Induction of mouse syngeneic MLR by in vivo xenogeneic immunization with HLA-DR antigens

To study in mice the effects of in vivo xenogeneic immunization with human major histocompatibility complex (MHC) class II antigens, the animals were injected with HLA-DR antigens and their proliferative responses tested in vitro. The results showed that small amounts of HLA-DR proteins, acting as nominal antigens, were not only able to prime mice for a secondary in vitro xenogeneic mixed lymphocyte reaction but also induced a syngeneic mixed lymphocyte reaction. In contrast, allogeneic or syngeneic immunization of mice with soluble MHC class II molecules failed to stimulate an autoreactive response. The syngeneic mixed lymphocyte reaction was primarily directed against syngeneic MHC class II molecules since the murine T lymphocytes reacted against MHC class II-positive dendritic spleen cells and MHC class II-transfected mouse fibroblasts. A self-reactive T-cell line induced under these experimental conditions did not react in xenogeneic and allogeneic mixed lymphocyte reactions. However, these T lymphocytes proliferated when human peripheral blood lymphocytes of various haplotypes were presented in the context of syngeneic mouse antigen presenting cells.     

Analysis of the immune response induced by a single xenoantigen in vivo

Transgenic mice expressing human major histocompatibility complex (MHC) class II molecules would provide a valuable model system for studying murine anti-human MHC immune response. We have previously shown that skin from HLA-DR1 transgenic mice was rejected by control littermates and spleen cells from rejecting mice were able to proliferate to donor cells. The aim of this paper is to analyze the mechanism of recognition of this xenoantigen and the possible involvement of antibody response in anti-HLA-DR1 immune response. Control littermates were immunized with spleen cells from HLA-DR1 transgenic (TG) mice; at indicated times, xenoantigen-specific proliferation and IFNgamma production was assessed using APC obtained from HLA-DR1 TG mice. Mixed direct-indirect pathway of xenoantigen recognition was suggested by the following findings: i)T cell response to HLA-DR1 was inhibited adding in culture monoclonal antibodies directed either to donor (HLA-DR) or to recipient MHC (I-A); ii) APC from control mice pulsed with purified DR1 molecules were able to induce proliferation by FVB/N mice immunized with transgenic spleen cells. HLA-DR1 recognition permits DR peptide-specific T cell response by lymphocytes of control littermates immunized with the xenoantigen. In addition, we detected xenoreactive IgM and IgG2 antibodies. Our data suggest that HLA-DR1 xenoantigen may be recognized through direct or indirect pathway and provide additional information on mouse anti-human HLA immune response.     

High precursor frequency of human T cells reactive to HLA-DQ molecules expressed on mouse L cell transfectants.

In humans, the HLA-DR molecule is a major stimulatory molecule of allogeneic mixed lymphocyte reactions (MLR) and a major restriction molecule for the presentation of soluble antigens to the T cell. Little is known of the biological function of HLA-DQ. To examine the size of the repertoire of precursor T cells recognizing the autologous or allogeneic HLA-DQ molecule, the frequency of T cells reactive to HLA-DQ was estimated in comparison with T cells reactive to HLA-DR. We made use of a limiting dilution analysis and mouse L cells transfectants expressing the HLA-DR or -DQ molecule, as stimulators. Human T cells recovered from a primary MLR stimulated with allogeneic peripheral blood lymphocytes (PBL) proliferated in response to L cell transfectants expressing HLA class II genes shared by the stimulator cells in the primary MLR. This observation suggested that the HLA class II molecules on L cell transfectants shared to some extent epitopes for alloreactive T cells with those expressed on human PBL. The precursor frequencies of CD4+ T cells reactive to allogeneic or autologous DQ molecules were as high as those of T cells reactive to allogeneic DR molecules and were estimated to be 1/800-1/1800. The frequency of the T cells reactive to autologous DR molecules was low (1/7200-1/16,000). The biological significance of the high frequency of HLA-DQ-reactive precursor T cells is discussed.     

