Cutting edge: the minor histocompatibility antigen H60 peptide interacts with both H-2Kb and NKG2D

Minor histocompatibility Ags elicit cell-mediated immune responses and graft rejection in individuals receiving MHC-matched tissues. H60 represents a dominant Ag that elicits a strong CTL response in C57BL/6 mice immunized against BALB.B. An 8-aa peptide in the H60 protein is presented by H-2K(b) and this is recognized by the TCR as an alloantigen. The intact H60 glycoprotein is a ligand for the costimulatory NKG2D receptor that is expressed by activated CD8(+) T cells. Thus, H60 may provide both an allogeneic peptide and its own costimulation. We show that mutation of an H-2K(b)-binding anchor residue in the H60 peptide completely abrogates binding of H60 glycoprotein to NKG2D and a synthetic H60 peptide partially blocks the binding of NKG2D to its ligand. Ligands of the human NKG2D receptor are remarkably polymorphic, suggesting that these may also serve as minor histocompatibility Ags.     

Two preferentially expressed proteins protect vascular endothelial cells from an attack by peptide-specific CTL

Vascular endothelial cells (EC) are an exposed tissue with intimate contact with circulating Ag-specific CTL. Experimental in vitro and clinical data suggested that endothelial cells present a different repertoire of MHC class I-restricted peptides compared with syngeneic leukocytes or epithelial cells. This endothelial-specific peptide repertoire might protect EC from CTL-mediated cell death. The HLA-A*02-restricted peptide profile of human EC and syngeneic B lymphoblastoid cells was biochemically analyzed and compared. For EC selective peptides, source protein expression, peptide binding affinity, and peptide-HLA-A*02 turnover were measured. The significance of abundant peptide presentation for target cell recognition by immunodominant CTL was tested by small interfering RNA treatment of EC to knock down the source proteins. High amounts of two peptides, PTRF(56-64) and CD59(106-114), were consistently detected in EC. This predominance of two endothelial peptides was explained by cell type-specific source protein expression that compensated for poor HLA-A*02 binding affinity and short half-live of peptide/HLA-A*02 complexes. Knocking down the source proteins containing the abundant endothelial peptide motifs led to a nearly 100-fold increase of surface expression of SMCY(311-319), an immunodominant minor histocompatibility Ag, as detected by cytotoxicity assays using SMCY(311-319)-specific CTL. We conclude that EC express and present preferentially two distinct HLA-A*02-restricted peptides at extraordinary high levels. These abundant self-peptides may protect EC from CTL-mediated lysis by competing for HLA-A*02 binding sites with immunodominant scarcely expressed antigenic peptides.     

Immune recognition by cytotoxic T lymphocytes of minor histocompatibility antigens expressed on a murine colon carcinoma line

We have characterized in vivo and in vitro responses of mice to the BALB/c-derived carcinoma, C26. BALB/c mice were highly susceptible, in a dose-dependent fashion, to local tumor development following subcutaneous injection of C26. Other strains of mice, including allogeneic strains and major histocompatibility complex compatible strains of different minor histocompatibility (H) backgrounds, were resistant to C26-induced tumors. The basis for resistance of mice to C26 was studied using an in vitro-derived C26 line as target cells in microcytotoxicity assays, and as a source of antigen for in vivo priming. An H-2d-specific alloreactive cytotoxic T lymphocyte (CTL) line was isolated from C57BL/6 mice primed with C26, demonstrating the expression, and immune recognition, of MHC class I antigens on C26. C26 also expressed minor H antigens of BALB background as demonstrated by the ability of CTL specific for BALB minor H antigens to selectively lyse C26. Conversely, minor H antigens on C26 were immunogenic across a minor H barrier as demonstrated by the ability to raise anti-minor H CTL to C26 from minor H disparate strains. Collectively, those experiments indicate that C26 may be useful for immunologic and biochemical studies of murine minor H antigens, and for in vivo and in vitro studies of local immunity.     

