Genetically-based olfactory signatures persist despite dietary variation

Individual mice have a unique odor, or odortype, that facilitates individual recognition. Odortypes, like other phenotypes, can be influenced by genetic and environmental variation. The genetic influence derives in part from genes of the major histocompatibility complex (MHC). A major environmental influence is diet, which could obscure the genetic contribution to odortype. Because odortype stability is a prerequisite for individual recognition under normal behavioral conditions, we investigated whether MHC-determined urinary odortypes of inbred mice can be identified in the face of large diet-induced variation. Mice trained to discriminate urines from panels of mice that differed both in diet and MHC type found the diet odor more salient in generalization trials. Nevertheless, when mice were trained to discriminate mice with only MHC differences (but on the same diet), they recognized the MHC difference when tested with urines from mice on a different diet. This indicates that MHC odor profiles remain despite large dietary variation. Chemical analyses of urinary volatile organic compounds (VOCs) extracted by solid phase microextraction (SPME) and analyzed by gas chromatography/mass spectrometry (GC/MS) are consistent with this inference. Although diet influenced VOC variation more than MHC, with algorithmic training (supervised classification) MHC types could be accurately discriminated across different diets. Thus, although there are clear diet effects on urinary volatile profiles, they do not obscure MHC effects.     

Effect of B2m gene disruption on MHC-determined odortypes

 Major histocompatibility complex (MHC) genes confer individual olfactory identity that can be detected with exquisite accuracy by mice. The fact that MHC genes themselves generate the characteristic odortype, rather than dedicated odor-determining genes, was supported in studies of point mutations in H2K and HLA transgenic mice, which evinced distinct odor profiles in olfactory assays. In this article we provide further evidence for a central role of MHC genes themselves in odortype specification by demonstrating that mice that are unable to express their genomic class I MHC genes because they lack β₂-microglobulin are distinguishable by scent from otherwise identical mice which possess an intact B2m gene. This odortype disparity appears at 9–12 days of gestational age, the period in which the MHC is first detectable in fetal cells of normal mice. Received: 15 October 1999 / Revised: 30 December 1999     

NK gene complex and chromosome 19 loci enhance MHC resistance to murine cytomegalovirus infection

An H-2^k MHC locus is critical for murine cytomegalovirus (MCMV) resistance in MA/My mice and virus control is abolished if H-2^k is replaced with H-2^b MHC genes from MCMV-susceptible C57L mice. Yet, H-2^k resistance varies with genetic background; thus, modifiers of virus resistance must exist. To identify non-MHC resistance loci, spleen and liver MCMV levels and genome-wide genotypes were assessed in (C57L × MA/My) and (MA/My × C57L) F₂ offspring (representing 550 meioses). Significantly, a non-Mendelian frequency of MHC genotypes was observed for offspring of the latter cross. Quantitative trait loci (QTL) and their interaction potential in MCMV resistance were assessed in R/qtl; QTL on chromosomes 17, 6, and 19 affected MCMV levels in infected animals. A chromosome 6 QTL was linked with the NK gene complex and acted in an additive fashion with an H-2^k MHC QTL to mitigate spleen MCMV levels. We provide biological confirmation that this chromosome 6 QTL provided MCMV control independent of H-2^k via NK cells. Importantly, both chromosome 6 and 19 QTLs contribute to virus control independent of H-2^k. Altogether, MHC and non-MHC MCMV-resistance QTL contribute in early resistance to MCMV infection in this genetic system.     

H2-E transgenic class II-negative mice can distinguish self from nonself in susceptibility to heterologous thyroglobulins in autoimmune thyroiditis

