Allogeneic recognition of class I molecules: Anti-H-2Ld repertoire of H-2b mice includes T cells recognizing mutant class II H-2b (Abm12) molecules

Two major histocompatibility complex (MHC) class I-reactive T cell clones derived from H-2^b mice, generated against the allogeneic L^d molecule, were found to recognize the H-2^b class II mutant A^bm12 molecule as well. In addition, these clones also recognize the class II A^s molecule, and display a class II-dependent reactivity to staphylococcal enterotoxin B. Neither the class I nor the class II alloreactivities of the clones were found to be dependent on other MHC molecules. Both clones express CD4+CD8^? phenotypes. The CD4 molecule appears to be involved in their class II reactivity, while little or no role for CD4 could be detected in the class I reactivity. This is the first report of a class I/class II cross-reactivity being mediated by CD4⁺ T cells. The structural basis for this cross-reactivity is discussed.     

Tracing interactions of thymocytes with individual stromal cell partners

Cellular interactions in T cell development can be analyzed using thymus chimeras prepared in vitro, in which stromal cells and T cell precursors are manipulated separately. In an earlier study, we showed that for optimal T cell maturation, most - if not all - stromal cells must display appropriate (selecting) major histocompatibility complex (MHC) molecules: the substituion of selecting by nonselecting stromal cells leads to a proportional decrease in mature T cell production. These data imply that the availability of selecting stromal micro-environments is rate limiting for positive selection, and that in positive selection, each thymocyte engages only one (rather than multiple) stromal cell partners. To test this hypothesis, we developed a tracing sytem for thymocyte/stromal cell interactions, based on the acquisition by thymocytes of stroma-derived MHC class II determinants. When MHC class II-deficient precursors are placed in H-2^{b × k} F1 environments (where all stromal cells co-express H-2^b and H-2^k), individual thymocytes acquire class II determinants of both haplotypes. In striking contrast, when placed in mosaic stromal environments (where stromal cells express either H-2^b or H-2^k evenly interspersed), individual thymocytes preferentially acquire MHC class II determinants of one or the other haplotypes, but rarely both. This provides strong evidence that thymocytes have intimate interactions with individual stromal cells: having engaged one stromal cell niche, thymocytes do not (or only rarely) have promiscuous liaisons with others.     

Poor loading of major histocompatibility complex class II molecules with endogenously synthesized short peptides in the absence of invariant chain

In normal antigen-presenting cells, newly synthesized major histocompatibility complex (MHC) class II molecules associate with the invariant chain (Ii) glycoprotein in the endoplasmic reticulum (ER). They are loaded with peptides only after proteolytic removal of the Ii in post-Golgi endocytic vesicles. Since the Ii inhibits peptide binding to MHC class II molecules, this association could protect MHC class II molecules from being loaded with endogenous peptides early after biosynthesis. If this were an important function of the Ii in vivo, MHC class II molecules synthesized in cells lacking the Ii should be loaded efficiently with short endogenous peptides in the ER; such peptides are known to be present there due to TAP-mediated import from the cytosol. To examine this possibility, we have studied peptide loading in HeLa transfectants expressing murine H-2A^k MHC class II molecules either alone or together with an excess of Ii. Endogenous peptides could readily be extracted from conformationally intact A^k αβ dimers of biosynthetically labeled Ii⁺ cells, whereas peptide loading was greatly (> 95%) diminished in the absence of Ii. Significant amounts of sodium dodecyl sulfate-(SDS) stable 55-kDa peptide: A^k complexes were only found in the Ii⁺ transfectants. In the absence of Ii, the MHC class II molecules instead formed stable complexes with long (20 and 50 kDa) polypeptides. Known A^k-binding peptides bound stably to A^k molecules on Ii^? cells, could be co-purified with them, and were resistant to release in SDS, suggesting that poor recovery of endogenous peptides was not due to decreased stability of A^k: peptide complexes in the absence of Ii. We conclude that protection of MHC class II molecules from endogenous short peptides does not appear to be a quantitatively important function of the Ii molecule, because peptide loading is inefficient in its absence.     

