MHC Ag Research Articles

Transcriptional profiling of gamma delta T cells.

Evolutionarily well-conserved, γδ T cells may have been precursors to modern B and αβ T cells ([1][1]). γδ T cells are distinct from αβ T cells, in part, by the genes that encode their TCRs, their ability to recognize Ag in the absence of classical MHC Ag presentation, and their role in

Regulation of the class II MHC pathway in primary human monocytes by granulocyte-macrophage colony-stimulating factor.

GM-CSF stimulates the growth and differentiation of hematopoietic progenitors and also affects mature cell function. These effects have led to the use of GM-CSF as a vaccine adjuvant with promising results; however, the mechanisms underlying GM-CSF-mediated immune potentiation are incompletely understood. In this study, we investigated the hypothesis that the immune stimulatory role of GM-CSF is in part due to effects on class II MHC Ag presentation. We find that, in primary human monocytes treated for 24-48 h, GM-CSF increases surface class II MHC expression and decreases the relative level of the invariant chain-derived peptide, CLIP, bound to surface class II molecules. GM-CSF also increases expression of the costimulatory molecules CD86 and CD40, but not the differentiation marker CD1a or CD16. Furthermore, GM-CSF-treated monocytes are better stimulators in a mixed leukocyte reaction. Additional analyses of the class II pathway revealed that GM-CSF increases total protein and RNA levels of HLA-DR, DM, and DOalpha. Expression of class II transactivator (CIITA) types I and III, but not IV, transcripts increases in response to GM-CSF. Furthermore, GM-CSF increases the amount of CIITA associated with the DR promoter. Thus, our data argue that the proinflammatory role of GM-CSF is mediated in part through increased expression of key molecules involved in the class II MHC pathway via induction of CIITA.

Tapasin-/- and TAP1-/- macrophages are deficient in vacuolar alternate class I MHC (MHC-I) processing due to decreased MHC-I stability at phagolysosomal pH.

Alternate class I MHC (MHC-I) Ag processing via cytosolic or vacuolar pathways leads to cross-presentation of exogenous Ag to CD8 T cells. Vacuolar alternate MHC-I processing involves phagolysosomal Ag proteolysis and peptide binding to MHC-I in post-Golgi compartments. We report the first study of alternate MHC-I Ag processing in tapasin(-/-) cells and experiments with tapasin(-/-) and TAP1(-/-) macrophages that characterize alternate MHC-I processing. Tapasin promotes retention of MHC-I in the endoplasmic reticulum (ER) for loading with high affinity peptides, whereas tapasin(-/-) cells allow poorly loaded MHC-I molecules to exit the ER. Hypothetically, we considered that a large proportion of post-Golgi MHC-I on tapasin(-/-) cells might be peptide-receptive, enhancing alternate MHC-I processing. In contrast, alternate MHC-I processing was diminished in both tapasin(-/-) and TAP1(-/-) macrophages. Nonetheless, these cells efficiently presented exogenous peptide, suggesting a loss of MHC-I stability or function specific to vacuolar processing compartments. Tapasin(-/-) and TAP1(-/-) macrophages had decreased MHC-I stability and increased susceptibility of MHC-I to inactivation by acidic conditions (correlating with vacuolar pH). Incubation of tapasin(-/-) or TAP1(-/-) cells at 26 degrees C decreased susceptibility of MHC-I to acid pH and reversed the deficiency in alternate MHC-I processing. Thus, tapasin and TAP are required for MHC-I to bind ER-derived stabilizing peptides to achieve the stability needed for alternate MHC-I processing via peptide exchange in acidic vacuolar processing compartments. Acidic pH destabilizes MHC-I, but also promotes peptide exchange, thereby enhancing alternate MHC-I Ag processing. These results are consistent with alternate MHC-I Ag processing mechanisms that involve binding of peptides to MHC-I within acidic vacuolar compartments.

IFN-gamma reverses the stop signal allowing migration of antigen-specific T cells into inflammatory sites.

