were not detected until 17 wk, and at 20 wk constituted 5% of the nucleated cell population. HLA-DR- and DP-positive lymphocytes and macrophages were detectable in fetal spleen from 12 wk onward; DR was expressed on more cells than DP, and the numbers of cells stained by HLA-DR-specific monoclonal antibodies exceeded the number of Ig-positive cells in all spleens examined. HLA-DQ was expressed by consistently fewer cells than HLA-DR and -DP in all spleens tested. The small number of DQ-positive cells in spleens from 12- to 13-wk fetuses had the morphology of macrophages; HLA-DQ expression by lymphoid cells followed a similar pattern to IgD expression and was associated mainly with follicular lymphocytes. It could be demonstrated by double-labeling experiments that all follicular IgM-positive cells in 17- to 20-wk spleens expressed HLA-DP, DQ, and DR antigens: IgM-positive cells in 12- to 16-wk spleens and interfollicular IgM-positive cells in 17- to 20-wk spleens all expressed HLA-DR, but only 59% and 43% expressed DP and DQ, respectively. Ninety-one to 100% of IgD-positive cells in all spleens examined expressed HLA-DQ in addition to DR and DP. In these experiments IgD-negative, DQ-positive cells had the morphologic appearance characteristic of macrophages. These data suggest that class II antigens are differentially expressed on developing lymphoid cells; DR and DP expression occurring in the earliest spleens examined, with expression of DP on a subpopulation of DR-positive cells; IgD and DQ expression appears to be coincident on maturing B cells as they begin to form follicles. An immunoregulatory role for HLA-DQ in B cell development is implicated and remains to be fully investigated.     

One NY-ESO-1-derived epitope that promiscuously binds to multiple HLA-DR and HLA-DP4 molecules and stimulates autologous CD4+ T cells from patients with NY-ESO-1-expressing melanoma

NY-ESO-1 is expressed by a broad range of human tumors and is often recognized by Abs in the sera of cancer patients with NY-ESO-1-expressing tumors. The NY-ESO-1 gene also encodes several MHC class I- and class II-restricted tumor epitopes recognized by T lymphocytes. In this study we report one novel pan-MHC class II-restricted peptide sequence, NY-ESO-1 87-111, that is capable of binding to multiple HLA-DR and HLA-DP4 molecules, including HLA-DRB1*0101, 0401, 0701, and 1101 and HLA-DPB1*0401 and 0402 molecules. We also demonstrate that peptide NY-ESO-1 87-111 stimulates Th1-type and Th-2/Th0-type CD4(+) T cells and clones when presented in the context of these HLA-DR and HLA-DP4 molecules. Both bulk CD4(+) T cells and CD4(+) T cell clones were capable of recognizing not only peptide-pulsed APCs, but also autologous dendritic cells, either loaded with the NY-ESO-1 protein or transfected with NY-ESO-1 cDNAs. Using IFN-gamma and IL-5 ELISPOT assays and PBL from patients with NY-ESO-1-expressing tumors, we observed the existence of Th1-type circulating CD4(+) T cells recognizing peptide NY-ESO-1 87-111 in the context of HLA-DP4 molecules. Taken together, these data represent the first report of an HLA-DR- and HLA-DP-restricted epitope from a tumor Ag. They also support the relevance of cancer vaccine trials with peptides NY-ESO-1 87-111 in the large number of cancer patients with NY-ESO-1-expressing tumors.     