Biochemical and immunogenetic analysis of an immunodominant peptide (B6dom1) encoded by the classical H7 minor histocompatibility locus

Of the many minor histocompatibility (H) Ags that have been detected in mice, the ability to induce graft vs host disease (GVHD) after bone marrow transplantation is restricted to a limited number of immunodominant Ags. One such murine Ag, B6dom1, is presented by the H2-Db MHC class I molecule. We present biochemical evidence that the natural B6dom1 peptide is indistinguishable from AAPDNRETF, and we show that this peptide can be isolated from a wide array of tissues, with highest levels from the lymphoid organs and lung. Moreover, we employ a novel, somatic cell selection technique involving CTL-mediated immunoselection coupled with classical genetics, to show that B6dom1 is encoded by the H7 minor H locus originally discovered approximately 40 years ago. These studies provide a molecular genetic framework for understanding B6dom1, and exemplify the fact that mouse minor H loci that encode immunodominant CTL epitopes can correspond to classical H loci originally identified by their ability to confer strong resistance to tumor transplantation. Additionally, these studies demonstrate the utility of somatic cell selection approaches toward resolving H Ag immunogenetics.     

A contraceptive peptide vaccine targeting sulfated glycoprotein ZP2 of the mouse zona pellucida

In this study, we have mapped and characterized a B cell epitope of sulfated glycoprotein ZP2 (ZP2) as a step toward the development of a multi-epitope zona pellucida (ZP) vaccine. Recombinant polypeptides expressed by random deoxyribonuclease-digested fragments of ZP2 cDNA were screened for binding to IE-3, a monoclonal antibody to murine ZP2. Positive clones contained cDNA inserts encoding polypeptide corresponding to ZP2(103-134). When normal or ovariectomized female mice were immunized with three overlapping peptides that span this region of ZP2 (101-120, 111-130, 121-140), only ZP2(121-140) elicited IgG antibodies that reacted with mouse ovarian ZP, indicative of the presence of native B epitope and helper T cell epitope in ZP2(121-140). To more finely map the ZP2 B cell epitope, a random peptide display library was screened with the IE-3 antibody, and a consensus tetramer sequence VxYK that matched the ZP2(123-126) sequence VRYK was located. Competitive immunofluorescence analysis with single alanine-substituted VxYK peptides ranked the relative contribution of the three critical B cell epitope residues as Y > V > K. A chimeric peptide was constructed that contained the YRYK motif of ZP2 and a bovine RNase T cell epitope. Although (C57BL/6xA/J) F1 (B6AF1) female mice immunized with the chimeric peptide developed ZP antibody response, this peptide elicited antibody only in mice of the histocompatibility complex (MHC) H-2(k or b) haplotype. In contrast, ZP2(121-140) peptide elicited antibody in inbred mice with three additional mouse MHC haplotypes. Moreover, although ZP2(121-140) contained a T cell epitope, no oophoritis was observed after immunization of B6AF1 mice with ZP2(121-140) in complete Freund's adjuvant (CFA). In a preliminary trial, female B6AF1 mice immunized with ZP2(121-140) in CFA had reduced litter sizes as compared with mice injected with CFA alone.     

Chicken major histocompatibility complex class II molecules of the B haplotype present self and foreign peptides

The chicken major histocompatibility complex (MHC), or B-complex, mediates genetic resistance and susceptibility to infectious disease. For example, the B19 haplotype is associated with susceptibility to Marek's disease. Here, we describe the sequencing and analysis of peptides presented by B19 MHC class II molecules. A B19/B19 B-cell line was used for the immunoaffinity purification of MHC class II molecules, which was followed by acid elution of the bound peptides. The eluted peptides were then analysed using tandem mass spectrometry. Thirty peptide sequences were obtained, ranging from 11 to 25 amino acids in length. Source protein cellular localization included the plasma membrane, cytosol and endosomal pathway. In addition, five peptides from the envelope glycoprotein of chicken syncytial virus (CSV) were identified. Chicken syncytial virus had been used as a helper virus along with reticuloendotheliosis virus strain T for transformation of B19/B19B cells. Alignment and analysis of the peptide sequence pool provided a putative peptide-binding motif for the B19 MHC class II.     