HLA class II transgenic mice as models of human diseases

Summary: Predisposition co develop Various autoimmune disorders has been associated with certain HLA class II molecules but there is a lack of information on che pathophysiological rule of HLA genes in conferring susceptibility Various experimental animal models of autoimmune disease have been studied to address the role of immune response genes. To study the interactions involved between class II molecules (DQ and DR) and define the immunologic mechanisms in various diseases, we generated HLA-DR and DQ transgenic mice that lacked endogenous class II molecules. The HLA molecules in these mice arc expressed on the cell surface and can positively select CD4+ T cells expressing Various Vβ T-cell receptors (TCR). A peripheral tolerance is maintained co transgenic HLA molecules thus indicating that these molecules act as self, Mouse co stimulatory and accessory molecules can interact with the HLA-peptide-TCR complex leading to efficient T-cell activation. In this review, we describe immunogenetic models for human diseases using these transgenic mice. Our studies show that HLA class II transgene-restricted T cells recognize the immunodominant antigens and peptide epitopes, similar to HLA class II-restricted human T cells. Thus these mice provide powerful tools to understand the role of HLA class II molecules in predisposition and onset of human diseases and to develop immunotherapy and vaccines.     

Functional difference between HLA-DR and HLA-DQ molecules in mixed lymphocyte reaction

In order to analyze the functional differences between HLA-DR and HLA-DQ molecules, we have established transfectants expressing HLA class II molecules. We investigated the contribution of these molecules in mixed lymphocyte reaction (MLR) using these transfectants. 1) The genomic clones encoding for DR alpha, DR beta, DQ alpha, and DQ beta from HLA-Dw 15 haplotype were isolated. These genes were introduced into murine L cells and two kinds of stable transfectants expressing either of HLA-DR4 and HLA-DQw4 were established. Expression of HLA class II molecules on transfectants was confirmed by FACS analysis using monoclonal antibodies specific for HLA class II molecules. 2) Primary MLR against class II transfectants and blocking experiments showed that DR molecules function as dominant stimulator molecules in allo MLR, whereas DQ molecules as well as DR molecules stimulate equally auto MLR. 3) We also determined the clone size of MLR reactive CD4+ T cells by the limiting dilution analysis. Frequencies of allo DR, auto DQ, and allo DQ reactive CD4+ T cells was estimated to be almost equal, but frequency of auto DR reactive CD4+ T cells was estimated to be far low. These results suggest the relatively high occurrence of auto DQ reactive clones which contribute significantly to auto MLR. These auto DQ reactive clones may not be eliminated as efficiently as DR reactive clones, because of lower expression of DQ molecules than DR molecules on bone marrow derived cells.     

Cytotoxic T lymphocyte recognition of HLA-A2 antigens in normal and HLA-Cw3-transgenic mice

It is well established that a large fraction of murine cytotoxic T cells can recognize allogeneic major histocompatibility complex (MHC) antigens, and that the majority of this response are not restricted by H-2 antigens of the responding host. In contrast, the murine response against the xenogeneic HLA class I antigens is relatively weak. Moreover, a large proportion of the responding murine T cells do not recognize the HLA antigen per se but only in an H-2-restricted manner, probably as an HLA peptide bound to H-2. Considerable evidence suggests that in mice the T cell repertoire is selected by thymic H-2 antigens. Therefore, we asked the question whether in transgenic mice expressing an HLA class I antigen the T cell repertoire would be shaped toward a more effective recognition of other HLA alleles. Normal C57BL/6 (B6) and HLA-Cw3-transgenic B6 mice were immunized with a B6-derived cell line transfected with HLA-A2. The resulting A2-specific CTL were tested on L cells transfected with either A2 alone, which should identify the H-2-unrestricted CTL, and on L cells transfected with A2 plus H-2b genes, which should identify the sum of H-2b-restricted and unrestricted CTL. Both bulk culture and limiting dilution experiments showed that the CTL precursor frequencies for A2-specific CTL were not increased in the transgenic mice, and that both strains produced comparable proportions of H-2b-restricted and of unrestricted A2-specific CTL. The B6.Cw3 mice seemed to respond less well to HLA-A2 than the normal B6 mice, suggesting the possibility of tolerance for peptides shared by the Cw3 and A2 molecules. In conclusion, the T cells in the B6.Cw3 transgenic mice did not seem to be selected towards a stronger and more unrestricted recognition of an allo-HLA antigen. The possible reasons are discussed.     