Immunochemical and functional analysis of HLA class II antigens induced by recombinant immune interferon on normal epidermal melanocytes

The effect of recombinant immune interferon (IFN-gamma) on the expression and shedding of HLA antigens and of melanoma-associated antigens (MAA) by epidermal melanocytes was investigated by using serologic and immunochemical techniques. IFN-gamma enhances the expression and/or shedding of HLA class I antigens and of the cytoplasmic MAA defined by monoclonal antibody (MoAb) 465.12S and induces a slight reduction in the expression of the high m.w. melanoma-associated antigen (HMW-MAA). In agreement with the data in the literature, melanocytes incubated with IFN-gamma acquire HLA-DR, -DQ, and -DP antigens. Contrary to previous information in the literature, the effect is not restricted to HLA class II antigens, since IFN-gamma also induces the expression of the 96-kDa MAA recognized by MoAb CL203. The effect of IFN-gamma on HLA class II antigens and 96-kDa MAA is dose and time dependent and is specific, because recombinant leukocyte interferon affects the expression of neither type of antigen. In spite of the expression of HLA class II antigens, IFN-gamma-treated melanocytes do not acquire the ability to stimulate the proliferation of allogeneic lymphocytes. HLA-DR antigens are more susceptible to induction by IFN-gamma than HLA-DQ and -DP antigens, since the percentage of melanocytes acquiring HLA-DQ and -DP antigens is lower than that acquiring HLA-DR antigens. Furthermore, the dose of IFN-gamma is higher and the time of incubation is longer to induce HLA-DQ and -DP antigens than to induce HLA-DR antigens. The differential susceptibility of HLA-DR, -DQ, and -DP antigens as well as of melanocytes from various donors to the modulating effect of IFN-gamma may provide an explanation for the more frequent detection of HLA-DR than of HLA-DQ and -DP antigens in melanoma lesions and for the expression of HLA class II antigens by some, but not all, melanoma lesions.     

IL-4 and granulocyte-macrophage colony-stimulating factor selectively increase HLA-DR and HLA-DP antigens but not HLA-DQ antigens on human monocytes

A number of cytokines were tested for their ability to modulate HLA-DR Ag expression on normal human monocytes. IL-4, granulocyte-macrophage (GM)-CSF as well as IFN-gamma were able to increase HLA-DR Ag expression on monocytes. IFN-alpha was also able to augment HLA-DR Ag expression, but to a lesser degree. Macrophage-CSF, granulocyte-CSF, TNF-alpha, TNF-beta, and IL-6 were not able to augment HLA-DR Ag expression. There were distinct patterns in the ability of different cytokines to augment class II histocompatibility Ag expression. IL-4 and GM-CSF selectively increased only HLA-DR and HLA-DP, but did not increase HLA-DQ antigens on monocytes. IFN-gamma, however, was able to augment the expression of HLA-DR, HLA-DP, and HLA-DQ Ag. Combinations of IFN-gamma with either IL-4 or GM-CSF did not show any synergy for the augmentation of any of the class II antigens on monocytes.     

Defective antigen presentation by human melanoma cell lines cultured from advanced, but not biologically early, disease

The present studies were undertaken to characterize Ag presentation by cultured human melanoma cell lines. Cell lines established from "biologically early" lesions of malignant melanoma were able to present the soluble Ag tetanus toxoid (TT) to autologous and HLA-DR-matched allogeneic, TT-immune T cell clones. Proliferation of T cell clones in response to Ag presented by primary melanoma peaked on day 2 of culture with Ag. Ag presentation was blocked by pretreatment of TT-pulsed and fixed melanoma cells with mAb against HLA-DR, but not HLA-DQ, HLA-DP, or HLA-ABC. Ag processing and presentation were inhibited by treating the melanoma cells with ammonium chloride. In parallel with previous findings from this laboratory demonstrating the inability of cell lines cultured from "advanced" primary or metastatic melanoma to induce autologous T cell proliferation, such cell lines also failed to present this exogenous Ag despite the presence of cell-surface HLA-class II molecules. Thus, in contrast to the finding in biologically early melanoma, none of the multiple TT-immune, T cell clones from autologous patients or HLA-DR matched donors was able to respond to TT presented by melanoma cells cultured from advanced disease. Co-incubation studies revealed that metastatic melanoma cells did not secrete inhibitory substances during the APC assay, however, they were able to process TT, rendering it "immunogenic" in the presence of fixed, autologous non-T cells. When fixed, autologous melanoma cells were assayed for their ability to present processed Ag; fixed cells of early but not advanced disease were able to present Ag in this setting, indicating that the presenting limb becomes flawed in the evolution of the metastatic phenotype. Finally, studies of chloroquine inhibition of the capacity of melanoma cells derived from early primary disease to stimulate autologous peripheral blood T cells suggest that such cells process and present tumor-associated Ag in the same fashion as the "model" Ag TT.     

