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Interest in improving the speed of DNA analysis via capillary electrophoresis has led to efforts to integrate DNA amplification into microfabricated devices. This has been difficult to achieve since the thermocycling required for effective polymerase chain reaction (PCR) is dependent on an effective contact between the heating source and the PCR mixture vessel. We describe a noncontact method for rapid and effective thermocycling of PCR mixtures in electrophoretic chip-like glass chambers. The thermocycling is mediated through the use of a tungsten lamp as an inexpensive infrared radiation source, with cooling effected with a solenoid-gated compressed air source. With temperature ramping between 94 and 55 degrees C executed in glass microchambers as rapidly as 10 degrees C/s (heating) and 20 degrees C/s (cooling), cycle times as fast as 17 s could be achieved. Successful genomic DNA amplification was carried out with primers specific for the beta-chain of the T-cell receptor, and detectable product could be generated in a fraction of the time required with commercial PCR instrumentation. The noncontact-mediated thermocycling format was not found to be restricted to single DNA fragment amplification. Application of the thermocycling approach to both quantitative competitive PCR (simultaneous amplification of target and competitor DNA) and cycle sequencing reactions (simultaneous amplification of dideoxy terminated fragments) was successful. This sets the stage for implementing DNA thermocycling into a variety of microfabricated formats for rapid PCR fragment identification and DNA sequencing. more...

Minor histocompatibility Ags (HA) play prominent roles in stimulating allograft rejection and are recognized by CTLs that mediate this process. However, there is no information on the diversity of TCRs that are specific for single minor histocompatibility Ag peptides and expressed by CTLs in vivo. We have used the technique of spectratyping to study the diversity of Vbeta usage and beta complementarity-determining region 3 (CDR3) length of TCRs expressed by CTL-infiltrating skin allografts expressing the immunogenic H4 peptide during the process of rejection. Spectratyping revealed overall reduction in diversity of both Vbeta usage and CDR3 length, with sequential application of primary, second-set, and third-set H4-incompatible grafts. This dissection of the array of beta-chains expressed by graft-infiltrating CTLs allowed the direct sequencing of individual beta-chain PCR products. Beta CDR3s were characterized by a net negative charge, as we have observed previously with CDR3s expressed by H4-specific CTL clones selected in vitro. Identical and closely related CDR3 amino acid sequences could be identified that were shared by TCRs that 1) utilized different Vbeta genes, 2) derived from different mice, or 3) derived from different sequential sets of allografts on individual mice. Furthermore, a number of CDR3 sequences expressed by graft-infiltrating CTLs were identical or closely related to sequences we have identified previously in in vitro selected CTL clones that were specific for the H4 peptide. more...

Minor histocompatibility Ags (HA) and their associated immunogenic peptides provide a formidable barrier for successful transplantation, but there is virtually no information regarding the diversity of minor HA-specific TCRs. We have investigated the diversity of alpha- and beta-chains in TCRs specific for the CTT-1 and CTT-2 peptides that are immunodominant in CTL responses to multiple minor HA. CTLs were cloned after in vitro stimulation, and alpha- and beta-chain transcripts were amplified and sequenced to identify utilized V genes, complementarity-determining regions 3 (CDR3s), and joining region genes. Twenty-one unique CTT-2-specific TCRs were identified in 31 clones, and 22 CTT-1-specific TCRs were identified in 29 clones. A relatively limited number of V beta subfamilies were represented in these panels of TCRs, with Vbeta 5 and V beta8 genes expressed in multiple TCRs in each panel. Similar diversity was observed with V alpha usage, and V alpha4 subfamily usage was more prominent in CTT-2-specific TCRs than in CTT-1-specific TCRs. Neither alpha nor beta CDR3 regions exhibited prominent motifs or length restriction. However, CTT-1- and CTT-2-specific beta CDR3 regions included an excess of negatively over positively charged residues that were bimodally distributed among CDR3 positions. In fact, 50% of CTT-1-specific CDR3 regions included two negative charges separated by three to five amino acids. Despite the similarities in net charge of beta CDR3 regions, TCRs specific for these two minor HA peptides are relatively diverse and would expectedly withstand attempts at anti-TCR Ab-mediated immunosuppression. more...