In humans the majority of endothelial cells (EC) constitutively express MHC class II Ags. We know that in vitro ECs can activate CD45RO(+) B7-independent CD4(+) T cells to proliferate and produce IL-2. The in vivo correlate of this T cell response is not known, and here we have explored whether endothelial expression of MHC class II Ags affects the transendothelial migration of alloreactive CD4(+) CD45RO(+) B7-independent T cells. Alloreactive CD4(+) T cell clones and lines were generated against HLA-DR11, DR13, DR4, and DR1 MHC Ags, and their rates of migration across untreated EC line Eahy.926 (MHC class II negative) or Eahy.926 transfected with CIITA (EahyCIITA) to express DR11 and DR13 were investigated. The migrations of EahyCIITA-specific T cell clones and lines were retarded in a DR-specific manner, and retardation was reversed in the presence of mAb to DR Ag. When investigating the ability of T cells to proliferate in response to EahyCIITA before and after transmigration, migrated cells were still able to proliferate, but the frequency of EahyCIITA-specific cells was much reduced compared with that of nonmigrated cells. The use of fluorescently labeled T cells revealed that specific cells become trapped within the endothelial monolayer. Pretreatment of EahyCIITA with IFN-gamma restored the ability of DR11- or DR13-specific T cells to transmigrate and proliferate, thus abrogating DR-specific retardation. We conclude that cognate interaction between T cells and endothelial MHC class II initiates a stop signal possibly similar to an immunological synapse, but this is overcome in an inflammatory milieu.

Expression of antigen on mature lymphocytes is required to induce T cell tolerance by gene therapy.

Expression of a retrovirally encoded allogeneic MHC class I gene in bone marrow-derived cells can be used to induce tolerance to the product of the retrovirally transduced gene. In this work we examined whether expression of a retrovirally transduced allogeneic MHC class I gene in bone marrow-derived cells from recombinase-activating gene-1 (RAG-1)-deficient mice was sufficient to induce tolerance when transplanted into conditioned hosts together with bone marrow from MHC-matched wild-type mice. Reconstitution of mice with either MHC-matched RAG-1-deficient or wild-type bone marrow transduced with the allogeneic MHC class I gene H-2K(b) led to long-term expression of K(b) on the surface of bone marrow-derived hematopoietic lineages. T cells from mice reconstituted with H-2K(b)-transduced wild-type bone marrow were tolerant to K(b). In contrast, expression of K(b) in the periphery of mice reconstituted with a mixture of retrovirally transduced RAG-1-deficient bone marrow and mock-transduced wild-type bone marrow fell below detectable levels by 4 wk after transplantation. T cells that developed in these mice appeared to be hyporesponsive to K(b), demonstrating that expression of K(b) on bone marrow-derived APCs was not sufficient to induce tolerance. Our data suggest that induction of tolerance in molecular chimeras requires expression of the retrovirally transduced allogeneic MHC Ag on the surface of mature lymphocytes that populate the host thymus.

Processing of exogenous antigens for presentation by class I MHC molecules involves post-Golgi peptide exchange influenced by peptide-MHC complex stability and acidic pH.

Vacuolar alternate class I MHC (MHC-I) Ag processing allows presentation of exogenous Ag by MHC-I molecules with binding of antigenic peptides to post-Golgi MHC-I molecules. We investigated the role of previously bound peptides and their dissociation in generating peptide-receptive MHC-I molecules. TAP1-knockout macrophages were incubated overnight with an initial exogenous peptide, producing a large cohort of peptide-K(b) complexes that could influence subsequent peptide dissociation/exchange. Initial incubation with FAPGNYPAL, KVVRFDKL, or RGYVYQGL enhanced rather than reduced subsequent binding and presentation of a readout peptide (SIINFEKL or FAPGNYPAL) to T cells. Thus, K(b) molecules may be stabilized by an initial (stabilizing) peptide, enhancing their ability to bind readout peptide and implicating peptide dissociation/exchange. In contrast, incubation with SIINFEKL as stabilizing peptide reduced presentation of readout peptide. SIINFEKL-K(b) complexes were more stable than other peptide-K(b) complexes, which may limit their contribution to peptide exchange. Stabilizing peptides (FAPGNYPAL, KVVRFDKL, or RGYVYQGL) enhanced alternate MHC-I processing of HB101.Crl-OVA (Escherichia coli expressing an OVA fusion protein), indicating that alternate MHC-I Ag processing involves peptide dissociation/exchange. Stabilizing peptide enhanced processing of HB101.Crl-OVA more than presentation of exogenous OVA peptide (SIINFEKL), suggesting that peptide dissociation/exchange may be enhanced in the acidic phagosomal processing environment. Furthermore, exposure of cells to acidic pH increased subsequent binding and presentation of readout peptide. Thus, peptide dissociation/exchange contributes to alternate MHC-I Ag processing and may be influenced by both stability of peptide-MHC-I complexes and pH.