Interspecies cross-reactivity of Class II antigen of MHC determined by syngeneic, allogeneic and xenogeneic B and T cells

Although this chapter ought to summarize the role of MHC antigens in T cell activation, the immunobiological meaning of the polymorphism of Class II antigens, as well as that of Class I antigens, is still unresolved. The antigen-presenting ability of human APC is dominant over that of murine APC in the stimulation of antigen-specific xenogeneic T cells. In addition, xenoreactive murine T cells specific for human PBL failed to recognize the polymorphic determinant of Class II antigens of human MHC. On the basis of the data, Class II antigens may be seen to have some role as antigen-presenting molecules rather than as restricting molecules, at least, in the xenogeneic APC-T cell interaction or the xenogeneic MLR responses. These data together with the fact that the linkage disequilibrium found among the various groups of alleles encoding Class I and II antigens making up an MHC haplotypes suggest that the MHC may play a key role during evolution. These studies using xenogeneic cell interaction may shed some light on the immunobiological function of polymorphism of MHC antigens in the mechanisms of T cell activation, and the evolutional history of the polymorphism of the NHC in self or not-self recognition by T cells.     

Complementation between specific HLA-DR and HLA-DQ genes in transgenic mice determines susceptibility to experimental autoimmune encephalomyelitis

To investigate the contribution of human leukocyte antigen (HLA) class II molecules in susceptibility to inflammatory demyelination, we induced experimental autoimmune encephalomyelitis (EAE) in transgenic (tg) mice expressing the HLA-DR3, HLA-DQ8 and HLA-DQ6 molecules in the absence of endogenous class II (Ab(o)). Following immunization with mouse myelin, HLA-DR3 tg mice mounted strong T-cell proliferative responses, and developed inflammatory lesions and demyelination in the central nervous system with mild to moderate clinical symptoms of EAE. HLA-DQ8 and HLA-DQ6 tg mice elicited weak T-cell proliferative responses and did not develop clinical symptoms of EAE. HLA-DR3/DQ6 double tg mice immunized with mouse myelin experienced clinical disease similar to the single tg HLA-DR3 tg mice, indicating that expression of DQ6 in this line had no effect on disease. In contrast, HLA-DR3/DQ8 double tg mice developed severe inflammatory lesions and clinical disease in response to immunization with mouse myelin. Our data suggest that in the presence of two susceptible class II alleles, namely HLA-DR3 and DQ8, there is additional selection and expansion of potential autoreactive T cells, resulting in enhanced severity of disease.     

Presentation of soluble antigen to human T cells by products of multiple HLA-linked loci: analysis of antigen presentation by a panel of cloned, autologous, HLA-mutant Epstein-Barr virus-transformed lymphoblastoid cell lines

Epstein-Barr virus-transformed human B lymphoblastoid cell lines (EBV-LCL) can present soluble antigens to antigen-primed T lymphocytes. In this study, we used HLA antigen-loss mutants of an EBV-LCL line (LCL 721) to demonstrate that the presentation of a soluble antigen from Candida albicans (CAN) by EBV-LCL to primed T cells can be restricted by multiple HLA determinants. Haplotype-deletion mutants that contained only the maternal or only the paternal HLA-haplotype were used to demonstrate the preferential role of autologous HLA antigens in presenting soluble antigens to Candida-primed T cells from the donor of LCL-721, and to T cells from her mother and father. Immunoselected mutants of LCL-721 showing a variety of distinct phenotypes that are deficient in HLA-DR, DQ, or DP antigen expression were tested as antigen-presenting cells. The antigen-presenting ability of these class II deficient EBV-LCL variants weakened with progressive loss of class II HLA determinants expressed on the cell surface. Our study, therefore, provides evidence for multiple HLA restriction determinants, including HLA-DR, DQ, and DP. Furthermore, LCL lacking all HLA-DR, DQ, and DP expression because of homozygous deletion of these MHC class II genes still presented CAN and Tetanus toxid (TET), although to a much lesser degree than presented by LCL-721. This suggests that determinants other than DR, DQ, and DP which are expressed on these EBV-LCL may also function as restriction elements for the proliferative T-cell response to soluble antigens.     