Minor histocompatibility (H) antigens stimulate in vivo rejection of allografts compatible for the MHC and are recognized by CTLs in short term in vitro assays. CTLs generated by the in vivo priming and in vitro boosting of mice with spleen cells incompatible for multiple minor H Ags are specific for a limited number of dominant Ags (peptides). We have addressed the issue of the identity of the Ags that stimulate rejection of solid tissue allografts vs the H Ags recognized by CTLs. C57BL/6 recipients reject skin grafts from BALB.B and CXB recombinant inbred strains with no significant differences in survival times. Primary grafts from these same strains prime for accelerated rejection of second-set grafts from all CXB strains regardless of inheritance of dominant Ags detected by CTLs. However, CTLs primed by these allografts and boosted in vitro do not exhibit ranges of reactivity with lymphoblast targets from CXB strains predicted by in vivo rejection, suggesting that CTLs primed by multiple Ag-incompatible skin grafts do not recognize all H Ags that stimulate allograft rejection. The fact that first- and second-set BALB.B skin grafts prime for accelerated rejection of 11/13 congenic strains defining single BALB/c minor H Ags indicates that multiple H Ags stimulate allograft rejection. However, CTLs from C57BL/6 mice primed with BALB.B grafts and boosted with BALB.B spleen cells recognize only the H4 Ag from this panel of congenic strains. Limited diversity of the CTL response is corroborated by the recognition of three minor H peptides (including H4 as the most prominent) eluted from Kb molecules from a BALB.B tumor by these CTLs. These results indicate that CTLs recognize only a limited number of Ags operative in vivo and do not accurately reveal the complexity of the antigenic specificity of the in vivo response. more...

Minor histocompatibility (H) Ags, encoded by autosomal and sex-linked genes, have classically been identified by the cytolytic T lymphocyte response to class I-bound minor H Ags. A limited number of studies have identified minor H Ags (defined by congenic strains) that stimulate Th cells. We have selected a panel of minor H Ag-specific, CD4+ Th hybrids by the fusion of a BW5147 variant with a C57BL/6 anti-BALB.B T cell line. Studied hybrids secrete IL-2 following stimulation with BALB.B spleen cells and stimulation is blocked with anti-I-Ab Ab. These Th hybrids recognize a minimum of six different Ags (helper T target (HTT)) encoded by independently segregating genes as defined by stimulator cells from 11 CXB recombinant inbred strains. The CXB strain distribution patterns of detected Ags did not match the distribution pattern of the BALB/c MTV6 viral genome indicating that none of the detected Ags were known viral superantigens. Four tested HTT Ags appeared not to be acquired by stimulators as exogenous Ags as BALB.B spleen cells but not a mixture of BALB/c spleen cells and C57BL/6 APC stimulated IL-2 production by the respective Th hybrids. To determine whether Th hybrids recognized peptides bound to I-Ab, I-Ab molecules were immunoprecipitated from the LB27.4 cell line that stimulated the A6.5.5 hybrid (HTT-4 specific). Peptides were extracted from I-Ab molecules, ultrafiltered, and separated by HPLC. A single fraction (with flanking peaks of lower activity) sensitized syngeneic APC to stimulate A6.5.5 hybrid cells; sensitization was observed at pH 5.0 but not pH 7.0, consistent with HTT-4 peptide:I-Ab binding occurring in an endosomal compartment. These observations indicate that at least one class II-restricted minor H Ag is recognized by Th cells as a peptide bound to class II molecules. more...

Minor histocompatibility (H) Ag are recognized in the context of MHC class I (K/D) molecules and can constitute a strong barrier to tissue transplantation. The products encoded by the MHC (H-2 in mice) have been shown recently to be Ag-presenting molecules that bind foreign and self peptides in their peptide binding sites. Different class I molecules preferentially bind different arrays of endogenous peptides for presentation to CTL. Previous studies showed that the Kb-restricted CTL response to one minor histocompatibility Ag, H-4, varied in different Kb mutants. One possible basis for this variation might be that the response is regulated by the level of binding of an H-4 peptide by class I molecules. To analyze this possibility, we initiated studies to identify the H-4 peptide that is included in the array of self peptides. The complete mixture of peptides eluted from Kb molecules of H-4+ tumor cells was able to sensitize H-4- targets for lysis by H-4-specific CTL. The presence of a specific H-4 peptide was confirmed when the radiolabeled peptide mix eluted from Kb molecules was separated by HPLC. Only one peak in the profile of H-4+ tumor peptides was capable of sensitizing H-4- targets for lysis by H-4-specific CTL; this active peak was absent in H-4 mutant cells. Peptide mixtures eluted from a panel of Kb mutant molecules extracted from H-4+ lymphocytes varied in their capacities to sensitize targets for H-4-specific lysis and the relative sensitization correlated with the demonstrated capacity of mutant mice to generate H-4-specific CTL. Therefore, the highly specific genetic control of the T cell response to the H-4 minor histocompatibility Ag appears to be due to the differential binding of an H-4 peptide by class I molecules. more...