H2-Mbeta 1 and H2-Mbeta 2 heterodimers equally promote clip removal in I-A(q) molecules from autoimmune-prone DBA/1 mice

Antigen-presenting cells degrade endocytosed antigens, e.g. collagen type II, into peptides that are bound and presented to arthritogenic CD4(+) helper T cells by major histocompatibility complex (MHC) class II molecules. Efficient loading of many MHC class II alleles with peptides requires the assistance of H2-M (HLA-DM in humans), a heterodimeric MHC class II-like molecule that facilitates CLIP removal from MHC class II molecules and aids to shape the peptide repertoire presented by MHC class II to CD4(+) T cells. In contrast to the HLA-DM region in humans, the beta-chain locus is duplicated in mice, with the H2-Mb1 beta-chain distal to H2-Mb2 and the H2-Ma alpha-chain gene. H2-M alleles appear to be associated with the development of autoimmune diseases. Recent data showed that Mbeta1 and Mbeta2 isoforms are differentially expressed in isolated macrophages and B cells, respectively. The tissue expression and functional role of these heterodimers in promoting CLIP removal and peptide selection have not been addressed. We utilized the human T2 cell line, which lacks part of chromosome 6 encompassing the MHC class II and DM genes, to construct transgenic cell lines expressing the MHC class II heterodimer I-A(q) alone or in the presence of H2-Malphabeta1 or H2-Malphabeta2 heterodimers. Both H2-M isoforms facilitate the exchange of CLIP for cognate peptides on I-A(q) molecules from arthritis-susceptible DBA/1 mice and induce a conformational change in I-A(q) molecules. Moreover, I-A(q) cell-surface expression is not absolutely dependent on H2-M molecules. These data suggest that I-A(q) exhibits a high affinity for CLIP since virtually all I-A(q) molecules on T2 cells were found to be associated with CLIP in the absence of both H2-M isoforms.     

Immune responses to the 18-kDa protein of Mycobacterium leprae. Similar B cell epitopes but different T cell epitopes seen by inbred strains of mice.

Antibody responses to the 18-kDa protein of Mycobacterium leprae have been analyzed in different strains of mice. High, intermediate, and low responder strains have been identified and these response patterns show clear linkage to genes encoded in the H-2 complex. Three peptides, residues 1-50, 51-100, and 101-148 have been synthesized, as well as a series of 20-mer peptides, which span the entire 18-kDa protein. Repeated immunization of different strains of mice with the 18-kDa protein resulted in IgG responses to epitopes found on all three synthetic peptides. Immunization of BALB/cJ and B10.BR mice, two high responder strains, with 18-kDa protein resulted in high levels of IgG antibody to epitopes found on peptides 1-20, 16-35, 31-50, 46-65, and 76-95. B10.BR mice also contained IgG that bound peptide 61-80 and BALB/cJ mice produced IgG that bound peptide 91-110. Although B10.BR mice produced IgG that bound the 50-mer peptide 101-148, this IgG was not detected by binding to peptides 91-110, 106-125, 121-140, and 131-148. Immunization of B10.BR mice with individual overlapping 20-mer peptides as Ag revealed that peptides 1-20, 16-35, 31-50, and 76-95 elicited high titers of IgG that bound both the immunizing peptide as well as 18-kDa protein. As these peptides induce antibody synthesis they must contain both B cell and T cell epitopes. By contrast, immunization of BALB/cJ mice with the same 20-mer peptides, all of which contain B cell epitopes for this strain, failed to elicit IgG responses with one exception. Peptide 91-110 induced IgG that bound peptide 91-110, but not the intact 18-kDa protein. We conclude that peptides 1-20, 16-35, 31-50, and 76-95 either lack T cell epitopes for BALB/cJ mice, or activate different T cell subpopulations in the two strains. We suggest that the induction of IgG responses to small peptide Ag is an in vivo assay of the activity of Th2 cell subpopulations.     