 Susceptibility to experimental autoimmune thyroiditis (EAT) is linked to H2-A class II genes;k and s haplotypes are susceptible, while b and f are resistant. EAT is inducible with thyroglobulins (Tgs) from several mammalian species which share portions of identical sequences. But cross-activation and cross-tolerance studies with mouse (m), human (h), and porcine (p) Tg have indicated mTg-unique T-cell epitope(s), in addition to conserved, in EAT induction. The recent introduction of the HLA-DRB1*0301 (DR3) transgene rendered major histocompatibility complex (MHC) class II-negative (Ab⁰) mice susceptible to EAT induction by both hTg and mTg, suggesting usage of conserved epitopes. Here, we introduced the H2-Ea ^k transgene into resistant B10 (H2 ^b ) or Ab⁰ mice with a defective Ea gene to provide functional surface H2E (b haplotype) expression. Surprisingly, both transgenic strains showed severe inflammation only after hTg, but not mTg, immunization, although the moderating influence of the A ^b gene in B10 was evident. In proliferative assays, hTg-primed cells did not respond to mTg, nor to conserved 12mer peptides from three primary hormonogenic sites, two of which can activate T cells for thyroiditis transfer and cytotoxicity. The vigorous response to hTg stimulation was reduced only by Eβ^b-specific monoclonal antibody. EAT induction with bovine and pTg showed responses similar to hTg, suggesting thyroiditogenic epitopes shared with hTg, but not mTg. This is the first demonstration of: (1) nonpermissiveness for EAT induction with mTg, normally the most thyroiditogenic Tg and the one with unique epitopes for susceptible mice, and (2) the separation of hTg from mTg in EAT induction in H2-E-transgenic mice. Received: 15 January 1999 / Revised: 23 April 1999     

Homopoietic histocompatibility (Hh-1) phenotype and the regulation of its expression

Hybrid resistance (HR) is primarily controlled by the genes of the Hemopoietic histocompatibility-1 (Hh-1) locus within the H-2 complex. HR is a consequence of the Hh-1-controlled target determinants in homozygous parental strain mice and their absence in heterozygous F₁ hybrid mice. To examine the mechanism that controls the Hh-1 phenotype, three independent clones of somatic cell hybrids between parental lines EL-4 (C57BL/6 origin, H-2 ^b ) and R1 (C58 origin, H-2 ^k ) were studied. The line EL-4 is Hh-1^b-positive and is subject to HR by H-2 ^b heterozygous F₁ mice, but R1 lacks the Hh-1 ^b allele and is not susceptible to HR. Of the three hybrid clones, F263.2 is Hh-1^b-positive, whereas the other two, F262.2 and F264.2, are Hh-1-negative, as judged by these cells' capacity to compete in vivo with the grafted parental C57BL/6 bone marrow cells in the resistant (C57BL/6 × C3H)F₁ mice. All three clones express the H-2^b and H-2^k class I antigens equally well, are susceptible to activated NK cells to the same extent, and all carry four copies of chromosome 17. However, Southern analysis reveals that clone F263.2 contains three copies of H-2 ^b chromosome and one H-2 ^k , whereas the other two clones carry two copies each of the parental chromosome 17. The results suggest that the relative copy number of specific alleles is the crucial determinanr of the Hh-1 phenotype, and render unlikely both the gene dosage hypothesis and the trans-acting dominant suppression hypothesis to account for the noncodominant expression of the Hh-1 phenotype.     

Polymorphic peptide transporters in MHC class I monomorphic Syrian hamster

We have already shown that in species with highly polymorphic major histocompatibility complex (MHC) class I molecules (human, mouse) no functional polymorphism of the peptide transporters TaP1 and TAP2 is detectable (Lobigs and Müllbacher 1993).Investigating the antigen-presentation machinery of the class I MHC I expression via recombinant vaccinia viruses MHC class I expression via recombinant vaccinia viruses (VV) we found that six hamster cell lines fall into two phenotypic classes. Four cell lines (HaK, FF, MF-2, and HT-1) showed no defect in expressing four different H2 class I molecules (K^K, K^d, K^b, D^d) and the appropriate VV peptide recognized by mouse VV-immune cytotoxic T (Tc) cells on the cell surface. Two cell lines (BHK-21 and NIL-2) expressed D^d and K^b in association with VV peptides as recognized by VV-immune, H2-restricted Tc cells but not K^k and K^d. However, K^d was expressed on the cell surface, as shown by fluorescence-activated cell sorter (FACS) analysis and alloreactive Tc-cell recogniction. K^k is only surface-expressed in these two cell lines when superinfected with two VV recombinants encoding rat TAP1 (VV-mtp1) and TAP2 (VV-mtp2). Superinfection with VV-mtpl and VV-mtp2 rendered both cell lines, after infection with either VV-K^k or V-K^d, susceptible to lysis by either K^k-orK^d- restricted VV-immune Tc cells. Thus Syrian hamster cell lines express functionally polymorphic peptide transporters.The TAP2 gene from FF cells was cloned and sequenced; comparison with human, mouse, and rat TAP2 sequences show 78%, 88% and 87% similarity, respectively.     