Analysis of normal human tonsil class II antigen glycosylation by lectin affinity chromatography

Human class II molecules include the HLA-DR, -DQ, and -DP alloantigens. Each class II molecule consists of two glycosylated polypeptide chains, the alpha chain and the beta chain. We have used lectin affinity analysis to investigate the glycosylation pattern of [3H]mannose-labeled glycopeptides derived from isolated alpha and beta chains of HLA-DR and -DQ molecules of normal tonsil cells. Glycopeptides obtained by Pronase digestion of each isolated chain were passed sequentially over columns of immobilized concanavalin A, Lens culinaris lectin, and phytohemagglutinins E and L in a prescribed manner to generate a lectin affinity profile which could be used to assign a minimal oligosaccharide structure for each glycopeptide studied. The data presented here demonstrate that a given class II polypeptide chain can bear several different oligosaccharides. Comparison of the glycosylation patterns of the HLA-DR and -DQ molecules shows that they are similar in most respects. However, there are qualitative differences in the oligosaccharides borne by HLA-DQ and -DR molecules. In addition, comparison between HLA-DQ and the homologous murine I-A molecules shows species-specific glycosylation patterns.     

Human myoblasts as antigen-presenting cells.

Human myoblasts, cultured from muscle and purified to greater than 95%, were investigated for their capacity to act as facultative APC. The myoblasts reacted with antidesmin mAb and had the capacity to fuse into multinucleated myotubes in appropriate medium. The expression of HLA class I, HLA-DR, HLA-DP, HLA-DQ, intercellular adhesion molecule-1 (ICAM-1/CD54), lymphocyte function-associated (LFA) molecules LFA-1 (CD11a/CD18), LFA-2 (CD2), and LFA-3 (CD58) was investigated by FACS analysis before and after induction for various times with human rIFN-gamma, TNF-alpha, or both. Without cytokine induction, myoblasts expressed only HLA-class I and LFA-3. IFN-gamma alone or in combination with TNF-alpha induced the expression of HLA-DR and ICAM-1 reaching a plateau after 48 h, followed by HLA-DP and even later HLA-DQ. TNF-alpha alone induced only ICAM-1. The functional capacity of myoblasts to present Ag to CD4+ T cells was investigated using autologous T cell lines specific for tuberculin, tetanus toxoid, and human myelin basic protein. Noninduced myoblasts or myoblasts treated with TNF-alpha alone could not present any of these Ag to the T cells. However, myoblasts treated with IFN-gamma induced Ag-specific proliferation. In the presence of relevant Ag, myoblasts were killed by the T cells as observed by microscopy and measured by 51Cr release. Ag-specific T cell proliferation and myoblast killing was inhibited in the presence of anti-DR mAb. These results suggest that human myoblasts may act as facultative APC during local immune reactions in muscle.     

T cell recognition of allopeptides in context of syngeneic MHC.

We have analyzed the ability of T cells to recognize peptides corresponding in sequence to an allogeneic HLA-DR molecule, in context of syngeneic MHC. PBMC from a responder with the HLA-DR beta 1*1101/DR beta 1*1201 genotype were stimulated in vitro with a mixture of four synthetic peptides derived from the first domain of the DR beta 1*0101 chain (amino acid residue 1-20, 21-42, 43-62, and 66-90). An alloreactive T cell line, TCL-LS, which proliferates only in response to peptide 21-42 presented by HLA-DR beta 1*1101, was obtained. The blastogenic response of the line was inhibited by anti-HLA-DR and CD4 antibodies but was not affected by antibodies to HLA-DQ, HLA-DP, HLA-ABC, and CD8. In the presence of irradiated, autologous APC, TCL-LS displayed specific proliferative responses to stimulating cells obtained from individuals carrying the DR beta 1*0101 allele. In the absence of autologous APC, TCL-LS recognized HLA-DR1 on allogeneic cells only when expressed together with HLA-DR beta 1*1101, the restrictive element. This indicates that TCL-LS recognizes processed HLA-DR1 molecule presented as nominal Ag. Study of TCR-V beta gene repertoire expressed by TCL-LS showed that only two V beta genes were used (V beta 13.2 and V beta 12). Two T cell clones (TCC) derived from this line, TCC-A5 and B4, exhibited a similar pattern of reactivity and expressed V beta 13.2. These results indicate that T cells recognizing peptides, which are derived from the breakdown of allogeneic MHC class II proteins and are presented by self-HLA-DR molecules, participate in allorecognition.     