CD4+ T cells engrafted with a recombinant immunoreceptor efficiently lyse target cells in a MHC antigen- and Fas-independent fashion.

T cells engrafted by a recombinant immunoreceptor with predefined Ag specificity can efficiently lyse Ag-positive target cells in a MHC Ag-independent manner. It is yet unresolved how receptor-grafted CD4+ T cells contribute to MHC Ag-independent target cell lysis. To address this issue, we grafted isolated CD8+ and CD4+ T cells from the peripheral blood with recombinant anti-carcinoembryonic Ag and anti-CD30 receptors, respectively. Cytotoxicity analyses revealed that grafted CD4+ T cells exert cytolysis of Ag-positive target cells with an efficiency similar to that of grafted CD8+ T cells. Lysis by receptor-grafted CD4+ T cells is Ag specific and is inhibited by blocking the target Ag or the Ag binding site of the recombinant receptor. Both Fas-sensitive and Fas-resistant target cells are lysed with equal efficiency, and lysis of Fas-sensitive target cells is not blocked by an anti-Fas ligand Ab, indicating that cytolysis by receptor-grafted CD4+ T cells is independent of the Fas pathway. We conclude that cytolysis by CD4+ T cells equipped with a recombinant immunoreceptor is MHC Ag and Fas independent and likely to be mediated by perforin present in receptor-grafted CD4+ T cells.

Fas-mediated apoptosis causes elimination of virus-specific cytotoxic T cells in the virus-infected liver.

Immunity to allogeneic MHC Ags is weak in rodent livers, raising questions as to the mechanisms that might control responses in this organ. Infection with an adenovirus vector reveals that T cell-mediated immunity to nonself-Ags in the liver is self-limiting. Virus-induced liver injury decreases and coincides with disappearance of virus-specific CTL, concomitant to an increase of apoptotic T cells early after infection. But whereas death in CD4 cells is independent of Fas, perforin, and TNF-alpha, that of CD8 cells requires Fas and not perforin or TNF-alpha pathways. Fas ligand is expressed on liver-infiltrating cells, pointing to death by fratricide that causes almost complete disappearance of virus-specific CTL 4 wk after infection. CTL elimination is virus dose dependent, and high doses induced high alanine aminotransferase values, elevated expression of Fas ligand on CD8 cells, and increased CD8 cell migration into the infected liver.

Structural and Functional Consequences of Altering a Peptide MHC Anchor Residue

To better understand TCR discrimination of multiple ligands, we have analyzed the crystal structures of two Hb peptide/I-E(k) complexes that differ by only a single amino acid substitution at the P6 anchor position within the peptide (E73D). Detailed comparison of multiple independently determined structures at 1.9 A resolution reveals that removal of a single buried methylene group can alter a critical portion of the TCR recognition surface. Significant variance was observed in the peptide P5-P8 main chain as well as a rotamer difference at LeuP8, approximately 10 A distal from the substitution. No significant variations were observed in the conformation of the two MHC class II molecules. The ligand alteration results in two peptide/MHC complexes that generate bulk T cell responses that are distinct and essentially nonoverlapping. For the Hb-specific T cell 3.L2, substitution reduces the potency of the ligand 1000-fold. Soluble 3.L2 TCR binds the two peptide/MHC complexes with similar affinity, although with faster kinetics. These results highlight the role of subtle variations in MHC Ag presentation on T cell activation and signaling.

Donor MHC class II antigen is essential for induction of transplantation tolerance by bone marrow cells.