Attachment of oligosaccharides to peptide antigen profoundly affects binding to major histocompatibility complex class II molecules and peptide immunogenicity

To investigate the immunogenicity of glycopeptides, a peptide fragment from hen egg lysozyme, HEL(81–96)-Y (here named 1) which is immunogenic in H-2^k mice and known to bind to the murine major histocompatibility complex (MHC) class II molecule E^k, was synthesized in five different glycosylated forms. The N-terminal serine of HEL(81–96)-Y was derivatized with D -glucose (2), maltotriose (3), and a branched D -glucose pentasaccharide (4). Furthermore, 1 was prepared with a central serine or asparagine derivatized with the branched D -glucose pentasaccharide (5) and GlcNAc (6), respectively. The ability of the five glycopeptides and the non-glycosylated peptide, labeled with ¹²⁵I, to bind to the two MHC class II molecules, A^k and E^k, was studied using a gel filtration assay. None of them could bind to A^k. Neither 5 nor 6 were able to bind to E^k. Surprisingly 2, 3 and 4 bound better to E^k than did the non-glycosylated peptide 1. The increased binding varied depending on the type of oligosaccharide attached to the N terminus of the peptide. The better binding to E^k of glycopeptide 4 was found to be due to an increased association rate. The binding of 1 as well as 4 was optimal at pH 5.0. Functional studies showed that 4 was able to elicit a heteroclitic proliferative response from T cells of mice immunized with the native non-glycosylated peptide. Circular dichroism studies of 1 and 4 indicated a more unordered structure of 4 and a predominant α-helical conformation of 1, suggesting that the MHC class II molecule may bind to peptides which are in a non-α-helical conformation. These results demonstrate that glycosylation has considerable influence on peptide immunogenicity for T lymphocytes.     

A defect in bone marrow derived dendritic cell maturation in the nonobesediabetic mouse

The pathogenesis of diabetes in the nonobese diabetic (NOD) mouse is characterized by a selective destruction of the insulin-producing β-cells in the islets of Langerhans mediated by autoreactive T cells. The function of T cells is controlled by dendritic cells (DC), which are not only the most potent activators of naïve T cells, but also contribute significantly to the establishment of central and peripheral tolerance. In this study, we demonstrate that the NOD mouse (H2: K^d, A^g7, E°, D^b) shows selective phenotypic and functional abnormalities in DC derived from bone marrow progeny cells in response to GM-CSF (DC^NOD). NOD DC, in contrast to CBA DC, have very low levels of intracellular I-A molecules and cell surface expression of MHC class II, CD80, CD86 and CD40 but normal β2-microglobulin expression. Incubation with the strong inflammatory stimulus of LPS and IFN-γ does not increase class II MHC, CD80 or CD86, but upregulates the level of CD40. The genetic defect observed in the DC^NOD does not map to the MHC, because the DC from the MHC congenic NOD.H2^h4 mouse (H2: K^k, A^k, E^k, D^k) shares the cell surface phenotype of the DC^NOD. DC from these NOD.H2^h4 also fail to present HEL or the appropriate HEL-peptide to an antigen-specific T cell hybridoma. However all the DC irrespective of origin were able to produce TNF-α, IL-6, low levels of IL-12(p70) and NO in response to LPS plus IFN-γ. A gene or genes specific to the NOD strain, but outside the MHC region, therefore must regulate the differentiation of DC in response to GM-CSF. This defect may contribute to the complex genetic aetiology of the multifactorial autoimmune phenotype of the NOD strain.     