The interaction defect of accessory molecules is responsible for the poor ex vivo response to human antigens of mouse T helper cells

Mouse T cells fail to respond to xenogeneic pig and human antigens using the direct antigen‐presenting pathway. The poor response by mouse CD8 cells is because of multiple defects in the molecular interactions between mouse CD8 cells and xenogeneic antigen‐presenting cells (APCs). Using human CD4/DR3⁺, mouse CD4^?/major histocompatibility complex (MHC) class II ^? mice, we investigated the defects in molecular interaction responsible for the poor response to xenogeneic antigens by naïve mouse CD4⁺ cells. Mouse CD4 cells failed to respond to human leucocyte antigen (HLA)‐DR3 expressed on mouse APCs but developed a strong response to alloantigens, indicating a defect in the interaction between mouse CD4 and HLA‐DR3 molecules. Human CD4⁺/mouse CD4^– mouse T cells respond poorly to human and pig APCs but not allogeneic APCs, indicating that accessory molecular interactions are deficient across highly separated species. Adding mouse interleukin‐2 (IL‐2) to the mixed lymphocyte reaction system did not improve the poor response to human or pig antigens by mouse or human CD4⁺/mouse CD4^– mouse T cells. Therefore, multiple molecular interactions deficient between mouse CD4 cells and human or pig APCs may lead to the poor response to xenogeneic antigens by naïve mouse CD4 cells.     

HLA and TLX antigen expression on the human oocyte, zona pellucida and granulosa cells

The expression of human leukocyte antigen (HLA) class I and class II molecules and one trophoblast-lymphocyte crossreactive (TLX) antigen by human oocytes and granulosa cells was investigated. Well-defined monoclonal antibodies directed against HLA class I and class II molecules as well as beta 2-microglobulin and a TLX molecule were used in a standard indirect immunofluorescence test and some immunogold techniques at the electron microscopic level. Single unfertilizable or multiply fertilized oocytes and granulosa cells obtained from an in-vitro fertilization programme as well as oocytes in primary, secondary and tertiary follicles of ovaries were studied. Neither HLA class I or class II molecules, nor beta 2-microglobulin, nor a TLX molecule were detected on cultured oocytes or oocytes in human ovarian follicles or in their zona pellucida. Granulosa cells taken from a culture medium and those in follicles at various stages of development expressed class I antigens, while granulosa cells from tertiary follicles also expressed HLA-DR antigens. These results confirm that the female human gamete belongs to that very small group of cells that lack major histocompatibility complex antigens. Since spermatozoa also lack HLA antigens, human germ cells are entirely different from other nucleated human cells with regard to the antigenic structures expressed on the cell surface. This would prevent recognition of these cells by the cellular immune system. Furthermore, HLA and TLX antigens are not involved in fertilization and early differentiation.     

Comparison of HLA-DR1-restricted T cell response induced in HLA-DR1 transgenic mice deficient for murine MHC class II and HLA-DR1 transgenic mice expressing endogenous murine MHC class II molecules