Immunodominance controls the generation of CTL in the C57BL/6By (B6) anti-BALB.B H-2b-matched strain combination. Despite the potential of responding to numerous individual minor histocompatibility (H) Ag on BALB.B APC, the focus of the CTL response is largely specific for only a limited number of target Ag. These minor H Ag could be distinguished by their differential expression on a panel of target cells from the CXB recombinant inbred strains, the E, G, I, J, and K (all H-2b), which express different composites of the original BALB minor H Ag. A hierarchy was observed in which first-order immunodominant Ag were present on both CXBK and CXBG cells, whereas second-order dominant Ag were found on CXBE, CXBJ, and CXBI cells. To test whether immunodominance also plays a role in the development of lethal graft-vs-host disease (GVHD) directed to multiple minor H Ag, B6 T cells were transplanted along with T cell depleted bone marrow, to irradiated (825 rad) recipients of either the BALB.B or CXB recombinant inbred strains. The results indicate that a hierarchy of immunodominance does exist in GVHD, but it differs from that predicted from the in vitro CTL studies. GVHD was observed in BALB.B, CXBE, CXBI, and CXBJ recipients, but not in CXBG and CXBK recipients. Presensitization of B6 donor mice to CXBG or CXBK splenocytes 3 wk before transplant did not significantly increase the overall GVHD potential in the corresponding CXBG or CXBK recipients. Evidence for second-order immunodominance was provided by the transfer of CXBE T cells and ATBM to irradiated CXBG and BALB.B recipients with resultant, potent GVHD. more...

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. more...

Perforin, Fas ligand (FasL), and tumor necrosis factor-alpha (TNF-alpha) have been implicated in cytolytic T lymphocyte (CTL) effector function. However, the relative roles of these effector molecules in allograft rejection are unclear, and there has been no rigorous quantitation of transcription of the respective genes throughout the period from transplantation to rejection.We orthotopically transplanted mouse tail skin allografts and estimated the numbers of transcripts of these genes expressed by graft-infiltrating T cells with rigorous quantitative, competitive reverse transcribed PCR (QC-RT-PCR) that enabled the comparison of transcription of different genes.Perforin and FasL mRNA levels correlated closely with the rejection of allografts by normal hosts over the 4 days preceding rejection. Antibody-mediated depletion of host CD4+ T cells retarded perforin transcription and significantly suppressed FasL transcription, suggesting FasL was not required for allograft rejection. TNF-alpha transcription was the highest of these genes in this time period, but these levels were dwarfed by TNF-alpha transcription at 24 hr posttransplant when transcription in both autografts and allografts was 30-fold higher than in allografts on the day before rejection. Elimination of the function of these single or paired genes through genetic mutation or antibody treatment had no significant effect on the speed of rejection.The levels of perforin and FasL transcription appeared to be related to the process of allograft rejection in normal hosts. However, TNF-alpha transcription was highest during the posttransplant period suggesting that the principal role of TNF-alpha is in wound-healing rather than the effector phase of rejection. more...

Minor histocompatibility Ags (HA) play prominent roles in stimulating allograft rejection and are recognized by CTLs that mediate this process. However, there is no information on the diversity of TCRs that are specific for single minor histocompatibility Ag peptides and expressed by CTLs in vivo. We have used the technique of spectratyping to study the diversity of Vbeta usage and beta complementarity-determining region 3 (CDR3) length of TCRs expressed by CTL-infiltrating skin allografts expressing the immunogenic H4 peptide during the process of rejection. Spectratyping revealed overall reduction in diversity of both Vbeta usage and CDR3 length, with sequential application of primary, second-set, and third-set H4-incompatible grafts. This dissection of the array of beta-chains expressed by graft-infiltrating CTLs allowed the direct sequencing of individual beta-chain PCR products. Beta CDR3s were characterized by a net negative charge, as we have observed previously with CDR3s expressed by H4-specific CTL clones selected in vitro. Identical and closely related CDR3 amino acid sequences could be identified that were shared by TCRs that 1) utilized different Vbeta genes, 2) derived from different mice, or 3) derived from different sequential sets of allografts on individual mice. Furthermore, a number of CDR3 sequences expressed by graft-infiltrating CTLs were identical or closely related to sequences we have identified previously in in vitro selected CTL clones that were specific for the H4 peptide. more...