Structural definition of the H-2Kd peptide-binding motif

Classic major histocompatibility complex (MHC) proteins associate with antigen- and self-derived peptides in an allele-specific manner. Herein we present the crystal structure of the MHC class I protein H-2K(d) (K(d)) expressed by BALB/c mice in complex with an antigenic peptide derived from influenza A/PR/8/34 nucleoprotein (Flu, residues 147-155, TYQRTRALV). Analysis of our structure in conjunction with the sequences of naturally processed epitopes provides a comprehensive understanding of the dominant K(d) peptide-binding motif. We find that Flu residues Tyr(P2), Thr(P5), and Val(P9) are sequestered into the B, C, and F pockets of the K(d) groove, respectively. The shape and chemistry of the polymorphic B pocket make it an optimal binding site for the side chain of Tyr(P2) as the dominant anchoring residue of nonameric peptides. The non-polar F pocket limits the amino acid repertoire at P9 to hydrophobic residues such as Ile, Leu, or Val, whereas the C pocket restricts the size of the P5-anchoring side chain. We also show that Flu is accommodated in the complex through an unfavorable kink in the otherwise extended peptide backbone due to the presence of a prominent ridge in the K(d) groove. Surprisingly, this backbone conformation is strikingly similar to D(b)-presented peptides despite the fact that these proteins employ distinct motif-anchoring strategies. The results presented in this study provide a solid foundation for the understanding of K(d)-restricted antigen presentation and recognition events.     

Presentation of type B peptide-MHC complexes from hen egg white lysozyme by TLR ligands and type I IFNs independent of H2-DM regulation

In APCs, presentation by MHC II molecules of the chemically dominant peptide from the protein hen egg white lysozyme (HEL) generates different conformational isomers of the peptide-MHC II complexes (pMHC). Type B pMHCs are formed in early endosomes from exogenous peptides in the absence of H2-DM, whereas in contrast, type A pMHC complexes are formed from HEL protein in late vesicles after editing by H2-DM. Thus, H2-DM edits off the more unstable pMHC complexes, which are not presented from HEL. In this study, we show that type B pMHC complexes were presented from HEL protein only after stimulation of dendritic cells (DC) with TLR ligands or type I IFN. Type I IFN contributed to most TLR ligand-induced type B pMHC generation, as presentation decreased in DC lacking the receptor for type I IFNs (IFNAR1(-/-)). In contrast, presentation of type A pMHC from HEL and from peptide was minimally affected by TLR ligands. The relative effectiveness of CD8α(+) DC or CD8α(-) DC in presenting type B pMHC complexes varied depending on the TLR ligand used. The mechanisms of generation of type B pMHC from HEL protein with TLR stimulation did not involve H2-DM or release of peptides. DC from H2-DM-deficient mice in the presence of TLR ligands presented type B pMHC. Such DC showed a slight enhancement of HEL catabolism, but peptide release was not evident. Thus, TLR ligands and type I IFN alter the pathways of presentation by MHC II molecules of DC such that type B pMHCs are generated from protein Ag.     

Quantitative analysis of the immune response to mouse non-MHC transplantation antigens in vivo: the H60 histocompatibility antigen dominates over all others

Minor histocompatibility Ags (minor H Ags) are substantial impediments to MHC-matched solid tissue and bone marrow transplantation. From an antigenic standpoint, transplantation between MHC-matched individuals has the potential to be remarkably complex. To determine the extent to which the immune response is simplified by the phenomenon of immunodominance, we used peptide/MHC tetramers based on recently discovered minor H Ags (H60, H13, and HY) and monitored in vivo CD8 T cell responses of female C57BL/6 mice primed with MHC-matched, but background-disparate, male BALB.B cells. CD8 T cells against H60 overwhelmed responses to the H13 and HY throughout primary and secondary challenge. H60 immunodominance was an inherent quality, overcoming a lower memory precursor frequency compared with that of H13 and evoking a T cell response with diverse TCRV beta usage. IFN-gamma staining examining congenically defined minor H Ags extended H60 dominance over additional minor H Ags, H28, H4, and H7. These four minor H Ags accounted for up to 85% of the CD8 T cell response, but H60 stood out as the major contributor. These findings show that immunodominance applies to antigenically complex transplantation settings in vivo and that the responses to the H60 minor H Ag dominates in this model. We suggest that immunodominant minor H Ags are those that result from the absence of a self analog.     