Transgenic mice expressing human HLA class II molecules provide a useful model for identifying HLA-restricted CD4+ epitopes. However, the influence of endogenous murine H-2-restricted T cell responses on HLA-restricted responses is not known. In the present study, we show that HLA-DR1 transgenic mice deficient for H-2 class II expression (HLA-DR1+/+/IAbeta0/0) exhibit an equivalent expression level of the transgene HLA-DR1 and a similar diversity in the TCR repertoire, but a slightly different number of CD4+ peripheral T cells, when compared to HLA-DR1 transgenic mice in which H-2 class II molecules were retained (HLA-DR1+/+/IAbeta+/+). More importantly, a strong antigen-specific HLA-DR1-restricted response was observed in nearly all HLA-DR1+/+/IAbeta0/0 mice immunized with HBV envelope protein (HBs) or capsid protein (HBc), whereas weak HBs- or HBc-specific HLA-DR1-restricted responses were detected in half of the immunized HLA-DR1+/+/IAbeta+/+ mice. Conversely, strong HBs- or HBc-specific H-2-restricted T cell responses were detected in HLA-DR1+/+/IAbeta+/+ mice but not in HLA-DR1+/+/IAbeta0/0 mice. Our results indicate that the coexpression of endogenous H-2 class II molecules reduces the intensity of HLA-DR1-restricted antigen-specific responses in transgenic mice, by favoring murine over human MHC recognition and education. Thus, HLA-DR1+/+/IAbeta0/0 mice represent a better model for identifying and characterizing HLA-DR1-restricted epitopes relevant for human disease.     

HLA-peptide complex and immunity-related diseases]

The class II HLA molecule (HLA-DR, -DQ or -DP) is expressed on various antigen presenting cells (APC). Proteolytic fragments of peptides processed by APC that match the physiochemical character of the peptide-binding grooves formed by alpha and beta chains of class II HLA molecules are expressed on the surface of APC and are recognized by T cells. Many class II-binding peptide motifs reported to date follow the 9-mer peptide pattern AxxBxCDxE, with A, B, C, D, and E residues functioning to bind to HLA, thereby designated as "anchors". On the contrary, the x residues are recognized by T cells through the T cell receptor (TCR). Analog peptides with single residue substitutions at x residues changed signals in T cells, leading to quantitative and qualitative (anergy/survival, etc.) changes in human T cell clonal responses. Certain analogs changed signals in monocytes, leading to the upregulation of IL-12. Furthermore, cross-linking of class II HLA molecules on monocytes using mAbs leads to upregulation of monokines, in which HLA-DR, -DQ, and -DP play differential roles. Thus, HLA molecules when recognized by TCR, not only present peptide antigens to T cells but also transmit signals to APC, where the polymorphism and heterogeneity of HLA molecules may play important roles.     

Role of HLA class II genes in susceptibility/resistance to inflammatory arthritis: studies with humanized mice

Summary: Predisposition to develop rheumatoid arthritis (RA) has been associated with certain human leukocyte antigen (HLA) class II molecules, although the mechanism is still unknown. Various experimental animal models of inflammatory arthritis have been studied to address the role of major histocompatibility complex (MHC) genes in pathogenesis. We have generated transgenic mice expressing HLA class II molecules (DR and DQ) lacking complete endogenous class II molecules to study the interactions involved between class II molecules (DQ and DR) and to define the immunologic mechanisms in inflammatory arthritis. The HLA transgene can positively select CD4⁺ T cells expressing various Vβ T-cell receptors, and a peripheral tolerance is maintained to transgenic HLA molecules. The expression of HLA molecules on various cells in these mice is similar to that known in humans. In this review, we describe collagen-induced arthritis as a model for human inflammatory arthritis using these transgenic mice. The transgenic mice carrying RA-susceptible haplotype develop gender-biased inflammatory arthritis with clinical and histopathological similarities to RA. Our studies show that polymorphism of HLA class II genes determine the predisposition to rheumatoid/inflammatory arthritis and the epistatic interactions between HLA-DQ and HLA-DR molecules dictate the severity, progression, and modulation of the disease.     

To B or not to B: role of B cells in pathogenesis of arthritis in HLA transgenic mice