Posttransplant infusion of donor bone marrow cells (BMC) induces tolerance to allografts in adult mice, dogs, nonhuman primates, and probably humans. Here we used a mouse skin allograft model and an allogeneic radiation chimera model to examine the role of MHC Ags in tolerance induction. Infusion of MHC class II Ag-deficient (CIID) BMC failed to prolong C57BL/6 (B6) skin grafts in ALS- and rapamycin-treated B10.A mice, whereas wild-type B6 or MHC class I Ag-deficient BMC induced prolongation. Removal of class II Ag-bearing cells from donor BMC markedly reduced the tolerogenic effect compared with untreated BMC, although graft survival was significantly longer in mice given depleted BMC than that in control mice given no BMC. Infusion of CIID BMC into irradiated syngeneic B6 or allogeneic B10.A mice produced normal lymphoid cell reconstitution including CD4+ T cells except for the absence of class II Ag-positive cells. However, irradiated B10.A mice reconstituted with CIID BMC rejected all B6 and a majority of CIID skin grafts despite continued maintenance of high degree chimerism. B10.A mice reconstituted with B6 BMC maintained chimerism and accepted both B6 and CIID skin grafts. Thus, expression of MHC class II Ag on BMC is essential for allograft tolerance induction and peripheral chimerism with cells deficient in class II Ag does not guarantee allograft acceptance.

Cloning and Characterization of the Bovine MHC Class I-Like Fc Receptor

In the cow, maternal immunity is exclusively mediated by colostral Igs, but the receptor responsible for the IgG transport has not yet been identified. The role of an IgG-Fc receptor (FcRn) that resembles a class I MHC Ag in transporting IgGs through epithelial cells was recently shown in selected species. We now report the cloning and characterization of the bovine FcRn (bFcRn). The cDNA and deduced amino acid sequences show high similarity to the FcRn in other species, and it consists of three extracellular domains, a hydrophobic transmembrane region, and a cytoplasmic tail. Despite the high similarity of the extracellular domains with other species, the bovine cytoplasmic tail is the shortest thus far analyzed. Aligning the known FcRn sequences, we noted that the bovine protein shows a 3-aa deletion compared to the rat and mouse sequences in the alpha1 loop. Furthermore, we found a shorter transcript of the bFcRn reflecting an exon 6-deleted mRNA, which results from an inadequate splice acceptor site in intron 5 and produces a transmembrane-deficient molecule, as was previously demonstrated in the related MHC class I gene family in mouse and humans. The presence of bFcRn transcripts in multiple tissues, including the mammary gland, suggests their involvement both in IgG catabolism and transcytosis.

Ly49I NK cell receptor transgene inhibition of rejection of H2b mouse bone marrow transplants.

The Ly49 family of genes encode NK cell receptors that bind class I MHC Ags and transmit negative signals if the cytoplasmic domains have immunoregulatory tyrosine-based inhibitory motifs (ITIMs). 5E6 mAbs recognize Ly49C and Ly49I receptors and depletion of 5E6+ NK cells prevents rejection of allogeneic or parental-strain H2d bone marrow cell (BMC) grafts. To determine the function of the Ly49I gene in the rejection of BMC grafts, we transfected fertilized eggs of FVB mice with a vector containing DNA for B6 strain Ly49I (Ly49IB6). Ly49IB6 is ITIM+ and is recognized by 5E6 as well as Ly49I-specific 8H7 mAbs. Normal FVB H2q mice reject H2b but not H2d BMC allografts, and the rejection of H2b BMC was inhibited partially by anti-NK1.1 and completely by anti-asialo GM1, but not by anti-CD8, Abs. In FVB mice, NK1.1 is expressed on only 60% NK cells. FVB. Ly49IB6 hosts failed to reject H2d or H2b BMC, but did reject class I-deficient TAP-1-/- BMC, indicating that NK cells were functional. Nondepleting doses of anti-Ly49I Abs reversed the acceptance of H2b BMC by FVB.Ly49IB6 mice. FVB.Ly49IB6+/- mice were crossed and back-crossed with 129 mice-H2b, 5E6-, poor responders to H2d BMC grafts. While transgene-negative H2b/q F1 or first-generation back-crossed mice rejected H2b marrow grafts (hybrid resistance), transgene-positive mice did not. Thus B6 strain Ly49I receptors transmit inhibitory signals from H2b MHC class I molecules. Moreover, Ly49IB6 has no positive influence on the rejection of H2d allografts.