A synthetic peptide to the E glycoprotein of Murray Valley encephalitis virus defines multiple virus-reactive T- and B-cell epitopes.

Synthetic peptides from the envelope glycoprotein sequence of Murray Valley encephalitis (MVE) virus were previously evaluated in various strains of mice for both the induction of antibody and the in vitro proliferation of peptide-primed T-helper (Th) cells. MVE peptide 6 (amino acids 230 to 251) elicited reciprocal Th- and B-cell reactivity with native MVE virus after primary inoculation of C57BL/6 mice. In this study, we prepared overlapping subunit peptides of MVE peptide 6 and evaluated their immunogenicity. Analysis of these peptides delineated at least two B-cell epitopes that induced antibody reactive with MVE and other Japanese encephalitis serocomplex viruses. This antibody at low titer neutralized MVE virus. Genetic restriction of the antibody response to various T-cell elements within peptide 6 was observed in C3H, BALB/c, C57BL/6, and B10 congenic mice. One element demonstrable after primary immunization, located in the carboxy terminus, associated only with major histocompatibility complex class II IAb and IAbiEk glycoproteins. Functional stimulation with the peptides in association with IAkIEk and IAdIEd molecules was observed only after in vivo secondary stimulation. Peptide 6-1 (amino acids 230 to 241) was nonimmunogenic but could be recognized by Th cells from peptide 6-immunized mice. Further association of peptide 6 with the IAkIEk and IAdIEd subregions was demonstrated by the finding that T cells from MVE peptide 6-inoculated C3H and BALB/c mice primed for an antibody response to MVE virus. These results suggest that the peptide 6 sequence, which is relatively conserved among a number of flaviviruses, should be given consideration when synthetic immunogens for vaccine purposes are designed.     

Hepatitis B virus splice-generated protein induces T-cell responses in HLA-transgenic mice and hepatitis B virus-infected patients

Hepatitis B virus splice-generated protein (HBSP), encoded by a spliced hepatitis B virus RNA, was recently identified in liver biopsy specimens from patients with chronic active hepatitis B. We investigated the possible generation of immunogenic peptides by the processing of this protein in vivo. We identified a panel of potential epitopes in HBSP by using predictive computational algorithms for peptide binding to HLA molecules. We used transgenic mice devoid of murine major histocompatibility complex (MHC) class I molecules and positive for human MHC class I molecules to characterize immune responses specific for HBSP. Two HLA-A2-restricted peptides and one immunodominant HLA-B7-restricted epitope were identified following the immunization of mice with DNA vectors encoding HBSP. Most importantly, a set of overlapping peptides covering the HBSP sequence induced significant HBSP-specific T-cell responses in peripheral blood mononuclear cells from patients with chronic hepatitis B. The response was multispecific, as several epitopes were recognized by CD8(+) and CD4(+) human T cells. This study provides the first evidence that this protein generated in vivo from an alternative reading frame of the hepatitis B virus genome activates T-cell responses in hepatitis B virus-infected patients. Given that hepatitis B is an immune response-mediated disease, the detection of T-cell responses directed against HBSP in patients with chronic hepatitis B suggests a potential role for this protein in liver disease progression.     

How H13 histocompatibility peptides differing by a single methyl group and lacking conventional MHC binding anchor motifs determine self-nonself discrimination.