Autologous B lymphoblastoid cell lines and long-term cultured T cells as stimulators in the mixed lymphocyte reaction: analysis of the role of HLA class II antigens as stimulatory molecules

Human activated T cells, long-term cultured in the presence of interleukin 2 (IL 2), were compared with autologous Epstein Barr virus-transformed B lymphoblastoid cell lines for expression of human leukocyte (HLA)-HLA-DR and -DQ antigens and for ability to induce proliferative responses in autologous and allogeneic lymphocytes. Immunofluorescence analysis performed with a panel of monoclonal antibodies (mAb) specific for HLA-DR or -DQ antigens did not reveal any significant difference in the expression of HLA-DR antigens but revealed reduced expression of HLA-DQ antigens on two out of four T cell lines tested. No obvious difference could be detected in the two-dimensional gel electrophoretic profile of HLA-DR and -DQ beta-chains synthesized by the autologous pairs of B and T cell lines. In contrast with previous reports, the IL 2-dependent cell lines consistently induced alloproliferative responses in standard 6-day mixed lymphocyte cultures; however, these responses were severalfold lower than those elicited by the autologous B lymphoid lines. Both anti-HLA-DR and anti-HLA-DQ mAb blocked the proliferative responses induced by the B cell lines but did not affect those generated by the T cell lines, suggesting that the latter cells induce T lymphocyte activation via a mechanism independent of HLA-DR or -DQ antigen expression on their surface. Addition of IL 2 to the mixed cultures with B cell lines as stimulators did not affect the outcome of the proliferative responses but partially or completely reversed the blocking activity of the mAb. In contrast, IL 2 significantly enhanced the alloproliferation induced by the T lymphoblastoid cell lines, and the anti-HLA class II mAb partially antagonized this effect. Taken together, these data suggest that unlike the HLA-DR and -DQ gene products on B cells, those on IL 2-dependent long-term cultured T cells do not play a direct or primary stimulatory role in the mixed lymphocyte reaction; the reduced levels of alloproliferation induced by the T cell lines are, at least in part, due to a defective production of endogenous IL 2 by the responder lymphocytes rather than to a defective expression of IL 2 receptors by the alloproliferative T cell subset; and the anti-HLA class II mAb in these cultures act only at the responder cell level, since they can efficiently block the enhancement of T cell proliferation triggered by exogenous IL 2, but not the proliferative responses induced by T cell lines in standard conditions.     

Human endometrial epithelial cell lines for studying steroid and cytokine actions