Presentation of a horse cytochrome c peptide by multiple H-2b class I major histocompatibility complex (MHC) molecules to C57BL/6- and bm1-derived cytotoxic T lymphocytes: Presence of a single MHC anchor residue may confer efficient peptide-specific CTL recognition

In this study the immunogenic tryptic fragment from a horse cytochrome c (cyt c) digest recognized by cytotoxic T lymphocytes (CTL), induced by in vitro peptide stimulation from C57BL/6 (B6) and mutant B6.C-H-2^bm1 (bm1) mice is identified. An identical sequence, p40—53, is recognized by CTL from both B6 and bm1 mice. In addition, both B6 and bm1 cloned CTL lines display unusual major histocompatibility complex (MHC) class I-restricted recognition of this peptide in that they respond to it in the context of H-2K^b, H-2D^b, and H-2K^bm1 class I molecules, although the sequence lacks the usual structural K^b and D^b peptide-binding motifs. Truncated analogues which resemble the lengths of naturally processed MHC class I-presented peptides, confer reactivity for B6 and bm1 CTL against EL4 (H-2^b) targets as well as the L cell transfectants, L + K^b, L + D^b, and L + K^bm1. The antigenic peptide with the greatest potency is p41—49, which appears to be generated by angiotensin converting enzyme cleavage of the full-length p40—53 tryptic peptide. The minimum antigenic peptide recognized by both B6 and bm1 CTL, and which targets lysis on each of the transfectants, is the hexamer p43—48 peptide from horse cyt c. Residues Pro⁴⁴ and Thr⁴⁷, which occupy polymorphic positions with respect to other species-variant cyt c molecules, influence recognition of these peptides differently for the B6 and bm1 CTL. The ability of H-2K^b, H-2D^b, and mutant H-2K^bm1 class I molecules to present the same peptide to a single cloned CTL is discussed in the context of current knowledge of peptide anchor residues and side chain-specific binding pockets in the MHC class I peptide-binding site.     

Impact of donor MHC class I or class II antigen deficiency on first- and second-set rejection of mouse heart or liver allografts

The influence of donor major histocompatibility complex (MHC) class I- or class II-deficiency on the initiation of first- and second-set rejection of mouse heart and liver allografts was examined. C3H (H-2^k) mice received heterotopic cardiac or orthotopic liver grafts from unmodified B10 (H-2^b), B6 (H-2^b), b2m (H-2^b; class I deficient) or AB⁰ (H-2^b; class II deficient) donors. Organ survival was also investigated in C3H recipients that had been presensitized by a normal B10 skin graft 2–3 weeks before heart or liver transplantation. The absence of cell surface MHC class I or class II resulted in significant prolongation of primary cardiac allograft survival. Three of seven (43%) MHC class I-deficient, and two of five (40%) class II-deficient heart grafts were accepted indefinitely (survival time >100 days). Thus both MHC class I and class II molecules appear to be important for the elicitation of first-set rejection in the heart allograft model. All liver allografts survived >100 days in normal recipients. In C3H recipients that had been presensitized by a B10 skin graft, however, both heart and liver grafts from AB⁰ (class II deficient) donors underwent accelerated rejection (median survival time [MST] 3 and 4 days, respectively). In contrast, liver grafts from class I-deficient mice (b2m) were still accepted indefinitely by B10 skin-presensitized C3H recipients, whereas class I-deficient hearts survived significantly longer than those from class II-deficient or normal donors. These data demonstrate that the expression of donor MHC class I, and not class II is crucial in initiating second-set organ allograft rejection. In vitro monitoring revealed that at the time of organ transplant, both splenocytes and serum of the skin-presensitized animals displayed high cytotoxicity against AB⁰ (class II-deficient) but not against b2m (class I-deficient) targets.     

The structural basis of MHC control of collagen-induced arthritis; binding of the immunodominant type II collagen 256 – 270 glycopeptide to H-2Aq and H-2Ap molecules