Minor histocompatibility Ags (HA) play prominent roles in stimulating allograft rejection and are recognized by CTLs that mediate this process. There is limited information regarding the sequences of minor HA peptides and the diversity of minor HA-specific TCRs. In the case of the male minor HA (HY), a peptide presented by H2Db molecules has been sequenced. We have used spectratyping to study the diversities of Vbeta usage and beta complementarity-determining region 3 (CDR3) lengths of TCRs expressed by CTLs that infiltrate HY-disparate skin allografts during rejection. Spectratyping of RNA from second- and third-set male allografts on CD4-depleted, female recipients showed a reduction in Vbeta usage and beta CDR3 length diversity with prominent representation of Vbeta8 genes. CDR3 sequences, as a group, were characterized by net negative charges resulting from negatively charged residues at positions 5-6 and 10-11. The effects of in vivo anti-Vbeta8 Ab treatment on rejection of second-set male allografts were investigated. This Ab treatment had no effect on allograft rejection time and resulted in increased Vbeta7 usage in recipients with complete Vbeta8 depletion. More interestingly, the net charges of beta CDR3s derived from Vbeta8-depleted recipients were altered by the inclusion of positively charged and polar residues at positions 4-6. These results indicate that Vbeta-specific T cell depletion has no effect on HY-disparate allograft survival, but it alters Vbeta usage and changes the characteristics of beta CDR3s that facilitate class I:peptide recognition. more...

Indirect presentation of minor histocompatibility antigens (HA) as revealed by cross-priming of H2 heterozygous recipients effectively primes minor HA-specific cytolytic T lymphocytes (CTLs). However, it is not known if indirect priming generates CTLs specific for the same set of immunodominant minor HA recognized by CTLs primed by direct spleen cell injections.In order to indirectly prime minor HA-specific CTLs, we implanted (C57BL/6 x B6.C-H2d)F1 recipients with BALB.B and BALB/c splenocytes loaded into immunoisolation devices that effectively preclude direct donor:host contact. Responder spleen cells from these recipients were stimulated in vitro to expand BALB.B- and BALB/c-specific CTLs to reveal classical cross-priming.Tests of CTL specificity using (1) CXB recombinant inbred strain targets that express different arrays of BALB/c minor HA and (2) high-performance liquid chromatography fractions of peptides from BALB.B Kb and Db molecules revealed that anti-BALB.B CTLs were specific for two previously identified dominant peptides, CTT-2 and CTT-5, presented by Kb molecules. Variation of responders and priming cells resulted in CTL responses to additional dominant peptides that had been identified previously with CTLs generated by direct priming with spleen cell injections. Indirect priming was not limited to this set of peptides recognized by CTLs in vitro because devices loaded with cells devoid of the CTL-detected peptides primed for accelerated skin allograft rejection.Indirect presentation of minor HA in vivo stimulates the generation of CTLs specific for a subset of dominant minor HA peptides recognized by CTLs primed by direct presentation. more...

C57BL/6 (B6) mice generate cytolytic T lymphocytes (CTLs) to a limited number of immunodominant cytotoxic T cell target (CTT) antigens and associated peptides when primed with H2-matched BALB.B spleen cells, despite multiple minor histocompatibility antigen (HA) differences. We previously showed that these CTLs recognize four Kb-bound minor HA peptides derived from CTT antigens. Here, we describe the identification of Db-bound minor HA peptides recognized by B6 anti-BALB.B CTLs.Peptides were extracted from Db molecules immunoprecipitated from lysates of T lymphoblasts from BALB.B mice and mice from the CXB recombinant inbred strains that express H2b and segregate minor HA from BALB/c and B6. Peptides were separated by reverse-phase high-performance liquid chromatography and tested for sensitization of targets for lysis by CTLs specific for BALB.B and the CXB strains.B6 anti-BALB.B CTLs recognized a single Db-bound peptide whose distribution in CXB strains matched that of the previously reported CTT-1 minor HA. An additional Db-bound peptide (CTT-7) was recognized by B6 anti-CXBG CTLs. CTT-1 was expressed by independently derived inbred mouse strains that express H2b. CTT-1 was recognized by B6 CTLs specific for these inbred strains, except for the LP and 129 strains that stimulated CTL specific for the CTT-8 peptide expressed by these two strains.These results demonstrate that B6 CTLs primed and boosted with multiple minor HA recognize a maximum of two minor HA peptides regardless of the strain of origin of H2b-matched stimulating lymphoid cells. more...