H-2 class I antigen expression on mouse teratocarcinoma cell lines

Immunity against PCC3 teratocarcinoma cells (129, H-2 ^b) was induced in allogeneic (C3H, H-2 ^k) mice by preimmunization with L cells (C3H, H-2 ^k) expressing cosmid-introduced K ^b or D ^b genes, but not with nontransfected L cells. In addition, the growth of PCC3 cells in sublethally irradiated (C3H × B6-H-2 ^bm1)F₁ and (C3H × B6-H-2 ^bm13 )F₁ mice bearing the K ^bm1 and D ^bm13 mutations, respectively, was either prevented, stopped, or delayed in comparison with the (C3H × B6)F₁ (k × b) mice, which failed to reject the PCC3 cells. The teratocarcinoma line OC15S was exceptional because it reacted specifically with K^b- and D^b-specific (but not I^b-specific) alloantisera, and because K^b- and D^b-specific antibodies could be absorbed by OC15S cells. The subpopulation of OC15S cells bearing the ECMA-7 antigen characteristic for embryonic carcinoma (EC) cells was isolated by the fluorescence-activated cell sorter and was shown to react specifically with K^b- and D^b-specific antisera. These experiments show that teratocarcinoma cells express antigens similar or identical to the K-and D-region products of differentiated cells. The lack of expression of class I antigens is thus neither a condition nor a consequence of the pluripotentiality of the EC cells. The exact nature of the major histocompatibility complex antigens on EC cells has yet to be established using the methods of molecular biology and biochemistry.     

Immune suppression genes control the anti-F antigen response in F1 hybrids and recombinant inbred sets of mice

The immune response to the liver protein F antigen which, in the mouse, occurs in two allelic forms, is under sharp immunogenetic control in that only mice that possess the A^k molecule can respond to allo-F antigen. This response has been studied in a number of F₁ hybrids between inbred strains and with recombinant inbred lines all of which express A^k, and which thus enable immune suppression effects to be detected. In the AKXL and AKXD sets, the hybrids with CBA are responders if H-2 ^k/H-2^k, and usually nonresponders if H-2 ^k/H-2^b or H-2 ^k/H-2^d. Although this may be due to gene dosage effects, this cannot be the explanation for the low responsiveness of the H-2 ^k/H-2^b relative to the H-2 ^k/H-2^d mice found in CBA × BXD hybrids. For this, and other reasons, it seems likely that low responsiveness in any mouse possessing a responder A ^k allele is due to suppression, and that this is mediated by the immune suppression effects of the non-H-2 ^k haplotype. These H-2-mediated effects can be modified, both positively and negatively, by background genes. Thus, of the ten H-2^k/H-2^d members of the CBA × AKXD cross, seven are low responders and three are high responders. No other typed marker has the same strain distribution pattern at present. Major unresolved questions, therefore, concern the location and mechanism of action of the background genes and the mechanism of action of the H-2 immune suppression genes.     

Variable spread of X inactivation affecting the expression of different epitopes of the Hya gene product in mouse B-cell clones

Cloned B-cell lines from a female T16H/XSxr mouse in which Tdy expression was suppressed due to X inactivation and from a male X/XSxr mouse, both of the (kxb)F₁ haplotype, were examined for H-Y expression. This was determined both by their ability to act as targets for H-2^k and H-2^b-restricted H-Y-specific cytotoxic T cells and by their ability to stimulate the proliferation of H-2K^k, H-2D^b (class I) and A^b (class II)-restricted T-cell clones. In B-cell clones from the T16H/XSxr mouse, expression of H-Y/D^b exhibited partial X inactivation and only a proportion ( 30%) of the cells were targets for or stimulated H-2D^b-restricted H-Y-specific T cells. In contrast, H-Y eiptopes restricted by H-2^k (H-Y/K^k, H-Y/D^k) and A^b (H-Y/A^b) exhibited no X inactivation. Furthermore, no inactivation of H-Y/D^b, H-Y/A^b, or H-Y^k was observed in the male X/XSxr mouse. These results indicate that the T16H/XSxr female is a mosaic, as a result of the variable spread of X inactivation into the Sxr region. They further suggest that the H-Y antigen recognized in association with H-2^k and H-2D^b class I molecules and A^b class II molecules may be the product of more than one gene.     