The mouse H13 minor histocompatibility (H) Ag, originally detected as a barrier to allograft transplants, is remarkable in that rejection is a consequence of an extremely subtle interchange, P4(Val/Ile), in a nonamer H2-D(b)-bound peptide. Moreover, H13 peptides lack the canonical P5(Asn) central anchor residue normally considered important for forming a peptide/MHC complex. To understand how these noncanonical peptide pMHC complexes form physiologically active TCR ligands, crystal structures of allelic H13 pD(b) complexes and a P5(Asn) anchored pD(b) analog were solved to high resolution. The structures show that the basis of TCRs to distinguish self from nonself H13 peptides is their ability to distinguish a single solvent-exposed methyl group. In addition, the structures demonstrate that there is no need for H13 peptides to derive any stabilization from interactions within the central C pocket to generate fully functional pMHC complexes. These results provide a structural explanation for a classical non-MHC-encoded H Ag, and they call into question the requirement for contact between anchor residues and the major MHC binding pockets in vaccine design.     

The model B6(dom1) minor histocompatibility antigen is encoded by a mouse homolog of the yeast STT3 gene

The B6(dom1) minor histocompatibility antigen (MiHA) is a model antigen, since it is both the epitome of an immunodominant epitope and an ideal target for adoptive cancer immunotherapy. Based on DNA sequencing and MS/MS analyses, we report that B6(dom1) corresponds to amino acids 770-778 (KAPDNRETL) of a protein we propose to call SIMP (source of immunodominant MHC-associated peptides) that is encoded by a mouse homolog of the yeast STT3gene. STT3, a member of the oligosaccharyltransferase complex, is essential for cell proliferation. Phenotypic and genotypic analyses among eight strains of mice revealed a precise correlation between susceptibility or resistance to B6(dom1)-specific cytotoxic T lymphocytes (CTLs) and the presence of a Glu vs Asp amino acid at position 776 of the SIMP protein, respectively. Strikingly, while the difference in the amino acid sequence 770-778 encoded by the two SIMP alleles represents a very conservative substitution, these allelic peptides were not crossreactive at the CTL level, and both peptides were immunodominant when presented to mice homozygous for the opposite allele. In addition, we have cloned a human ortholog of SIMP whose predicted protein shares 97% amino acid identity with mouse SIMP. These results strengthen the concept that MHC class-I-associated MiHAs originate as a consequence of rare polymorphisms among highly conserved genes. Furthermore, the notion that a peptide differing from a self analog by a single methylene group can be immunodominant has implications regarding our understanding of the mechanisms of immunodominance.     

The H4b minor histocompatibility antigen is caused by a combination of genetically determined and posttranslational modifications

Minor histocompatibility (H) Ag disparities result in graft-vs-host disease and chronic solid allograft rejection in MHC-identical donor-recipient combinations. Minor H Ags are self protein-derived peptides presented by MHC class I molecules. Most arise as a consequence of allelic variation in the bound peptide (p) that results in TCR recognizing the p/MHC as foreign. We used a combinational peptide screening approach to identify the immune dominant H2K(b)-restricted epitope defining the mouse H4(b) minor H Ag. H4(b) is a consequence of a P3 threonine to isoleucine change in the MHC-bound peptide derived from epithelial membrane protein-3. This allelic variation also leads to phosphorylation of the H4(b) but not the H4(a) epitope. Further, ex vivo CD8(+) T lymphocytes bind phosphorylated Ag tetramers with high efficiency. Although we document the above process in the minor H Ag system, posttranslational modifications made possible by subtle amino acid changes could also contribute to immunogenicity and immune dominance in tumor immunotherapeutic settings.     

Primary vascularization of the graft determines the immunodominance of murine minor H antigens during organ transplantation