Minor histocompatibility Ags (HA) and their associated immunogenic peptides provide a formidable barrier for successful transplantation, but there is virtually no information regarding the diversity of minor HA-specific TCRs. We have investigated the diversity of alpha- and beta-chains in TCRs specific for the CTT-1 and CTT-2 peptides that are immunodominant in CTL responses to multiple minor HA. CTLs were cloned after in vitro stimulation, and alpha- and beta-chain transcripts were amplified and sequenced to identify utilized V genes, complementarity-determining regions 3 (CDR3s), and joining region genes. Twenty-one unique CTT-2-specific TCRs were identified in 31 clones, and 22 CTT-1-specific TCRs were identified in 29 clones. A relatively limited number of V beta subfamilies were represented in these panels of TCRs, with Vbeta 5 and V beta8 genes expressed in multiple TCRs in each panel. Similar diversity was observed with V alpha usage, and V alpha4 subfamily usage was more prominent in CTT-2-specific TCRs than in CTT-1-specific TCRs. Neither alpha nor beta CDR3 regions exhibited prominent motifs or length restriction. However, CTT-1- and CTT-2-specific beta CDR3 regions included an excess of negatively over positively charged residues that were bimodally distributed among CDR3 positions. In fact, 50% of CTT-1-specific CDR3 regions included two negative charges separated by three to five amino acids. Despite the similarities in net charge of beta CDR3 regions, TCRs specific for these two minor HA peptides are relatively diverse and would expectedly withstand attempts at anti-TCR Ab-mediated immunosuppression. more...

Minor histocompatibility (H) antigens stimulate in vivo rejection of allografts compatible for the MHC and are recognized by CTLs in short term in vitro assays. CTLs generated by the in vivo priming and in vitro boosting of mice with spleen cells incompatible for multiple minor H Ags are specific for a limited number of dominant Ags (peptides). We have addressed the issue of the identity of the Ags that stimulate rejection of solid tissue allografts vs the H Ags recognized by CTLs. C57BL/6 recipients reject skin grafts from BALB.B and CXB recombinant inbred strains with no significant differences in survival times. Primary grafts from these same strains prime for accelerated rejection of second-set grafts from all CXB strains regardless of inheritance of dominant Ags detected by CTLs. However, CTLs primed by these allografts and boosted in vitro do not exhibit ranges of reactivity with lymphoblast targets from CXB strains predicted by in vivo rejection, suggesting that CTLs primed by multiple Ag-incompatible skin grafts do not recognize all H Ags that stimulate allograft rejection. The fact that first- and second-set BALB.B skin grafts prime for accelerated rejection of 11/13 congenic strains defining single BALB/c minor H Ags indicates that multiple H Ags stimulate allograft rejection. However, CTLs from C57BL/6 mice primed with BALB.B grafts and boosted with BALB.B spleen cells recognize only the H4 Ag from this panel of congenic strains. Limited diversity of the CTL response is corroborated by the recognition of three minor H peptides (including H4 as the most prominent) eluted from Kb molecules from a BALB.B tumor by these CTLs. These results indicate that CTLs recognize only a limited number of Ags operative in vivo and do not accurately reveal the complexity of the antigenic specificity of the in vivo response. more...