Population studies have shown that amongst all the genetic factors linked with autoimmune disease development, MHC class II genes are the most significant. Experimental autoimmune arthritis resembling human rheumatoid arthritis (RA) can be induced in susceptible strains of mice following immunization with type II collagen (CIA). We generated transgenic mice lacking endogenous class II molecules and expressing various HLA genes including RA-associated, HLA-DRB1*0401 and HLA-DQ8, and RA-resistant, DRB1*0402, genes. The HLA molecules in these mice are expressed on the cell surface and can positively select CD4+ T cells expressing various Vβ T cell receptors. Endogenous class II invariant chain is required for proper functioning of the class II transgene. Arthritis development in transgenic mice is CD4+ and B cells dependent. Studies in humanized mice showed that B cells are required as antigen presenting cells in addition to antibody producing cells for the development of CIA. The transgenic mice expressing *0401 and *0401/DQ8 genes developed sex-biased arthritis with predominantly females being affected, similar to that of human RA. Further, the transgenic mice produced autoantibodies like rheumatoid factor and anti-cyclic antibodies. Antigen presentation by B cells leads to a sex-specific immune response in DRB1*0401 mice suggesting a role of B cells and HLA-DR in rendering susceptibility to develop arthritis in females.     

Major histocompatibility complex status in breast carcinogenesis and relationship to apoptosis

Major histocompatibility complex (MHC) molecules are of central importance in regulating the immune response against tumors. In this study we used immunohistochemistry to study human leukocyte antigen (HLA) class I and II antigen expression in normal breast tissues and benign, preneoplastic, primary, and metastatic breast lesions using antibodies against beta-2-microglobulin (beta2-m), heavy-chain, and HLA-DR antigens. Whereas all normal tissues and benign lesions were positive for beta2-m and HLA-A, -B, and -C antigens, total loss of HLA class I antigens was found in 37% (11 of 30) of in situ carcinomas, in 43% (56 of 131) of the primary tumors, and in 70% (31 of 45) of the lymph node metastases. HLA-DR was also underexpressed in breast cancer cells; thus 20% (6 of 30) of in situ carcinomas, 15% of invasive carcinomas (20 of 131), and only 1 metastatic case were positive for this antigen. Both HLA class I and II antigen expression were more frequently down-regulated in metastatic lesions than in primary breast lesions (P <0.05), and a tendency toward a simultaneous defective expression of HLA class I and II antigens was observed in primary carcinomas (P = 0.07). However, no correlation was found between the expression of any of the aforementioned molecules and pathological parameters or survival. Interestingly, HLA class I expression was expressed more frequently in tissues with high apoptotic activity and was significantly associated with the expression of the proapoptotic bax gene (P = 0.02), and was inversely associated with expression of the antiapoptotic bcl-2 gene (P = 0.03). We conclude that alterations in HLA class I and II antigen expression are early events in breast carcinogenesis and play significant roles in metastatic progression. In addition, their expression is correlated with apoptosis-regulating proteins, which may influence the cytotoxicity of T cells against HLA class I-specific tumor antigens.     

HLA-class II restriction of the proliferative T lymphocyte responses to nickel, cobalt and chromium compounds

Peripheral blood mononuclear cells from nickel and/or cobalt or chromium sensitive patients were stimulated with the appropriate metal compound for 7 days in culture. The transformed blasts were separated from non-transformed small cells on Percoll density gradients. The blasts, maintained in interleukin-2 containing medium and enriched for T cells, were both antigen specific and devoid of alloreactivity. The metal compound primed T cell blasts from all patients showed a good proliferative response on restimulation with the priming compound in the presence of autologous adherent cells (AC). Analysis of HLA class II phenotypes of allogeneic AC that could serve as antigen presenting cells indicated that responsiveness of T cell blasts from most but not all patients could be explained on the basis of HLA-DR as a restricting element. We conclude that products of other class II loci (HLA-DQ and/or HLA-DP) might also serve as restricting elements for a secondary in vitro proliferative response to these metal compounds.     

Restriction molecules involved in the interaction of human T cell clones with antigen-presenting cells

Two of three distinct human Ia molecules detected by murine monoclonal antibodies (MoAb) have been suggested to be involved in antigen presentation for T cell responses to purified protein derivatives (PPD) and herpes simplex virus (HSV). This observation was first suggested from studies on the inhibition of proliferative responses of whole T cell populations with MoAb against human Ia molecules. To determine whether a single T cell recognizes the antigen in the context of both Ia molecules or in the context of each one of two Ia molecules, we isolated and propagated PPD-reactive T cell clones from an HLA-DR heterozygous individual. They showed four different restriction patterns: type I and type II clones each appeared to be restricted to one of two HLA-DR antigens, type III clone gave anomalous patterns of response and seemed to be restricted to non-DR antigens, and type IV clone recognized antigen when both DR antigens were presented on the same antigen-presenting cells (APC) surface. Blocking study with monoclonal anti-Ia antibodies suggested that type I, II and IV clones are restricted to DR molecules and type III clones are restricted to 1B4 molecules distinct from DR or MB1 molecules. These data imply that human T cell clones recognizing PPD in the context of each one of two Ia molecules are clonally distributed.     

