Specific prolongation of MHC class II disparate skin allografts by in vivo administration of anti-IFN-gamma monoclonal antibody

In vivo rejection of MHC class II disparate skin allografts has been thought to involve IFN-gamma-induced expression of MHC class II alloantigens because less than 3% of skin epidermal cells express MHC class II alloantigens constitutively. In our study we directly tested this hypothesis by examining the effect of in vivo administered anti-IFN-gamma mAb on rejection of MHC class II disparate skin allografts, and comparing its effect on rejection of MHC class I disparate skin allografts placed on the same individual mice. We found that anti-IFN-gamma mAb blocked the rejection of MHC class II disparate skin allografts, but had no effect on the rejection of MHC class I disparate skin allografts. These results demonstrate that endogenously produced IFN-gamma is critical for rejection of MHC class II disparate skin allografts, but not for rejection of MHC class I disparate skin allografts. Thus, this study strongly supports the concept that MHC class II rejection responses require IFN-gamma induced MHC class II expression on keratinocytes of the allograft.     

Recognition of MHC class I allodeterminants regulates the generation of MHC class II-specific CTL

We have analyzed the signals influencing the generation of major histocompatibility complex (MHC) class II allospecific cytolytic T lymphocytes (CTL) and have found that the development of these CTL is actively regulated in primary in vitro cultures by Lyt-2+ T cells triggered in response to MHC class I alloantigens. Class II allospecific CTL can be readily stimulated in primary cultures, but the presence of a simultaneous class I MHC stimulus in these cultures causes a marked reduction of class II-specific CTL activation. This reduction can be prevented by adding to culture a dose of monoclonal anti-Lyt-2 antibody (in the absence of complement) that does not block the generation of class I-specific CTL. The role of MHC class I alloantigens in the regulation of class II allospecific responses illustrates that T cells recognizing class I and class II MHC antigens in mixed leukocyte cultures interact in a complex and nonreciprocal manner to influence the final effector T cell repertoire elicited by this complex immunogenic challenge.     

Some cloned murine CD4+ T cells recognize H-2Ld class I MHC determinants directly. Other cloned CD4+ T cells recognize H-2Ld class I MHC determinants in the context of class II MHC molecules.

Murine T lymphocytes recognize nominal Ag presented by class I or class II MHC molecules. Most CD8+ T cells recognize Ag presented in the context of class I molecules, whereas most CD4+ cells recognize Ag associated with class II molecules. However, it has been shown that a proportion of T cells recognizing class I alloantigens express CD4 surface molecules. Furthermore, CD4+ T cells are sufficient for the rejection of H-2Kbm10 and H-2Kbm11 class I disparate skin grafts. It has been suggested that the CD4 component of an anti-class I response can be ascribed to T cells recognizing class I determinants in the context of class II MHC products. To examine the specificity and effector functions of class I-specific HTL, CD4+ T cells were stimulated with APC that differed from them at a class I locus. Specifically, a MLC was prepared involving an allogeneic difference only at the Ld region. CD4+ clones were derived by limiting dilution of bulk MLC cells. Two clones have been studied in detail. The CD4+ clone 46.2 produced IL-2, IL-3, and IFN-gamma when stimulated with anti-CD3 mAb, whereas the CD4+ clone 93.1 secreted IL-4 in addition to IL-2, IL-3, and IFN-gamma. Cloned 46.2 cells recognized H-2Ld directly, whereas recognition of Ld by 93.1 apparently was restricted by class II MHC molecules. Furthermore, cytolysis by both clones 46.2 and 93.1 was inhibited by the anti-CD4 mAb GK1.5. These results demonstrate that CD4+ T cells can respond to a class I difference and that a proportion of CD4+ T cells can recognize class I MHC determinants directly as well as in the context of class II MHC molecules.     