The A^q major histocompatibility complex (MHC) class II molecule is associated with susceptibility to murine collagen-induced arthritis (CIA), whereas the closely related H-2A^p molecule is not. To understand the molecular basis for this difference, we have analyzed the ability of H-2A^q and H-2A^p molecules (referred to as A^q and A^p) to bind and present collagen type II (CII)-derived glycosylated and non-glycosylated peptides. T cell clones specific for the immunodominant CII 256 – 270 peptide and restricted to both A^q and A^p molecules were identified. When these clones were incubated with CII protein and either A^q- or A^p-expressing antigen-presenting cells (APC), only A^q-expressing APC were able to induce stimulation. With the use of A_β transgenic mice this could be shown to be solely dependent on the MHC class II molecule itself and to be independent of other MHC- or non-MHC genes. Peptide binding studies were performed using affinity-purified MHC class II molecules. The CII 256 – 270 peptide bound with lower affinity to the A^p molecule than to the A^q molecule. Using a set of alanine-substituted CII 256 – 270 peptides, MHC class II and T cell receptor (TCR) contacts were identified. Mainly the side chains of isoleucine 260 and phenylalanine 263 were used for binding both the A^q and A^p molecule, i. e. the peptide was orientated similarly in the binding clefts. The major TCR contact amino acids were lysine 264, which can be posttranslotionally modified, and glutamic acid 266, which is the only amino acid in the heterologous peptide which differs from the mouse sequence. Glycosylation at positions 264 and 270 of the CII 256 – 270 peptide did not change the anchor positions used for binding to the A^q or A^p molecules. The autologous form of the peptide (with aspartic acid at position 266) bound with lower affinity to the A^q molecule as compared with the heterologous peptide. The variable affinity displayed by the immunodominant CII 256 – 270 peptide for different MHC class II molecules, the identification of MHC and TCR contacts and the significance of glycosylation of these have important implications for the understanding of the molecular basis for inherited MHC class II-associated susceptibility to CIA and in turn, for development of novel treatment strategies in this disease.     

Presentation of viral antigens restricted by H-2Kb,Db or Kd in proteasome subunit LMP2-and LMP7-deficient cells

In the class II region of the major histocompatibility complex (MHC), four genes implicated in MHC class I-mediated antigen processing have been described. Two genes (TAP 1 and TAP 2) code for multimembrane-spanning ATP-binding transporter proteins and two genes (LMP 2 and LMP 7) code for subunits of the proteasome. While TAP 1 and TAP 2 have been shown to transport antigenic peptides from the cytosol into the endoplasmic reticulum, where the peptides associate with MHC class I molecules, the role of LMP 2/7 in antigen presentation is less clear. Using antigen processing mutant T2 cells that lack TAP 1/2 and LMP 2/7 genes, it was recently shown that expression of TAP 1/2 alone was sufficient for processing and presentation of the influenza matrix protein M1 as well as the minor histocompatibility antigen HA-2 by HLA-A2. To understand if presentation of a broader range of viral antigens occurs in the absence of LMP 2/7, we transfected T2 cells with TAP 1, TAP 2 and either of the H-2K^b, D^b or K^d genes and tested their ability to present vesicular stomatitis vires and influenza virus antigens to virus-specific cytotoxic T lymphocytes. We found that T2 cells, expressing TAP 1/2 gene products, presented all tested viral antigens restricted through either the H-2K^b, D^b or K^d class I molecules. We conclude that the proteasome subunits LMP 2/7 as well as other gene products in the MHC class II region, except from TAP 1/2, are not generally necessary for presentation of a broader panel of viral antigens to cytotoxic T cells. However, the present results do not exclude that LMP 2/7 in a more subtle way may, or in rare cases completely, affect processing of antigen for presentation by MHC class I molecules.     

Efficacy of HLA-DRB1∗03:01 and H2E transgenic mouse strains to correlate pathogenic thyroglobulin epitopes for autoimmune thyroiditis