We addressed the contributions of persistent virus infection and immune cells to the pathogenesis of Theiler's virus-induced demyelination, a model for human multiple sclerosis. We developed a model involving the transfer of spleen cells into immunodeficient C.B-17-scid (SCID) mice, which normally die of overwhelming virus encephalitis without demyelination when infected with Theiler's virus. Adoptive transfer of nonimmune spleen cells from BALB/c mice into SCID mice resulted in the survival of all mice. However, these mice developed extensive demyelination and virus Ag/RNA persistence in the spinal cord white matter. The most demyelination was observed when mice received an intermediate number of spleen cells (1.8-7.5 x 10(6)), whereas too few cells (0.5 x 10(6)) did not ameliorate the SCID phenotype, and too many cells (30 x 10(6)) resulted in almost complete viral clearance with minimal demyelination. Adoptive transfer of spleen cells depleted of either CD4+ or CD8+ T cells produced vacuolar demyelination associated with virus persistence. In contrast, reconstitution with both CD4+ and CD8+ T cells produced less severe demyelination and partial clearance of virus. These experiments support the hypothesis that demyelination is the result of a balance between persistent virus infection and immune injury mediated by either CD4+ or CD8+ T cells. more...

C57BL/6 mice preferentially generate cytolytic T lymphocytes (CTL) to a limited number of immunodominant minor antigens and associated immunogenic peptides when primed with H2-matched Balb.B spleen cells despite multiple minor histocompatibility (H) antigen differences. We have examined the complexity of dominant H antigens recognized by these CTLs to estimate the number of peptides associated with single antigens. Peptides eluted from Kb molecules of lymphoblasts from Balb.B and CXB recombinant inbred (RI) strains were tested for sensitization of RMA-S cells for lysis by short-term C57BL/6 CTL lines specific for Balb.B and CXB strains. Anti-Balb.B CTLs recognized four Kb-bound peptides; subsets of these peptides were recognized by anti-CXB CTLs when tested with peptides from the respective CXB strains. Single peptides segregated independently among the CXB strains, confirming that single peptides were encoded by independently segregating alleles. These peptides were expressed in diverse inbred mouse strains and were recognized preferentially by C57BL/6 CTLs stimulated by different inbred mouse strains. This set of peptides was subclassified by their capacity to sensitize targets when presented in unfractionated mixtures of Kb-bound peptides. The peptide associated with the previously classified dominant CTT-2 antigen was the only peptide to strongly sensitize RMA-S cells for lysis under these conditions. These results suggest that dominant peptides have a wide strain distribution and may have a distinct advantage over dominated peptides in binding to class I molecules and/or in presentation to CTLs. more...

The tassel-eared squirrel, Sciurus aberti, is separated into six subspecies which occupy restricted and comparable habitats in ponderosa pine forests in the south-western United States and Mexico. These forests and squirrel populations are currently isolated by large arid areas and, as such, S. aberti appears to offer an example of incipient speciation. We sequenced the complete mitochondrial cytochrome b gene to construct a molecular phylogeny for S. aberti and to determine whether subspecific genetic structure and geographic patterns are correlative. Twenty alleles were identified among 612 squirrels throughout the species' range. Nucleotide divergence between alleles ranged from 0.009 to 0.0233, whereas average sequence divergence between S. aberti and an outgroup species, Sciurus niger, was 0.1823. Neighbor-joining and maximum parsimony analyses identified three major genetic assemblages composed of the following subspecies groups: (1) barberi and durangi; (2) aberti and kaibabensis; and (3) ferreus. The chuscensis samples were the only population with divergent sequences; one sequence was identical to an aberti sequence and a second unique sequence clustered with the ferreus sequences. The presence of divergent sequences in the chuscensis population, coupled with its central geographic position between aberti and ferreus, suggests a relatively recent influx of aberti mtDNA. Estimates of the times separating sequences in subspecies within different groups ranged from 0.94 to 1.52 x 10(6) years, based on a rate estimate of 7.15 x 10(-9) substitutions/year/site. The limited divergence observed between (1) aberti and kaibabensis as well as (2) barberi and durangi suggests relatively recent separations of subspecies within each assemblage. In fact, populations defined morphologically and geographically as ferreus exhibited greater sequence divergence than the aforementioned groups, identifying ferreus as the subspecies with the greatest genetic substructuring. The levels of cytochrome b divergence observed for the three distinct groups argues against a significant role for late Pleistocene glaciation in dispersal of this particular species; however, the proposed intermixing of aberti and chuscensis populations may well have been associated with such glacial events. Nucleotide diversity within subspecies ranked chuscensisabertibarberi approximately kaibabensis approximately ferreus subpopulations; the relatively high level of diversity of chuscensis samples likely results from the apparent introgression of an aberti haplotype. The comparative levels of diversity in the aberti, barberi, kaibabensis, and ferreus sample populations do not correlate with respective habitat size (and presumably population size), suggesting that relatively recent forces, e.g., glaciation and inconsistent timber harvests, may have influenced diversity in these populations without apparent alterations in population size. more...