Specificity, phenotype, and precursor frequency of primary cytolytic T lymphocytes specific for class II major histocompatibility antigens

Most cytolytic T lymphocytes (CTL) recognize class I rather than class II MHC determinants, and relatively little is known about those CTL that do recognize class II MHC determinants. The present study was undertaken to document the specificity, phenotype, and precursor frequency of primary class II allospecific CTL. It was found that class II-allospecific CTL could be consistently generated in vitro from unprimed spleen or thymus populations in the presence of exogenously added helper factors. The class II MHC specificity of both the precursor and CTL effectors activated in primary cultures by Ia-disparate stimulator cells was documented both by blocking experiments with anti-Ia mAb and by the use of L cell transfectants. The mechanism by which primary allospecific CTL effectors lysed their targets appeared to involve direct cell-cell contact, because they failed to lyse bystander target cells. The frequency in unprimed spleen populations of precursor CTL specific for class II alloantigens was examined by limiting dilution analysis and was found to be as high as 1/15,000 splenocytes and approximately 10% of the frequency reported for primary class I allospecific CTL. Finally, the Lyt phenotype of primary class II allospecific CTL precursors and effectors was determined. It was found that anti-class II CTL derive from at least two distinct precursor subpopulations that are either L3T4+Lyt-2- or L3T4-Lyt-2+, and that the Lyt phenotype expressed by the CTL effectors are concordant with that of their precursors. No correlation was found between the I subregion gene products recognized by CTL effectors and the Lyt phenotype they expressed in that both I-A- and I-E-specific CTL were both L3T4+Lyt-2- and L3T4-Lyt-2+.     

Syrian hamsters express diverse MHC class I gene products

MHC class I glycoproteins are highly diverse in most species. The Syrian hamster has long been thought to express monomorphic MHC class I molecules and thus be an exception to this rule. Here we show that Syrian hamsters express diverse MHC class I gene products. The nucleotide sequences of the alpha 1 and alpha-2 domains of classical Syrian hamster MHC class I molecules are highly variable and show evidence of having been under selective pressures at their Ag recognition sites. Interestingly, none of the Syrian hamster class I genes was closely related to their counterparts in the mouse. These observations suggest that Syrian hamsters in the wild may express diverse MHC class I molecules.     

A quantitative analysis of lymphokine release from activated T cells. Evidence for a novel form of T-T collaboration in vitro

We previously developed a simple mathematical model describing Ag-triggered lymphokine release from activated T cells. Previous test of this model revealed qualitative differences in the antigenic requirement for lymphokine release between activated T cell populations with the same apparent specificity when activated under different conditions. We now have found a case where class I MHC-reactive T cells (class I T cells) can modulate the nature of Ag-triggered lymphokine release from class II MHC-reactive T cells (class II T cells). Two significant requirements for this modulation event are: 1) Linked recognition/presentation of class I and class II Ag; that is, class I and class II MHC alloantigens must be presented on the same APC, and 2) active participation of the APC in this process; metabolic inactivation of the APC abrogates the class I T cell modulation of the class II activated T cell. These results suggest a novel form of T-T collaboration that involves the active participation of the APC, and provides evidence that T cells of one MHC specificity (class I) can influence the function of T cells of another MHC specificity (class II).     

L3T4+ T-cell-independent reactivity of Lyt2+ T cells in vivo

The aim of this study was to analyze in vivo the L3T4+ T-cell-subset-independent reactivity of Lyt2+ T cells toward transplantation alloantigens. To this end, we depleted normal mice of L3T4+ T cells by injection of monoclonal antibodies to the L3T4 antigen. This procedure not only led phenotypically to a disappearance of L3T4+ T cells, but also effectively abolished reactivity toward class II MHC antigens in vitro and in vivo. However, L3T4+ T-cell-depleted mice still reacted to class I MHC alloantigens in vivo: after immunization with class I MHC alloantigens Il-2 receptor-bearing T cells appeared in the draining lymph nodes, and developed antigen-specific cytolytic activity. Moreover, upon in vivo priming the frequencies of class I MHC-specific precursors of Il-2-producing and cytolytic Lyt2+ T lymphocytes increased up to 20-fold. L3T4+ T-cell-depleted mice rejected class I MHC-bearing skin grafts promptly. We conclude that not only in vitro but also in vivo Lyt2+ T cells remain reactive toward class I MHC antigens in the absence of L3T4+ T helper cells.     