Thyroglobulin (Tg), a homodimer of 660 kD comprising 2748 amino acids, is the largest autoantigen known. The prevalence of autoimmune thyroid disease, including Hashimoto’s thyroiditis and Graves’ disease, has provided the impetus for identifying pathogenic T cell epitopes from human Tg over two decades. With no known dominant epitopes, the search has long been a challenge for investigators. After identifying HLA-DRB1∗03:01 (HLA-DR3) and H2E^b as susceptibility alleles for Tg-induced experimental autoimmune thyroiditis in transgenic mouse strains, we searched for naturally processed T cell epitopes with MHC class II-binding motif anchors and tested the selected peptides for pathogenicity in these mice. The thyroiditogenicity of one peptide, hTg2079, was confirmed in DR3 transgenic mice and corroborated in clinical studies. In H2E^b-expressing transgenic mice, we identified three T cell epitopes from mouse Tg, mTg179, mTg409 and mTg2342, based on homology to epitopes hTg179, hTg410 and hTg2344, respectively, which we and others have found stimulatory or pathogenic in both DR3- and H2E-expressing mice. The high homology among these peptides with shared presentation by DR3, H2E^b and H2E^k molecules led us to examine the binding pocket residues of these class II molecules. Their similar binding characteristics help explain the pathogenic capacity of these T cell epitopes. Our approach of using appropriate human and murine MHC class II transgenic mice, combined with the synthesis and testing of potential pathogenic Tg peptides predicted from computational models of MHC-binding motifs, should continue to provide insights into human autoimmune thyroid disease.     

Tolerance induction as a multi-step process

Tolerant T cells are characterized by their partial or full resistance to activation by antigen. We investigated whether tolerant T cells were still receptive to further tolerogenic signals. T cells expressing a transgenic T cell receptor (TCR) specific for the major histocompatibility complex (MHC) class I molecule K^b were deleted in mice carrying K^b but not in mice expressing the mutant K^b-molecule K^bm1 [TCR (H-2^{bm1 × k}) mice]. These T cells were tolerant in vivo but could be activated in vitro by the K^b antigen. This in vitro reactivity was abolished after the tolerant T cells encountered K^b-positive cells that had been intravenously injected. Furthermore, in TCR (H-2^{bm1 × k}), mice expressing K^b only on hepatocytes, no T lymphocytes bearing the transgenic TCR could be found in the periphery, indicating that the additional contact with K^b on hepatocytes led to deletion of the tolerant T cells. These findings demonstrate that tolerance induction can be a multi-step process.     

The T/B cell interaction involved in induction of the mouse IgG2ah suppression is restricted by major histocompatibility complex class I,but not class II molecules

To determine the major histocompatibility complex (MHC) restriction of the T/B cell interaction involved in a negative regulation of Ig production, we used mouse model of T cell-induced IgG2a^b suppression in vivo. Normal or specifically triggered T splenocytes from mice of the Igh^a haplotype, when neonatally transferred into histocompatible Igh^a/b heterozygotes, are able to induce a specific and total suppression of the IgG2a^b allotype. Nevertheless, only transfer of IgG2a^b-primed Igh^a T splenocytes induces this suppression in Igh^b/b homozygous congenic mice in which the whole IgG2a isotype production is inhibited. This suppression is chronically maintained by CD8⁺ T cells, but can be experimentally reversed. We have established that the suppression induction required a CD4⁺CD8⁺ T cell cooperation and operated via the recognition by the involved TCR of Cγ2a^b-derived peptides presented by the target B cells in an MHC haplotype-restricted manner. Here, by using Igh^b mice genetically deficient for MHC class I (β2-microglobulin^%, or β2m^%) or class II (I-Aβ^%) molecules, we demonstrate functionally that the suppression induction implicates an MHC class I-, but not class II-restricted interaction. Indeed, the anti-IgG2a^b T cells transferred into Igh^b H-2^b I-Aβ^% mice carry out the suppression process normally, while in Igh^b H-2^b β2m^% recipients, their suppression induction capacity is significantly inhibited. Moreover, the Cγ2a^b 103–118 peptide, identified as the sole Cγ2a^b-derived peptide able to amplify the anti-IgG2a^b T cell reactivity in Igh^a H-2^b mice, is also able to stabilize the H-2D^b, but not the H-2K^b class I molecules at the surface of RMA-S (TAP2^?, H-2^b) cells. These results indicate that, despite the CD4⁺/CD8⁺ T cell cooperation during the induction phase of suppression only MHC class I molecule expression is required at the surface of IgG2a^b+ B cells for suppression establishment.     