Histocompatibility gene mutation rates in the mouse: a 25-year review

 Mutation rates of H2 and non-H2 histocompatibility genes in the mouse are examined over a 25-year period. Detected by skin graft rejections, the mutations were screened in inbred and hybrid mice from a continuously maintained and monitored colony and from a regularly supplied set of mice provided from the National Cancer Institute for monitoring of genetic integrity. Twenty-five H2 mutations were recovered, involving the K, D, L, and Ab loci, as well as over 80 mutations of non-H2 histocompatibility genes. Aside from a single allele at a single locus (H2-K ^b ), the spontaneous mutation rate of H2 class I genes appears to be equivalent to that found estimated for non-H2 histocompatibility genes, and comparable to rates reported for a variety of mouse genes. This is in contrast with previous suggestions that H2 genes mutate at orders of magnitude greater than do “average” mammalian genes. The discrepancy is attributed to the H2-K ^b gene which accounts for over half of all reported H2 mutations and which mutates spontaneously at a rate of 1–2×10^–4 per gene per generation. Furthermore, over half of the spontaneous H2-K ^b mutations result in a single mutant phenotype (the “bg” group) which involve similar changes at amino acid residues 116 and 121. Thus, the high spontaneous mutation rate for H2-K ^b appears to be the exception among major histocompatibility genes, rather than the rule. Received: 18 April 1997 / Revised: 22 May 1997     

Genetic analysis of immune suppression

We have analyzed the genetic control of susceptibility to suppression by 1-J⁺, suppressor-T-cell derived factors (TsF) specific for the synthetic polymer L-glutamic acid⁵⁰-L-tyrosine⁵⁰ (GT). GT-TsF activity was measured as specific inhibition of proliferative responses to GT developed in cultures of lymph-node T cells from mice primed with GT complexed to methylated bovine serum albumin (GT-MBSA). These experiments demonstrated that there is no MHC-encoded genetic restriction between donors and recipients of GT-TsF in suppression of proliferative responses. We have also confirmed the observations that mice of the H-2 ^b, H-2 ^d, and H-2 ^khaplotypes can produce GT-TsF, whereas H-2 ^amice do not, and that H-2 ^b, H-2 ^d, and H-2 ^kmice are sensitive to GT-TsF from all producer strains, whereas H-2 ^bmice are not sensitive to GT-TsF from any strain. Analysis of the effect of GT-TsF on responses by mice bearing recombinant haplotypes suggests that at least two genes are required for susceptibility to GT-TsF and that these genes show coupled complementation.Abbreviations used in this paper GAT random linear terpolymer of L-glutamic acid⁶⁰-L-alanine³⁰-L-tyrosine¹⁰ - GAT-MBSA GAT complexed to methylated bovine serum albumin - GATTsF GAT-specific-T-cell derived suppressor factor - GT random linear copolymer of L-glutamic acid⁵⁰-L-tyrosine⁵⁰ - GT-MBSA GT complexed methylated bovine serum albumin - GT-TsF GT, specific, T-cell derived suppressor factor - ³H-TdR tritiated thymidine - Ir gene immune response gene - MBSA methylated bovine serum albumin - MEM minimal essential media - MHC major histocompatibility complex - PFC plaque-forming cell(s) - PPD purified protein derivative of M. tuberculosis H37Ra     

Class I MHC molecules rather than other mouse genes dictate influenza epitope recognition by cytotoxic T cells

Influenza nucleoprotein (NP) is an important target antigen for influenza A virus cross-reactive cytotoxic T cells (Tc). Here we examine the NP epitope recognized by cloned and polyclonal BALB/c Tc and the genetics of this recognition pattern. We can define NP residues 147–161 as the epitope seen in conjunction with K ^d , the only H-2^d class I responder allele for NP restriction. H-2 ^d /H-2 ^b F₁ mice (C57BL × DBA/2) primed by influenza infection lyse only H-2^d target cells treated with peptide 147–161 while H-2^b targets are recognized only after treatment with NP residues 365–379 (previously found to be recognized by D^b restricted Tc cells). Tc cell recognition of NP peptide 147–161 is entirely dictated by expression of K ^d and not by other B10 or OH background genes of congenic mice. Restriction of a unique NP sequence by each responder class I major histocompatibility complex (MHC) allele suggests that antigen and class I MHC interact for Tc recognition.     