Frequency analysis of class I MHC-reactive Lyt-2+ and class II MHC-reactive L3T4+ IL 2-secreting T lymphocytes

The reactivity of Lyt-2+ or L3T4+ T cells stimulated with either mutant class I or class II MHC alloantigens was studied. Whereas stimulation with class I MHC antigens induced only Lyt-2+ T cells to proliferate and to secrete IL 2, stimulation with class II MHC alloantigens induced L3T4+ but not Lyt-2+ T cells. When the frequencies of precursors of IL 2-secreting T lymphocytes (IL 2TL-p) were determined by limiting dilution analyses, class I MHC-reactive Lyt-2+ T cells displayed frequencies (f = 1/200) as high in magnitude as those within class II MHC-reactive L3T4+ (f = 1/100). Clonally developing IL 2TL of either T cell subset were antigen-specific, as shown in split-culture experiments. Whereas L3T4+ helper TL could be induced to specific IL 2 secretion over a long time period (days 3 to 9), Lyt-2+ TL showed a marked time optimal on day 4; thereafter, the number of TL colonies inducible to secrete IL 2 decreased steadily. IL 2 production and IL 2TL-p frequencies of unseparated T responder cells were not the numerical superposition of the two individual T cell subsets (Lyt-2+ + L3T4+); the latter finding is likely to reflect regulatory influences of Lyt-2+ T cells on IL 2-secreting L3T4+ T cells.     

Regulation of MHC expression in vivo. Bacterial lipopolysaccharide induces class I and II MHC products in mouse tissues by a T cell-independent, cyclosporine-sensitive mechanism

The effect of injections of bacterial LPS on the expression of class I and II products of the MHC in mouse tissues was investigated. MHC products were assessed in tissue homogenates by radiolabeled antibody binding and in tissue sections by indirect immunoperoxidase (IIP) staining. In mice given two i.p. injections of LPS from Escherichia coli or Salmonella minnesota, there were increases in class I and II MHC products in kidney, liver, heart, lung, and pancreas. Focusing on the changes in kidney, we demonstrated that the increase in MHC expression occurred in tubules and, in the case of class I, in glomeruli. LPS treatment also increased the deposition of Ig in glomeruli. Expressed on a standard curve, the total kidney class I and II expression was elevated approximately 10-fold. Time course studies indicated that increased class I expression could be induced by a single LPS injection, whereas class II induction required a second injection. The induction was influenced by the LPS sensitivity of the mice, being much greater in LPS-sensitive C3H/HeSn mice than in LPS-resistant C3H/HeJ mice. LPS induced class I and II Ag in nude mice and in mice with severe combined immunodeficiency, indicating that T cells were not required. Nevertheless, the effect of LPS was inhibitable by cyclosporine and by a mAb against IFN-gamma indicating that IFN-gamma was required for the MHC induction. We conclude that LPS induces an increase in expression and a redistribution of MHC products in kidney and in other tissues by a T cell-independent, cyclosporine-sensitive pathway. These findings are probably related to the known ability of LPS to mediate release of IFN-gamma and other cytokines.     

Hamster T cells participate in MHC alloimmune reactions but do not effect virus-induced cytotoxic activity

The participation of hamster T cells in a variety of putative MHC-determined reactions was studied utilizing a well-characterized, highly selective goat anti-hamster thymocyte (GHT) serum. Hamster lymphoid cell suspensions treated with GHT lose much of their capacity to induce local graft-versushost reactions and to function as responder cells in mixed lymphocyte reactions. In contrast to the participation of hamster T cells in alloimmune reactions (MLR and GVHR), virus-induced, cytotoxic activity in hamsters undergoing acute virus infection is not T-cell-mediated. This latter finding was rather surprising in view of the major role played by cytotoxic T effector cells in comparably infected mice and rats. These results suggest that, although hamsters are able to respond to putative class II MHC disparities in allogeneic reactions, MHC-encoded molecules, presumably class I, are not utilized for induction of effective cytotoxic activity in response to acute virus infection in this species. The implications of these findings are discussed in relation to our present understanding of the hamster MHC.     