Identification of an HLA-DQ6-derived peptide recognized by mouse MHC class I H-2Db-restricted CD8+ T cells in HLA-DQ6 transgenic mice

Summary CD8⁺ T cells from C57BL/6(B6) mice show cytotoxicity to B cell blasts prepared from syngeneic transgenic mice expressing HLA-DQ6 molecules in a mouse MHC class I H-2D^b restricted manner. Although these results suggest that CD8⁺ T cells recognize peptides derived from DQ6 molecule bound to H-2D^b on target cells, no direct evidence so far has been obtained. To clarify this, we synthesized 23 peptides corresponding to DQ6α orβ chain and carrying the motifs of D^b-binding peptides, and examined their capacity to induce cytotoxicity in the CD8⁺ T cell line. We show here that DQA1-2, one of these peptides, induced cytotoxicity of the CD8⁺ T cells when this peptide was pulsed to H-2D^b expressing target cells, as efficiently as HLA-DQ6 expressing target cells did. Thus, our results suggest that DQA1-2 can be naturally processed from DQ6 molecules and recognized by the CD8⁺ T cells in the context of H-2D^b molecules. These results suggest that allogeneic HLA class II molecules are involved in the rejection not only as the ligand for T cell receptor of alloreactive CD4⁺ T cells but also as self-peptides bound to HLA class I molecules recognized by CD8⁺ T cells.     

Qa-1 interaction and T cell recognition of the Qa-1 determinant modifier peptide

The peptide-binding properties of the nonclassical major histocompatibility complex (MHC) class 1b molecule Qa-1 were investigated using a transfected hybrid molecule composed of the α1 and α2 domains of Qa-1^b and the α3 domain of H-2D^b. This allowed the use of a monoclonal antibody directed against H-2D^b whilst retaining the peptide-binding groove of Qa-1^b. By comparison with classical MHC class I molecules, intracellular maturation of the chimeric molecule was inefficient with weak intracellular association with β2-microglobulin. However, at the cell surface the hybrid molecules were stably associated with β2-microglobulin and were recognized by cytotoxic T lymphocyte (CTL) clones specific for the Qa-1^b -presented peptide Qdm (AMAPRTLLL). A whole-cell binding assay was used to determine which residues of Qdm were important for binding to Qa-1^b and CTL clones served to identify residues important for T cell recognition. Substitutions at position 1 and 5 did not reduce the efficiency of binding and had little effect on CTL recognition. In contrast, substitutions at position 9 resulted in loss of MHC class I binding. Mass spectrometric analysis of peptides eluted from immunopurified Qa-1^b/D^b molecules indicated that Qdm was the dominant peptide. The closely related peptide, AMVPRTLLL, which is derived from the signal sequence of H-2D^k, was also present, although it was considerably less abundant. The mass profile suggested the presence of additional peptides the majority of which consisted of eight to ten amino acid residues. Finally, the finding that a peptide derived from Klebsiella pneumoniae can bind raises the possibility that this non-classical MHC class I molecule may play a role in the presentation of peptides of microorganisms.     

Analysis of intraepithelial lymphocytes from major histocompatibility complex (MHC)-deficient mice: No evidence for a role of MHC class II antigens in the positive selection of Vδ4+γδ T cells

Three-color flow cytometric analysis was carried out with intraepithelial lymphocytes from mice deficient in expression of major histocompatibility complex (MHC) antigens. These experiments were done to address the possible role of MHC class II molecules in the positive selection of Vδ4⁺ γδ T cells. By analyzing mice deficient MHC class II antigens alone or in combination with MHC class I antigens, no evidence was found for positive selection of Vδ4⁺ cells among CD8a⁺ or CD4^?CD8^? subpopulations of γδ T cell receptor-positive cells. Because V54⁺, CD8a⁺ cells were reported to be positively selected on I-E^k and hybrid I-E^k/b molecules, class II-deficient animals were crossed with I-E^k transgenic mice and progeny examined for Vδ4 expression. Again, no evidence for positive selection was found. Interestingly, in MHC class I-deficient animals, the total number of γδ T cells was about twofold higher than in control and MHC class II-deficient mice and the proportion of V8δ-expressing cells was correspondingly decreased. Taken together, these results cast doubt on a major role for conventional MHC antigens in shaping the γδ T cell repertoire of intraepithelial lymphocytes.     