A single nucleotide polymorphism in the<Emphasis Type="Italic"> Emp3</Emphasis> gene defines the H4 minor histocompatibility antigen

Minor histocompatibility antigens (minor H antigen) elicit strong T-cell-mediated responses during both graft rejection and graft versus leukemia (GvL) among MHC-matched individuals (where MHC is major histocompatibility complex). Employing expression-cloning methodology, we have identified a cDNA clone, MI-35, encoding the immunodominant H4^b minor H antigen within the classical mouse H4 complex. The minimal antigenic epitope derived from H4^b presented on K^b class I MHC is SGIVYIHL (SYL8) and the polymorphism is due to CT nucleotide modification in p3 resulting in the change of threonine (ACT) to isoleucine (ATT). The results presented here demonstrate that amino acid variation in the allelic epitopes results in the low abundance of H4^a peptide. The differential peptide copy number resulted in an immunodominant cytotoxic T cells (CTL) response directed against H4^b while the anti-B6 response directed against H4^a was easily dominated. These results provide a molecular mechanism for the H4 minor H antigen and suggest a novel mechanism by which alloantigenic disparity caused by conservative amino acid changes can be augmented by posttranslational antigen processing events.     

Hierarchy ofH-2 haplotypes governs inheritance of immune responsiveness to TNP-MSA

Production of indirect TNP-specific plaque-forming cells (PFC) in response to immunization with 2, 4, 6-trinitrophenyl (TNP) conjugated to autogenous mouse serum albumin (MSA) in complete Freund's adjuvant (CFA) is underH-2 control. On the C57BL/10 (B10) background,H-2 ^b andH-2 ^d strains of mice are high responders, whereasH-2 ^a ,H-2 ^k orH-2 ^y2 strains yield low levels of indirect TNP-specific PFC. An unusual pattern of inheritance has been revealed in B10 congenic mice: high responsiveness controlled byH-2 ^b is inherited recessively, while high responsiveness controlled byH-2 ^d is inherited dominantly. On the C3H and A strain backgrounds, high responsiveness controlled byH-2 ^b is partially recessive;H-2 ^b /H-2 ^a F₁ mice respond with 20%-40% of the high responderH-2 ^b response. Yet, high responsiveness controlled by theH-2 ^d haplotype remains dominant on the C3H background. A hierarchy of haplotypes in order of decreasing immune responsiveness to TNP-MSA is evident as follows:H-2 ^d >H-2 ^b >H-2 ^k ,H-2 ^a orH-2 ^y2 . The unusual patterns of inheritance in the TNP-MSA system reveal graded regulation of responsiveness attributable to bothH-2 and non-H-2 genes.     

MHC recognition by clones of Mls specific T-lymphocytes

To test whether M1s determinants, like other non-MHC or nominal antigens, are recognized by T-cells in association with H-2 determinants, the in vitro proliferative responses of T-cell lines and clones were studied. Lines and clones were prepared by soft agar cloning (B10.BR x BALB/c)F₁ (H-2^k/H-2^d, M1s^b/M1s^b) T-cells responding in a primary MLR to AKD2F1 (H-2^k/H-2^d, M1s^a/M1s^a) stimulator cells. All the T-cell clones obtained could respond equally well in a proliferative assay to the Mls^a determinant in association with the H-2 haplotype of either parent, i. e., DBA/2 (H-2^d, M1s^a), and AKR (H-2^k, M1s^a) both stimulated equally well. When the T-cell lines and clones were screened against stimulators from recombinant inbred (RI) strains, it became apparent that strains exhibiting the H-2^b, M1s^a genotype stimulated poorly or not at all. This shows that the T-cell response to M1s^a involves MHC recognition, and raises the possibility that the response to M1s^a can involve recognition of H-2 specificities shared between the H-2 ^k and H-2 ^d haplotypes.Abbreviations used in this paper MHC major histocompatibility complex - MLC mixed lymphocyte culture - IL-2 interleukin 2 - Con A concanavalin A - RI recombinant inbred Howard Hughes Medical Institute     

Isoforms of the nonclassical class I MHC antigen H2-Q5 are enriched in brain and encode Qdm peptide