MHC class II invariant chain homologues in rainbow trout (Oncorhynchus mykiss)

The MHC class II invariant chain (Ii or CD74) in higher vertebrates is necessary for normal MHC class II loading in endosomal compartments. Detection of an Ii chain in fish would greatly support the idea that MHC class II function in fish and higher vertebrates is similar. Before this study only Ii homologues had been reported in fish that are unlikely to perform true Ii function. In the present study two Ii-like genes, Onmy-Iclp-1 and Onmy-Iclp-2, were detected in rainbow trout. Conservation of elements, particularly in Onmy-Iclp-1, suggests that the encoded proteins may be involved in MHC class II transport and peptide loading as is the Ii protein. The expression pattern of both rainbow trout genes was similar to that of the MHC class II beta chain, with strong expression in the lymphoid tissues, gills and intestine. Analysis of separated peripheral blood leucocyte fractions indicated that expression of Onmy-Iclp-1, Onmy-Iclp-2 and the MHC class II beta chain were all highest in B lymphocytes. This agrees with the expectation that the functions of the products of the new genes are closely associated with MHC class II. It is interesting why in rainbow trout there are two proteins that may function similar to Ii in higher vertebrates.     

CD8 is needed for development of cytotoxic T cells but not helper T cells.

A mutant mouse strain without CD8 (Lyt-2 and Lyt-3) expression on the cell surface has been generated by disrupting the Lyt-2 gene using embryonic stem cell technology. In these mice, CD8+ T lymphocytes are not present in peripheral lymphoid organs, but the CD4+ T lymphocyte population seems to be unaltered. Cytotoxic response of T lymphocytes from these mice against alloantigens and viral antigens is dramatically decreased. Proliferative response against alloantigens and in vivo help to B lymphocytes, however, are not affected. These data suggest that CD8 is necessary for the maturation and positive selection of class I MHC restricted cytotoxic T lymphocytes but is not required on any of the intermediate thymocyte populations (CD8+CD4-TcR- or CD4+CD8+TcRlow) during the development of functional class II MHC restricted helper T cells.     

Bap29/31 influences the intracellular traffic of MHC class I molecules

In this study, we examine the role of the putative cargo receptor B cell-associated protein (Bap)29/31 in the export of MHC class I molecules out of the endoplasmic reticulum (ER). We show that Bap31 binds to two allotypes of mouse class I molecules, with the interaction initiated at the time of H chain association with beta(2)-microglobulin and maintained until the class I molecule has left the ER. We also show that Bap31 is part of the peptide-loading complex, although is not required for its formation. Bap31 binds not only to class I molecules, but can bind to tapasin in the absence of class I. Consistent with an important role in recruiting class I molecules to transport vesicles, we show that in the absence of Bap29/31, there is a loss of class I colocalization with mSec31 (p137), a component of mammalian coat protein complex II coats. This observation is also associated with a delay in class I traffic from ER to Golgi. Our results are consistent with the view that class I molecules are largely recruited to ER exit sites by Bap29/31, and that Bap29/31 is a cargo receptor for MHC class I molecules.     

Alloreactive CD8 T cell tolerance requires recipient B cells, dendritic cells, and MHC class II

Allogeneic bone marrow chimerism induces robust systemic tolerance to donor alloantigens. Achievement of chimerism requires avoidance of marrow rejection by pre-existing CD4 and CD8 T cells, either of which can reject fully MHC-mismatched marrow. Both barriers are overcome with a minimal regimen involving anti-CD154 and low dose (3 Gy) total body irradiation, allowing achievement of mixed chimerism and tolerance in mice. CD4 cells are required to prevent marrow rejection by CD8 cells via a novel pathway, wherein recipient CD4 cells interacting with recipient class II MHC tolerize directly alloreactive CD8 cells. We demonstrate a critical role for recipient MHC class II, B cells, and dendritic cells in a pathway culminating in deletional tolerance of peripheral alloreactive CD8 cells.     