Grafts can be rejected even when matched for MHC because of differences in the minor histocompatibility Ags (mH-Ags). H4- and H60-derived epitopes are known as immunodominant mH-Ags in H2(b)-compatible BALB.B to C57BL/6 transplantation settings. Although multiple explanations have been provided to explain immunodominance of Ags, the role of vascularization of the graft is yet to be determined. In this study, we used heart (vascularized) and skin (nonvascularized) transplantations to determine the role of primary vascularization of the graft. A higher IFN-γ response toward H60 peptide occurs in heart recipients. In contrast, a higher IFN-γ response was generated against H4 peptide in skin transplant recipients. Peptide-loaded tetramer staining revealed a distinct antigenic hierarchy between heart and skin transplantation: H60-specific CD8(+) T cells were the most abundant after heart transplantation, whereas H4-specific CD8(+) T cells were more abundant after skin graft. Neither the tissue-specific distribution of mH-Ags nor the draining lymph node-derived dendritic cells correlated with the observed immunodominance. Interestingly, non-primarily vascularized cardiac allografts mimicked skin grafts in the observed immunodominance, and H60 immunodominance was observed in primarily vascularized skin grafts. However, T cell depletion from the BALB.B donor prior to cardiac allograft induces H4 immunodominance in vascularized cardiac allograft. Collectively, our data suggest that immediate transmigration of donor T cells via primary vascularization is responsible for the immunodominance of H60 mH-Ag in organ and tissue transplantation.     

CD3- bone marrow cells augment the generation of cytotoxic T lymphocytes showing a preference for the X-chromosome linked gene product of stimulator cells

Responder cells, composed of both a limited number of nylon wool-passed lymph node (NW-LN) cells and an excess number of CD3+ cell-depleted bone marrow (CD3- BM) cells from the same strain of mice, were stimulated with allogeneic spleen cells in vitro. The CD3- BM cells augmented the generation of allogeneic major histocompatibility complex (MHC) class I specific cytotoxic T lymphocytes (CTL) from NW-LN cells. C3H/He (H-2k, C3H background) responder cells were stimulated with either B10.D2 (H-2d, B10 background) or BALB/c (H-2d, BALB background) spleen cells. In the former stimulation, the CTL induced lysed B10.D2 target cells more efficiently than the BALB/c cells. Furthermore, these CTL lysed more (B10.D2 x BALB/c) F1 male target cells than (BALB/c x B10.D2) F1 male. In the latter stimulation, the CTL lysed more BALB/c than B10.D2 cells, and more (BALB/c) x B10.D2) F1 male than (B10.D2 x BALB/c) F1 male. The reciprocal mixed lymphocyte cultures (MLC) were carried out, in which BALB/c responder cells were stimulated with either C3H/He or B10.BR (H-2k, B10 background) spleen cells. In the former stimulation, the CTL induced lysed more C3H/He or (C3H/He x B10.BR) F1 male target cells than B10.BR or (B10.BR x C3H/He) F1 male, and in the latter, the reciprocal results were obtained. These results suggested that the CTL induced had a preference for the X-chromosome linked gene products (Xlgp), besides the specificity for the allogeneic MHC class I, of the mice used as stimulator.     

Expression and regulation of interferon gamma-inducible proteasomal subunits LMP7 and LMP10 in the bovine corpus luteum

The proteasome is a large, polymeric protease complex responsible for intracellular protein degradation and generation of peptides that bind to class I major histocompatibility complex (MHC) molecules. Interferon gamma (INFgamma) induces expression of alternative proteasomal subunits that affect intracellular protein degradation, thereby changing the types of peptides that bind to class I MHC molecules. These alterations in class I MHC peptides can influence whether cells and tissues are tolerated by the immune system. Expression of two INFgamma-inducible proteasomal subunits, LMP7 and LMP10, in bovine luteal tissue was examined in this study. Northern analysis revealed the presence of mRNA encoding LMP7 and LMP10 in luteal tissue. Steady-state amounts of LMP7 mRNA did not change during the estrous cycle, but LMP10 mRNA was low in early corpus luteum (CL) and elevated in midcycle and late CL. Tumor necrosis factor alpha alone and in the presence of LH and/or prostaglandin F2alpha elevated steady-state amounts of LMP10 mRNA but did not affect LMP7 mRNA in cultured luteal cells. Immunohistochemistry revealed the presence of LMP10 primarily in small luteal cells. Numbers of LMP10-positive cells were lower in early CL than in midcycle and late CL. The finding that INFgamma-inducible proteasomal subunits are expressed in luteal tissue when the CL is fully functional was unexpected and suggests that proteasomes in luteal cells may generate peptides capable of stimulating a class I MHC-dependent inflammatory response.