Both positive and negative effects on immune responses by expression of a second class II MHC molecule

It is perplexing why vertebrates express a limited number of major histocompatibility complex (MHC) molecules when theoretically, having a greater repertoire of MHC molecules would increase the number of epitopes presented, thereby enhancing thymic selection and T cell response to pathogens. It is possible that any positive effects would either be neutralized or outweighed by negative selection restricting the T cell repertoire. We hypothesize that the limit on MHC number is due to negative consequences arising from expressing additional MHC. We compared T cell responses between B6 mice (I-A⁺) and B6.E⁺ mice (I-A⁺, I-E⁺), the latter expressing a second class II MHC molecule, I-E^b, due to a monomorphic Eα^k transgene that pairs with the endogenous I-Eβ^b chain. First, the naive T cell Vβ repertoire was altered in B6.E⁺ thymi and spleens, potentially mediating different outcomes in T cell reactivity. Although the B6 and B6.E⁺ responses to hen egg-white lysozyme (HEL) protein immunization remained similar, other immune models yielded differences. For viral infection, the quality of the T cell response was subtly altered, with diminished production of certain cytokines by B6.E⁺ CD4⁺ T cells. In alloreactivity, the B6.E⁺ T cell response was significantly dampened. Finally, we observed markedly enhanced susceptibility to experimental autoimmune encephalomyelitis (EAE) in B6.E⁺ mice. This correlated with decreased percentages of nTreg cells, supporting the concept of Tregs exhibiting differential susceptibility to negative selection. Altogether, our data suggest that expressing an additional class II MHC can produce diverse effects, with more severe autoimmunity providing a compelling explanation for limiting the expression of MHC molecules.     

Vaccination with cytotoxic T lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16-transformed cells

Cytotoxic T lymphocyte (CTL) peptide epitopes can be used for immunization of mice against lethal virus infection. To study whether this approach can be successful against virus-induced tumors we generated a B6 (H-2^b) tumorigenic cell line transformed by human papillomavirus (HPV). This virus is detected in over 90% of all human cervical cancers. To identify vaccine candidates, we generated a set of 240 overlapping peptides derived from the HPV type 16 (HPV16) oncogenes E6 and E7. These peptides were tested for their ability to bind H-2K^b and H-2D^b MHC class I molecules. Binding peptides were compared with the presently known peptide-binding motifs for H-2K^b and H-2D^b and the predictive value of these motifs is shortly discussed. The high-affinity H-2D^b-binding peptide and putative CTL epitope E₇ 49-57 (RAHYNIVTF) was used in vaccination studies against HPV 16-transformed tumor cells. Immunization with peptide E₇ 49-57 rendered mice insensitive to a subsequent challenge with HPV 16-transformed tumor cells in vivo, and induced a CTL response which lysed the tumor cells in vitro.     

Determination of the allele-specific antigen-binding site on I-Ak and I-Ab molecules