Deficiency of Transporter for Antigen Presentation (TAP) in Tumor Cells Allows Evasion of Immune Surveillance and Increases Tumorigenesis

Proteins involved in class I MHC (MHC-I) Ag processing, such as the TAP, are deficient in some human tumor cells. This suggests that antitumor responses by CD8 T cells provide selection pressure to favor outgrowth of cells with defective processing of tumor Ags. Nonetheless, this evidence is only correlative, and controlled in vivo experiments have been lacking to demonstrate that TAP deficiency promotes survival of tumor cells. To explore the role of Ag processing defects in tumor progression, matched panels of TAP1-positive and TAP1-negative tumor cell lines were generated from a parental transformed murine fibroblast line. Inoculation of C57BL/6 mice with TAP1-negative cells produced large and persistent tumors. In contrast, TAP1-positive cells did not generate lasting tumors, although small tumors were detected transiently and regressed spontaneously. Both TAP1-positive and TAP1-negative cells produced tumors in athymic mice, confirming that TAP-dependent differences in tumorigenicity were due to T cell-dependent immune responses. Inoculation of C57BL/6 mice with mixtures of TAP1-positive and TAP1-negative cells produced tumors composed exclusively of TAP1-negative cells, indicating in vivo selection for cells with TAP deficiency. Thus, loss of TAP function allows some tumor cells to avoid T cell-dependent elimination, resulting in selection for tumor cells with deficient Ag processing.

Pretransplant Frequency of Donor-Specific, IFN-γ-Producing Lymphocytes Is a Manifestation of Immunologic Memory and Correlates with the Risk of Posttransplant Rejection Episodes

Abstract While matching for MHC Ags improves renal allograft survival, closely matched grafts sometimes fail due to rejection, and poorly matched allografts are often well tolerated by the recipient. The severity of the rejection process may partially depend on the presence of environmentally primed T cells in the recipient that cross-react with donor Ags. To test for the presence of primed, donor-specific T cells in humans before transplantation, we used an enzyme-linked immunospot assay for detection of allospecific cytokines produced by individual human PBLs. We demonstrate that this approach detects cytokine production at single cell resolution and detects production of IFN-γ only when there is defined immunologic priming, thus representing a measure of primed donor-specific immunity. Because the environmental Ag exposure of the recipient is not a function of the HLA mismatch between donor and potential recipient, the number of HLA mismatches may not correlate with the frequency of pretransplant, donor-specific IFN-γ-producing PBLs. Studies of donor-specific IFN-γ-producing lymphocytes in a cohort of patients being evaluated for renal transplantation corroborated this hypothesis. Moreover, for recipients of both living and cadaver renal allografts, the pretransplant frequency of donor-specific memory cells correlated with the posttransplant risk of developing acute rejection episodes. This improved ability to define the strength of the allospecific immune response by enzyme-linked immunospot assay may allow improved pairing of recipients with donors and identification of kidney allograft donor-recipient pairs at high risk for acute rejection, thus permitting targeted interventions aimed at prolonging graft survival.

Modulation of Allospecific CTL Responses During Pregnancy in Equids: An Immunological Barrier to Interspecies Matings?

Maternal immune recognition of the developing conceptus in equine pregnancy is characterized by the strongest and most consistent alloantibody response described in any species, a response directed almost exclusively against paternal MHC class I Ags. This work investigated the cellular immune response to paternal MHC Ags in pregnant and nonpregnant horses and donkeys, and in horses carrying interspecies hybrid mule conceptuses. We observed profound decreases in classical, MHC-restricted, CTL activity to allogeneic paternal cells in peripheral blood lymphocytes from both horse mares and donkey jennets carrying intraspecies pregnancies, compared with cells from nonpregnant controls. This is the first evidence in a randomly bred species for a generalized systemic shift of immune reactivity away from cellular and toward humoral immunity during pregnancy. Surprisingly, mares carrying interspecies hybrid mule conceptuses did not exhibit this transient, pregnancy-associated decrease in CTL activity. The failure of interspecies pregnancy to down-regulate cellular immune responses may be a heretofore-unrecognized, subtle barrier to reproductive success between species.