Involvement of ERK, p38 MAP kinase, and PKC in MHC class II-mediated signal transduction in a resting B cell line

Substantial evidence suggests that MHC class II molecules play a critical role in transducing signals during B cell activation and differentiation. In addition, we previously found that cross-linking of MHC class II molecules using anti-MHC class II antibodies inhibited NF-kappaB activation in resting B cells isolated from mouse spleen. In this study, we investigated the mechanism of anti-MHC class II antibody-mediated inhibition of LPS-induced NF-kappaB activation using a resting B cell line, 38B9. We found that treatment with a corresponding anti-MHC class II antibody reduced the activation of NF-kappaB in LPS-stimulated 38B9 cells, treatment of the antibody mediated down-regulation of PKC and ERK/p38 MAP kinase pathways, and treatment with PKC inhibitors caused down-regulation of ERK and p38 MAP kinase activities in LPS-stimulated 38B9 cells. Our results suggest that the PKC and ERK/p38 MAP kinase pathways are regulated by anti-MHC class II antibodies, and that MHC class II molecules are actively involved in the signal transduction pathway in the resting B cell line, 38B9. Consequently, disruption of these pathways might contribute to the inhibition of LPS-induced NF-kappaB activation in 38B9 cells.     

The definition of five B lymphocyte alloantigens closely linked to BoLA class I antigens

B lymphocyte alloantigens in cattle were identified by serological analysis. Alloantisera were raised by skin implant immunization or leucocyte immunization and were absorbed with platelets to reduce class I-specific antibody activity. Leucocyte absorptions were done to reduce the complexity of some antisera. A panning technique was used to prepare B-enriched and B-depleted lymphocytes. Antisera which displayed anti-B cell activity over a number of dilutions were tested against 115 Charolais cattle, and 13 antisera were used to define five B lymphocyte alloantigens. These antigens were present on B lymphocytes but did not appear to be present, at least at the same density, on the majority of T lymphocytes or platelets. Family studies suggested that these antigens are coded by one or two loci which are closely linked to the bovine class I loci. These results suggest the five antigens are class II antigens of the major histocompatibility complex (MHC) of cattle.     

Discrimination between major histocompatibility complex class II DQ and DR locus products in cattle

We have used a panel of anti-major histocompatibility complex (MHC) class II monoclonal antibodies (mAbs) and have assessed their specificity for the products of the individual bovine MHC (BoLA) class II subregions. The mAbs identified two distinct class II molecules by affinity purification and ELISA. Two-dimensional immunoblotting confirmed these data and NH₂-terminal sequencing of the purified class II α chains of one member of each group identified the subregion specificity of the mAbs. The mAbs VPM36, TH22A and TH81A are specific for BoLA DQ, whereas VPM54, TH14B and J11 are specific for BoLA DR. SW73.2 reacts with both MHC subgroups of all cattle tested.     

Prothymosin alpha enhances human and murine MHC class II surface antigen expression and messenger RNA accumulation.

Prothymosin alpha (ProT alpha) is an acidic polypeptide with potentiating effects on HLA-DR-restricted in vitro cellular immune response systems such as T cell proliferative responses to soluble proteins and cellular auto- or alloantigens. Experiments were performed to investigate the effect of ProT alpha on MHC class II Ag expression in human monocytes, murine splenocytes, and tumor cell lines at both protein and molecular levels. RIA and immunofluorescence analysis revealed that ProT alpha enhances HLA-DR surface Ag expression whereas Northern blot analysis demonstrated that ProT alpha causes significant accumulation of MHC class II mRNA. The enhancing effect of ProT alpha was demonstrated convincingly using precultured human peripheral monocytes, which are known to express decreased amounts of surface HLA-DR Ag, and HLA-DR-positive human cell lines. Moreover, ProT alpha was shown to induce HLA-DR Ag expression in a priori HLA-DR-negative tumor cells. Furthermore, ProT alpha was shown to be active in vivo. Splenocytes from mice pretreated with ProT alpha expressed more surface Ilpha Ag and contained more I alpha-specific mRNA. These findings suggest that ProT alpha may be important in the regulation of the immune response by enhancing MHC class II Ag expression in APC.