Summary Recent studies suggest that the proliferation and expression of HLA-DR molecules in endometrial epithelium may be regulated by systemic steroids and local cytokines. To test the interacting influences of cytokines and steroids on the expression of HLA-DR and proliferation of epithelial cells, an endometrial cell model is required that is sensitive to both signals. In this study, we characterize cells of carcinoma cell lines of endometrial lineage for their responsiveness to cytokines and steroids. Independently developed for its response to steroid hormones from a well-differentiated adenocarcinoma of human endometrium, EnCa101AE cell line is further cloned for the expression of progesterone receptor. Immunohistochemical localization using monoclonal antibodies demonstrates that both EnCa101AE cell line and cloned ECC1 cells are purely epithelial, as evidenced by the expression of cytokeratin and epithelial membrane antigen, express estrogen receptors, and concomitantly exhibit IFN-gamma receptor. Experiments using radioiodinated IL-1 reveal that these cell lines also possess high affinity receptors for IL-1. As indicated by the induction of HLA-DR molecules, and alterations in morphologic characteristics, these cell lines are sensitive to both IFN-gamma and IL-1 action. The class II molecules (HLA-DR, HLA-DP, and HLA-DQ) are differentially induced by IFN-gamma treatment in carcinoma cell lines, with HLA-DR being the prevailing induced molecule. IFN-gamma inhibits and estradiol-17β promotes growth of ECC1 cells in a dose-and time-dependent manner. These findings indicate that the interacting effect(s) of the cytokines and steroid hormones on endometrial epithelium may be studied in these unique steroid-and cytokine-sensitive epithelial cell lines.     

A HLA-DQ5 restricted Melan-A/MART-1 epitope presented by melanoma tumor cells to CD4+ T lymphocytes

Melan-A/MART1 is a melanocytic differentiation antigen expressed by tumor cells of the majority of melanoma patients and, as such, is considered as a good target for melanoma immunotherapy. Nonetheless, the number of class I and II restricted Melan-A epitopes identified so far remains limited. Here we describe a new Melan-A/MART-1 epitope recognized in the context of HLA-DQa1*0101 and HLA-DQb1*0501, -DQb1*0502 or -DQb1*0504 molecules by a CD4+ T cell clone. This clone was obtained by in vitro stimulation of PBMC from a healthy donor by the Melan-A51-73 peptide previously reported to contain a HLA-DR4 epitope. The Melan-A51-73 peptide, therefore contains both HLA-DR4 and HLA-DQ5 restricted epitope. We further show that Melan-A51-63 is the minimal peptide optimally recognized by the HLA-DQ5 restricted CD4+ clone. Importantly, this clone specifically recognizes and kills tumor cell lines expressing Melan-A and either HLA-DQb1*0501, -DQb1*0504 or -DQb1*0502 molecules. Moreover, we could detect CD4+ T cells secreting IFN-gamma in response to Melan-A51-63 and Melan-A51-73 peptides among tumor infiltrating and blood lymphocytes from HLA-DQ5+ patients. This suggests that spontaneous CD4+ T cell responses against this HLA-DQ5 epitope occur in vivo. Together these data significantly increase the fraction of melanoma patients susceptible to benefit from a Melan-A class II restricted vaccine approach.     

Staphylococcal enterotoxins direct and trigger CTL killing of autologous HLA-DR+ mononuclear leukocytes and freshly prepared leukemia cells

Attempts have been made to induce cytolytic T cells to kill target cells that do not express the appropriate target molecules by crosslinking the T cells and the target cells in various ways. One successful strategy has been to use heteroconjugates or bispecific monoclonal antibodies reacting with T cell molecules with activating properties (e.g., mab directed to CD3/TCR) and target cell surface antigens. In this report we show that Staphylococcal enterotoxins (SE) direct human T lymphocytes to execute cytotoxicity toward MHC class II-expressing Raji cells, but not against MHC class II-deficient Raji mutant RJ 2.2.5. Both HLA-DR+ and HLA-DR- effector T lymphocytes are effective in the killing of Raji cells coated with SE. The Staphylococcal enterotoxin-dependent cell-mediated cytotoxicity (SDCC) is a rapid T lymphocyte-mediated cytolytic mechanism killing the targets within an hour of incubation. HLA-DR+ target cells are sensitized to be killed within minutes of incubation with picomolar concentrations of SE. SE-sensitized Raji cells remain targets for SDCC after overnight culture at 37 degrees C, demonstrating that the sensitive state is relatively stable. SEA- and SEB-selective cytolytic T cell lines were established to illustrate the clonal variability of SDCC effectors with respect to SE specificity. We also demonstrate that autologous monocytes and activated T lymphocytes as well as B lymphocytes and freshly prepared HLA-DR+ leukemic cells are excellent targets in SDCC.     