Modulation of Monocytic Cell Activity and Virus Susceptibility During Differentiation into Macrophages

A major component of innate immune responses relies on monocytes and macrophages, virus infection of which will pose a particular problem for immunological defense. Consequently, the monocytic cell differentiation pathway was analyzed in terms of cellular modulations therein and their relation to monocytotropic virus infection. Differentiation was characterized by down-regulation of CD14, MHC Ags, the monocytic SWC1 marker, and p53; concomitant up-regulation of the SWC9 macrophage marker, a putative porcine CD80 (detected with anti-human CD80 Ab), and acid phosphatase secretion were also characteristic. Elevated phagocytic and endocytic activities as well as endosomal/lysosomal acidification were identified as being important to the macrophage. In contrast, monocytes possessed high accessory activity. This was multifactorial, concomitantly requiring 1) high MHC Ag expression; 2) enzyme activity of esterase, peroxidase, myeloperoxidase, and 5' nucleotidase in preference to glucosidase, galactosidase, and glucuronidase; and 3) elevated capacity for spontaneous IL-1 production. Only with all parameters was efficient stimulation of Ag-specific lymphocytes possible. These results point to a continuous process during differentiation, involving inter-related characteristics linking the more accessory monocyte to the scavenger macrophage, both in vitro and in vivo. Of particular interest was how these characteristics related to monocytotropic virus infection, and how a particular virus could show a clear preference for the differentiating macrophages. Such results not only further our understanding of porcine immunology, but also provide evidence and a potential model for the determination and characterization of monocytotropic virus-host cell interactions.

Cyclosporine A prevents antigen presentation by porcine endothelial cells by inhibiting TNFα-mediated class II MHC antigens expression

530 Cyclosporine A (CsA) has been so far required in the immunosuppression regimen of recipient of vascularized xenograft beyond the phase of hypercute rejection. Effects of CsA on non lymphoid immune cells such as endothelial cells (EC) are not well established and sometimes contradictory since both protective and adverse effects have been reported. In the present study, we have investigated whether CsA could contribute to alter the antigenicity of porcine aortic endothelial cells (PAEC) by reducing class I and class II MHC antigen expression. The ability of CsA to prevent MHC antigens expression during human TNFα- or lymphocytes-mediated EC activation has been evaluated by Facs and semiquantitative RT-PCR and correlated to the ability of EC to promote human lymphocyte proliferation. First, Facs analysis showed that overexpression of class II MHC antigens on PAEC, mediated by human TNFα, was strongly inhibited in the presence of CsA. The expression level of class I MHC antigens was reduced by CsA to less than 50 % of the value for EC treated with TNFα alone whereas the presence of CsA completely prevented the induction of class II MHC molecules. CsA-mediated inhibition was dose-dependent within drug concentrations ranging between 2.5 and 20 μg/ml and was consistently observed at all time points (24h to 72h) of the activation period. The presence of CsA during activation of EC with TNFα dramatically reduce the ability of EC to further promote human PBL proliferation. Indeed, when PAEC were pretreated with both TNFα and CsA, lymphocyte proliferation was below that induced by resting EC for all time points (proliferation index: 0.6, 0.2 and 0.23 for day 3, 5 and 7, respectively). Similar level of inhibition was achieved by using an anti-porcine class II MHC blocking mAb. We also observed that expression of E-selectin, as well as class I and class II porcine MHC Ag, was induced following coculture of porcine EC with human PBL. Pretreatment of porcine EC with CsA for 4h, before coculture experiments, efficiently prevented the induction of E-selectin and class II MHC antigens and inhibits overexpression of class I antigens. Semiquantitative RT-PCR experiments showed that the presence of CsA markedly reduced the steady-state level of porcine class II (SLADRA and SLADQA) mRNA at 16h as compared with PAEC stimulated with TNFα alone. This reduced level of mRNA for class II MHC was associated with the lack of class II transcriptional coactivator, CIITA, expresssion at this time point. The ability of CsA to promote the rapid decay of CIITA mRNA is analysed. Therefore, our data indicate that CsA could contribute to prevent direct presentation of porcine MHC antigens by xenogeneic EC to human T lymphocytes.