Human alloreactive CTL interactions with gliomas and with those having upregulated HLA expression from exogenous IFN-gamma or IFN-gamma gene modification.

By flow cytometry, a panel of 18 primary glioma cell explants exhibited high expression of class I HLA-A, B, C, but class II HLA-DR expression was absent. Freshly isolated normal brain cells displayed little or no HLA antigens. Alloreactive cytotoxic T lymphocytes (aCTL), sensitized to the HLA of the patient, were generated in a one-way mixed lymphocyte response (MLR). The specificity of aCTL was confirmed to be to target cells (patient glioma cells or lymphoblasts) expressing the relevant HLA antigens. However, nontumor patient-specific aCTL did not lyse normal brain cells. Titration of antibodies to HLA class I into cytotoxicity assays blocked lysis of gliomas by aCTL, confirming aCTL T cell receptor (TCR) interactions with the class I antigen on gliomas. Furthermore, aCTL interactions with glioma cells caused their apoptosis. Coincubations of aCTL with gliomas resulted in upregulated cytokine secretion. Importantly, dexamethasone, an immunosuppressive steroid used for brain edema, did not affect aCTL lytic function against tumor, indicating that steroid-dependent patients may benefit from the immunotherapy. We also explored the use of interferon-gamma (IFN-gamma) to increase aCTL tumor recognition. Coincubation of gliomas with exogenous IFN-gamma (500 U/ml, 48 h) caused a 3-fold upregulation of HLA class I and a slight induction of class II antigen expression. Gene-modified glioma cells producing IFN-gamma similarly displayed upregulated HLA expression. Glioma cells incubated with exogenous IFN-gamma or IFN-gamma-transduced glioma cells were more susceptible to lysis by aCTL than their parental counterparts, thus supporting the concept of combining IFN-gamma cytokine gene therapy with adoptive aCTL immunotherapy for brain tumor treatment.     

Activation of CD8+ T cells by allogeneic class II-deficient B-cell lines derived from patients with bare lymphocyte syndrome

The major histocompatibility complex (MHC) class II antigens (Ags) are known to carry the major stimulating determinants of the primary mixed lymphocyte reactions (MLR). We investigated the mechanism of generating HLA class I-directed alloreactive T-cells in primary MLR. With the use of class II-deficient EBV-transformed B-lymphoblastoid cell lines (B-LCLs) derived from patients with bare lymphocyte syndrome (BLS), we have demonstrated in the present study that class I disparity alone can trigger primary MLR in the absence of exogenous IL-2. The CD8+ T cells were primary MLR-responsive cells, and the CD4+ T cells seem to play no role in primary MLR when class II alloantigens are not involved in stimulation. Addition of autologous macrophages did not influence the primary MLR response. The primary MLR was completely blocked by anti-class I or anti-CD8 antibodies but not by anti-class II or anti-CD4 antibodies. The MLC-generated CD8+ T cells exhibited cytolytic activity as well as proliferative responses. The proliferative response of the CD8+ T cells was specifically directed against class I antigens, demonstrated by proliferative assays; and the helper-independent CD8+ T cells were generated only when the activation of CD4+ T cells did not occur. This observation suggests that functional recruitment of T-cell receptor (TCR) repertoire is under active regulation, and the suppression of CD8+ T-cell helper recruitment appears to be dictated by the CD4+ T-cell subset. Further analysis of the primed T-cell specificities showed that alloreactivity of the CD8+ T cells was mostly accounted for by the HLA-B Ags.(ABSTRACT TRUNCATED AT 250 WORDS)