Minor histocompatibility (H) Ags, encoded by autosomal and sex-linked genes, have classically been identified by the cytolytic T lymphocyte response to class I-bound minor H Ags. A limited number of studies have identified minor H Ags (defined by congenic strains) that stimulate Th cells. We have selected a panel of minor H Ag-specific, CD4+ Th hybrids by the fusion of a BW5147 variant with a C57BL/6 anti-BALB.B T cell line. Studied hybrids secrete IL-2 following stimulation with BALB.B spleen cells and stimulation is blocked with anti-I-Ab Ab. These Th hybrids recognize a minimum of six different Ags (helper T target (HTT)) encoded by independently segregating genes as defined by stimulator cells from 11 CXB recombinant inbred strains. The CXB strain distribution patterns of detected Ags did not match the distribution pattern of the BALB/c MTV6 viral genome indicating that none of the detected Ags were known viral superantigens. Four tested HTT Ags appeared not to be acquired by stimulators as exogenous Ags as BALB.B spleen cells but not a mixture of BALB/c spleen cells and C57BL/6 APC stimulated IL-2 production by the respective Th hybrids. To determine whether Th hybrids recognized peptides bound to I-Ab, I-Ab molecules were immunoprecipitated from the LB27.4 cell line that stimulated the A6.5.5 hybrid (HTT-4 specific). Peptides were extracted from I-Ab molecules, ultrafiltered, and separated by HPLC. A single fraction (with flanking peaks of lower activity) sensitized syngeneic APC to stimulate A6.5.5 hybrid cells; sensitization was observed at pH 5.0 but not pH 7.0, consistent with HTT-4 peptide:I-Ab binding occurring in an endosomal compartment. These observations indicate that at least one class II-restricted minor H Ag is recognized by Th cells as a peptide bound to class II molecules. more...

T cell responses to multiple minor histocompatibility antigens are governed by the complex phenomenon of immunodominance, as demonstrated clearly in the generation of CTL in the C57BL/6By (B6) anti-BALB.B strain combination. Immunodominance has also been found in lethal graft-versus-host disease (GVHD) responses directed to BALB.B minor histocompatibility antigens, after transplantation of B6 T cells and T cell-depleted bone marrow to irradiated (825 cGy) recipients of either the BALB.B or CXB recombinant inbred strains. However, previous results indicated that the hierarchy of immunodominance in GVHD differed from that predicted from the in vitro CTL studies. Lethal GVHD was observed in BALB.B, CXBE, CXBI, and CXBJ recipients, but not in CXBG and CXBK recipients, the latter 2 strains expressing immunodominant antigens for CTL generation. A major hypothesis to account for these discordant observations is that GVHD reflected the activity of CD4+, but not CD8+, T cell subsets, in contrast to only the in vitro cytolytic potential of CD8+ T cells. Therefore, the current study was undertaken to assess the GVHD potential of both T cell subsets in the B6--BALB.B, CXBE, CXBI, and CXBJ strain combinations and to analyze the early GVHD responses in the B6--CXBG and CXBK strains. The results indicate that lethal GVHD responses in the B6--BALB.B combination can be mediated by either CD4+ T cells or CD4-dependent CD8+ T cells; a similar observation was made with the B6--CXBI strain combination. Lethal GVHD in the B6--CXBE strain combination is mediated only by CD4-dependent CD8+ T cells, whereas GVHD in the B6--CXBJ combination involves either CD4+ T cells alone or CD4-independent CD8+ T cells. In the B6--CXBG and CXBK recipients, which do not develop lethal GVHD, the early phases of a GVHD response was detected with involvement of both CD4+ and CD8+ T cells. These results indicate that CD8+ T cells are active at some level in all of the strain combinations tested, that CD4+ T cells do not account for the GVHD immunodominant response in the CXBE recipients, and that the failure to obtain extensive clinical disease in the CXBG and CXBK strains is not due to a lack of a graft-versus-host response. more...