Normal Development in Porcine Thymus Grafts and Specific Tolerance of Human T Cells to Porcine Donor MHC

Abstract The induction of T cell tolerance is likely to play an essential role in successful xenotransplantation in humans. In this study, we show that porcine thymus grafts in immunodeficient mice support normal development of polyclonal, functional human T cells. These T cells were specifically tolerant to MHC Ags of the porcine thymus donor and responded to nondonor porcine xenoantigens and alloantigens. Exogenous IL-2 did not abolish tolerance, suggesting central clonal deletion rather than anergy as the likely tolerance mechanism. Our study suggests that the thymic transplantation approach to achieving tolerance with restoration of immunocompetence may be applicable to xenotransplantation of pig tissues to humans.

IL-1β Inhibits IFN-γ-Induced Class II MHC Expression by Suppressing Transcription of the Class II Transactivator Gene

AbstractClass II MHC Ags are critical for the initiation of immune responses by presenting Ag to T lymphocytes, leading to their activation and differentiation. The transcriptional activation of class II MHC genes requires the induction of the class II transactivator (CIITA) protein, a master regulator that is essential for both constitutive and IFN-γ-inducible class II MHC expression. The cytokine IL-1β has been shown to inhibit IFN-γ-induced class II MHC expression in various cell types. We investigated the underlying mechanism of this inhibitory effect of IL-1β using human astroglioma cell lines. Our findings demonstrate that IL-1β prevents the expression of class II MHC mRNA and protein upon treatment with IFN-γ. Furthermore, we demonstrate that IFN-γ induction of CIITA mRNA expression is inhibited by treatment of cells with IL-1β. IL-1β suppressed IFN-γ activation of the type IV CIITA promoter in astroglioma cells, indicating that the inhibitory influence of IL-1β is mediated by inhibition of CIITA transcription. IL-1β did not interfere with IFN-γ receptor signal transduction, since tyrosine phosphorylation, nuclear translocation, and DNA binding of STAT-1α to an IFN-γ activation sequence of the type IV CIITA promoter were not affected by IL-1β. As well, IL-1β treatment did not affect the ability of IFN-γ-induced interferon-regulatory factor-1 (IRF-1) to bind the IRF-1 element within the type IV CIITA promoter. This study suggests that IL-1β may play a role in regulating immunoreactivity by inhibiting transcription of the CIITA gene, thereby reducing subsequent class II MHC expression.

HLA (A*0201) Mimicry by Anti-Idiotypic Monoclonal Antibodies

Soluble MHC Ags and anti-Id (anti-anti-MHC) Abs have both been shown to inhibit MHC alloantigen-specific B cell responses in vivo. We hypothesized that some anti-idiotypic Abs function as divalent molecular mimics of soluble HLA alloantigen. To test this idea, we studied two well-defined anti-idiotypic mAbs, T10-505 and T10-938, elicited in syngeneic BALB/c mice by immunization with CRll-351, an HLA-A2,24,28-specific mAb. Each anti-Id induced "Ab-3" Abs in rabbits that cross-reacted with HLA-A2 but not with HLA-B Ags. Furthermore, each anti-Id could bind to and block Ag recognition by Ha5C2.A2, a human homologue of mAb CRll-351. Both anti-Id mAb displayed weak reactivity with the human mAb SN66E3, which recognized an overlapping but distinct determinant of HLA-A2 Ags; neither reacted with human mAb MBW1, which recognized a nonoverlapping HLA-A2 determinant. Amino acid sequence comparison of mAb CRII-351 heavy and light chain variable region complementarity-determining regions (CDRs) with those of mAb Ha5C2.A2 and SN66E3 revealed short regions of homology with both human mAb; a large insert in the light chain CDR1 of mAb SN66E3 distinguished it from both CRll-351 and Ha5C2.A2. The amino acid sequences of mAb T10-505 and T10-938, which differed markedly from each other, revealed no homology to the alpha2 domain sequence of HLA-A*0201 that contains the CRll-351 mAb-defined epitope. We conclude that structurally different anti-Id Abs can mimic a polymorphic conformational epitope of an HLA Ag. In the case of T10-505 and T10-938 mimicry was not based on exact replication of the epitope by the hypervariable loops of the anti-Id mAb.