Residues 46 and 54 on a pigeon cytochrome c 43–58 analog, 50E, function as major histocompatibility complex class II contact sites. A peptide, 46F50E54A, with phenylalanine (F) at position 46 and alanine (A) at 54 on 50E bound to A^b and a peptide, 46D50E54A, with aspartic acid (D) at 46 and alanine at 54, bound to A^k. To determine the allele-specific peptide contact sites on I-A molecules corresponding to the I-A contact sites of the peptides, we analyzed responses of A^k- and/or A^b-restricted T cell hybridomas to 46F50E54A or 46D50E54A using L cell transfectants expressing recombinant I-A molecules between A^k and A^b or point mutants of A^k as antigen presenting cells. It was shown that the N-terminal half of the α helix of the Aα chain determined the allele-specific T cell responses. Furthermore, with arginine (k type amino acid) or alanine (b type amino acid) at position 56 of the A^k α chain, these T cell hybridomas were stimulated predominantly by 46D50E54A (A^k binding peptide) or 46F50E54A (A^b binding peptide), respectively. Thus, the amino acid at position 56 of the Aα chain determines allele-specific antigen presentation. This postulate was confirmed by direct binding analysis of 50E analogs of various I-A molecules. A single amino acid change (arginine to alanine) at position 56 of the A^k α chain altered the peptide binding specificity (46D50E54A to 46F50E54A).     

Invriant chain peptides enhancing or inhibiting the presentation of antigenic peptides by major histocompatibility complex class II molecules

Two soluble invariant chain (Ii) peptides with overlapping sequences had contrasting effects on the presentation of antigenic peptides by murine A^d, A^k, E^d, and E^k major histocompatibility complex (MHC) class II molecules. Naturally produced class II-associated invariant chain peptides human (h)Ii81–104/murine (m)Ii80–103 inhibited antigen presentation on these MHC class II alleles in a manner consistent with competitive inhibition. The Ii-4 peptides hIi77–92/mIi76–91 enhanced presentation of antigenic peptides on I-E class II alleles by promoting the exchange of peptides at the cell surface. Treatment of antigenpresenting cells (APC) with Ii-4 before the addition of antigenic peptide greatly enhanced subsequent T cell responses, while treatment of APC with Ii–4 after antigenic peptide binding decreased subsequent T cell responses. The hIi81–104 and mIi80–103 peptides inhibited T cell responses in both types of assays. The binding of biotinylated antigenic peptide to MHC class II-transfected L cells, as measured by flow cytometry, was inhibited by mIi80-103 and enhanced by mIi-4. Segments of Ii fragments remaining associated with MHC class II, or released Ii peptides, appear to regulate the formation of stable antigenic peptide/MHC class II complexes either positively or negatively through interactions at or near the antigenic peptide binding site. These findings open a pathway for the design of novel therapeutics based on the structure and function of natural and rationally designed fragments of Ii.     

SPECIFICITY OF ANTI-H-2 CLASS I ANTIBODIES INDUCED BY SYNGENEIC IMMUNIZATION WITH SENDAI VIRUS-TREATED CELLS IS REGULATED BY THE MOUSE MHC AND VIRAL ANTIGENS. NO EVIDENCE FOR MHC-RESTRICTED VIRUS- SPECIFIC ANTIBODIES

In a previous study, we searched for Sendai virus (SV)-specific antibodies that were restricted in their binding by self-major histocompatability complex (MHC) antigens. In C57BL/6 (B6; H-2^b) mice, most of the sera obtained after i.p. injections with syngeneic SV-coated (SV⁺) spleen cells contained auto- and alloreactive lymphocytotoxic antibodies directed against H-2 class I molecules, but no viral-specific, MHC-restricted antibodies. Here we report that syngeneic immunization with SV⁺ cells regularly induced H-2-specific antibodies in various mouse strains. From a total of 12 strains tested, only the B10.S (H-2^s) strain appeared to be a low responder. The immune responses are of two types: (i) mice of some strains produce autoreactive antibodies and a broad variety of alloreactive antibodies; and (ii) mice of some strains produce only narrow or widely alloreactive antibodies. Because most of the strains differ only in the H-2 region, the patterns observed are regulated by the MHC. To locate the genes involved in the induction of H-2-specific antibodies more precisely, two B6 mutant strains, bml (K^b mutant) and bm13 (D^b mutant), were immunized with syngeneic SV⁺ cells. The results suggest that the H-2D^b region plays an important role in the induction and specificity of the lymphocytotoxic H-2 class I-specific antibodies present in sera of H-2^b mice after syngeneic immunization with SV⁺ cells.