Although the human nonclassical class Ib major histocompatibility complex (Mhc) locus, HLA-G, is known to act as an immune suppressor in immune-privileged sites, little is currently known regarding participation of the rodent class Ib Mhc in similar pathways. Here, we investigated the expression properties of the mouse nonclassical Mhc H2-Q5 ^k gene, previously detected in tumors and tissues associated with pregnancy. We find that H2-Q5 ^k is alternatively spliced into multiple novel isoforms in a wide panel of C3H tissues. Unlike other known class I MHC, it is most highly transcribed in the brain, where the classical class Ia Mhc products are scarce. The truncated isoforms are selectively enriched in sites of immune privilege and are translated into cell surface proteins in neural crest-derived transfected cells. Furthermore, we present data supporting a model whereby Q5^k isoforms serve an immune-protective role by donating their Qdm leader peptide to Qa-1, in a pathway homologous to the HLA-G leader fragment binding HLA-E and inhibiting CD94/NKG2A-positive cytotoxic cells. In addition, we report a previously unknown homolog of H2-Q5 ^k in the C57BL/6 mouse, which encodes Qdm, but is transcribed solely into noncanonical isoforms. Collectively, these studies demonstrate that H2-Q5 ^k , and its homologous class I-like H2 ^b gene may play tissue-specific roles in regulating immune surveillance.     

Structural and genetic properties of the Eb recombinational hotspot in the mouse

The Eb gene of the mouse contains a recombinational hotspot which plays a predominant role in meiotic crossing-over within the I region of the mouse major histocompatibility complex (MHC). The nucleotide sequences of five recombinants derived from H-2 ^k /H-2 ^b heterozygotes at the Eb locus placed the sites of recombination in each recombinant haplotype within a 2.9 kilobase (kb) segment located fully within the second intron of the Eb gene. Further resolution of the crossover sites was not possible since the nucleotide sequences of the parental and recombinant haplotypes are identical within this segment. The molecular characterization of these five recombinants considered in conjunction with three previously reported intra-Eb recombinants indicates that there are at least two distinct sites of recombination within the Eb recombinational hotspot. In a related study, an examination of the nucleotide sequence of the H-2 ^p allele of the Eb gene revealed a major genetic rearrangement in the 5' half of the intron in this haplotype. A 597 base pair (bp) nucleotide sequence found in the H-2 ^p haplotype is replaced by a 1634 bp segment found in the H-2 ^b and H-2 ^k haplotypes. Sequence analysis of this 1634 bp segment shows strong nucleotide sequence similarity to retroposon long terminal repeat (LTR), env, and pol genes indicating that this segment of the second intron has evolved through retroposon insertion. The location of these retroposon sequences within the 2.9 kb recombination segment defined by the five H-2 ^k /H-2 ^b recombinant haplotypes suggests a possible relationship between these retroviral elements and site-specific recombination within the second intron of the Eb gene. Offprint requests to: H. C. Passmore     

Mapping of thyroglobulin epitopes: Presentation of a 9mer pathogenic peptide by different mouse MHC class II isotypes

We have previously reported that the 17mer thyroglobulin (Tg) peptide TgP1 (a.a. 2495–2511) induces experimental autoimmune thyroiditis (EAT) inH-2 ^k mice, a process requiring expression ofE ^k genes, and inH-2 ^S mice that lack functional E molecules. To test whether this apparent discrepancy was due to recognition of distinct TgP1 determinants in each strain, we mapped in this study minimal T -cell epitopes within TgP1 and examined their pathogenicity in C3H (H-2 ^k) or SJL (H-2 ^S) mice. Truncation analysis using TgP1-specific, CD4+ hybridomas from C3H mice identified two overlapping determinants, (2496-2504) and (2499–2507), that were restricted by the E^k and A^k molecules, respectively. Subsequent challenge of C3H and SJL mice with these 9mer peptides revealed that the Ekrestricted (2496–2504) determinant elicited EAT and specific proliferative LNC responses in both strains, suggesting recognition in the context of A^s, since this is the only class II molecule expressed in SJL mice. This was further confirmed by blocking of the proliferative LNC response by an As-specific monoclonal antibody. In contrast, the A^k-restricted (2499–2507) determinant induced weak EAT and no proliferative LNC responses in either strain. These data 1) delineate the 9mer (2496–2504) peptide as a minimal Tg T-cell epitope with direct pathogenic potential in mice and 2) highlight the use of nonisotypic MHC class II molecules for the presentation of this peptide in mice of differentH-2 haplotypes.