NetMHCIIpan-3.0, a common pan-specific MHC class II prediction method including all three human MHC class II isotypes, HLA-DR, HLA-DP and HLA-DQ

Major histocompatibility complex class II (MHCII) molecules play an important role in cell-mediated immunity. They present specific peptides derived from endosomal proteins for recognition by T helper cells. The identification of peptides that bind to MHCII molecules is therefore of great importance for understanding the nature of immune responses and identifying T cell epitopes for the design of new vaccines and immunotherapies. Given the large number of MHC variants, and the costly experimental procedures needed to evaluate individual peptide–MHC interactions, computational predictions have become particularly attractive as first-line methods in epitope discovery. However, only a few so-called pan-specific prediction methods capable of predicting binding to any MHC molecule with known protein sequence are currently available, and all of them are limited to HLA-DR. Here, we present the first pan-specific method capable of predicting peptide binding to any HLA class II molecule with a defined protein sequence. The method employs a strategy common for HLA-DR, HLA-DP and HLA-DQ molecules to define the peptide-binding MHC environment in terms of a pseudo sequence. This strategy allows the inclusion of new molecules even from other species. The method was evaluated in several benchmarks and demonstrates a significant improvement over molecule-specific methods as well as the ability to predict peptide binding of previously uncharacterised MHCII molecules. To the best of our knowledge, the NetMHCIIpan-3.0 method is the first pan-specific predictor covering all HLA class II molecules with known sequences including HLA-DR, HLA-DP, and HLA-DQ. The NetMHCpan-3.0 method is available at http://www.cbs.dtu.dk/services/NetMHCIIpan-3.0.     

Interferon-gamma regulates the T cell response to precursor nevi and biologically early melanoma

To examine the potential regulatory role of interferon-gamma in the cellular immune response to melanoma and its precursor lesions, we have tested the capacity of this lymphokine to enhance HLA class II antigen-dependent T lymphocyte blastogenesis, its in vitro production by autologous T cells stimulated by melanoma, and its presence in melanocytic lesions in situ. Cell lines derived from a dysplastic nevus, a radial growth phase primary tumor, a vertical growth phase primary, and metastatic lesions were induced by recombinant interferon-gamma to express increased amounts of HLA class II antigens. Such cells were then examined in radioimmunoassay for expression of HLA-DR antigens and in co-culture for their ability to stimulate proliferation of autologous T cells. Interferon-gamma treatment of melanocytic cells increased their expression of HLA-DR antigens threefold to sixfold. In parallel with these findings, co-culture of T cells with interferon-treated cells of a dysplastic nevus and a radial phase melanoma led to augmented T cell incorporation of tritiated thymidine, and this stimulation was inhibited with a monoclonal antibody to HLA-DR antigens. Despite augmented expression of HLA class II antigens (HLA-DR, -DQ, and -DP), vertical growth phase and metastatic melanoma cells failed to stimulate autologous T cells. When T cells were co-cultured with stimulating melanoma cells, culture supernatants contained significantly increased amounts of interferon-gamma (12 U/ml) in comparison with supernatants of T cells alone (4 U/ml). No interferon was detectable in cultures of melanoma cells alone. To link these in vitro phenomena to in situ events, we used murine monoclonal antibodies to interferon-gamma, the interleukin 2 receptor, and HLA-DR antigens in an immunoperoxidase system to detect interferon production and lymphocyte activation in frozen sections of lesions representative of melanocytic tumor progression. In these studies, precursor dysplastic nevi and radial phase melanomas contained the highest numbers of activated lymphocytes and stained positively for interferon-gamma. These results suggest that interferon-gamma plays a central role in the regulation of the cellular immune response to melanoma. It is produced by T cells, likely activated by tumor antigens seen in the context of HLA class II antigens. In turn, interferon-gamma production enhances expression of HLA class II antigens by melanoma and precursor cells, and such enhancement is associated with additional T cell activation in a positive feed-back loop.