Minor histocompatibility (H) Ag are recognized in the context of MHC class I (K/D) molecules and can constitute a strong barrier to tissue transplantation. The products encoded by the MHC (H-2 in mice) have been shown recently to be Ag-presenting molecules that bind foreign and self peptides in their peptide binding sites. Different class I molecules preferentially bind different arrays of endogenous peptides for presentation to CTL. Previous studies showed that the Kb-restricted CTL response to one minor histocompatibility Ag, H-4, varied in different Kb mutants. One possible basis for this variation might be that the response is regulated by the level of binding of an H-4 peptide by class I molecules. To analyze this possibility, we initiated studies to identify the H-4 peptide that is included in the array of self peptides. The complete mixture of peptides eluted from Kb molecules of H-4+ tumor cells was able to sensitize H-4- targets for lysis by H-4-specific CTL. The presence of a specific H-4 peptide was confirmed when the radiolabeled peptide mix eluted from Kb molecules was separated by HPLC. Only one peak in the profile of H-4+ tumor peptides was capable of sensitizing H-4- targets for lysis by H-4-specific CTL; this active peak was absent in H-4 mutant cells. Peptide mixtures eluted from a panel of Kb mutant molecules extracted from H-4+ lymphocytes varied in their capacities to sensitize targets for H-4-specific lysis and the relative sensitization correlated with the demonstrated capacity of mutant mice to generate H-4-specific CTL. Therefore, the highly specific genetic control of the T cell response to the H-4 minor histocompatibility Ag appears to be due to the differential binding of an H-4 peptide by class I molecules. more...

Immunodominance controls the generation of CTL in the C57BL/6By (B6) anti-BALB.B H-2b-matched strain combination. Despite the potential of responding to numerous individual minor histocompatibility (H) Ag on BALB.B APC, the focus of the CTL response is largely specific for only a limited number of target Ag. These minor H Ag could be distinguished by their differential expression on a panel of target cells from the CXB recombinant inbred strains, the E, G, I, J, and K (all H-2b), which express different composites of the original BALB minor H Ag. A hierarchy was observed in which first-order immunodominant Ag were present on both CXBK and CXBG cells, whereas second-order dominant Ag were found on CXBE, CXBJ, and CXBI cells. To test whether immunodominance also plays a role in the development of lethal graft-vs-host disease (GVHD) directed to multiple minor H Ag, B6 T cells were transplanted along with T cell depleted bone marrow, to irradiated (825 rad) recipients of either the BALB.B or CXB recombinant inbred strains. The results indicate that a hierarchy of immunodominance does exist in GVHD, but it differs from that predicted from the in vitro CTL studies. GVHD was observed in BALB.B, CXBE, CXBI, and CXBJ recipients, but not in CXBG and CXBK recipients. Presensitization of B6 donor mice to CXBG or CXBK splenocytes 3 wk before transplant did not significantly increase the overall GVHD potential in the corresponding CXBG or CXBK recipients. Evidence for second-order immunodominance was provided by the transfer of CXBE T cells and ATBM to irradiated CXBG and BALB.B recipients with resultant, potent GVHD. more...

The immunization of C57BL/6 responder mice with spleen cells from H-2-matched BALB.B donors, which differ by multiple non-H-2 histocompatibility (H) antigens, results in the generation of cytotoxic T lymphocytes (CTL) that are specific for only a limited number of immunodominant antigens. Previous analysis of the genes encoding these dominant antigens has not mapped these genes to any of the non-H-2 H loci defined by congenic strains. It would have been expected that the histogenetic techniques employed for congenic strain selection would have preferentially identified the "strongest" H antigens. Therefore, we have investigated the possibility that immunodominant antigens do not belong to the class of non-H-2 H antigens encoded by genes mapping to H loci defined and mapped by congenic strains. The first experiments were aimed at identifying antigens that were expressed by independently derived inbred strains and were cross-reactive with the immunodominant cytotoxic T cell target (CTT-1) antigen of BALB.B. Strong cross-reaction with the C3H.SW (H-2b) strain was observed; the C3H gene encoding this antigen was mapped with BXH recombinant inbred strains. Contrary to the mapping of the CTT-1 gene to chromosome 1 in BALB.B, the C3H gene was shown to map to either chromosome 4 or chromosome 7. This result indicates that identical, or at least extensively cross-reactive, non-H-2 antigens may be encoded by genes mapping to independently segregating loci in different inbred strains. The tissue distribution of immunodominant antigens was approached by determining the reactivity of CTL specific for these antigens with either lymphoid-derived or fibroblast-derived targets. These CTL effectively lysed lymphoblast and lymphoid tumor targets but did not lyse an SV40-transformed fibroblast line that was shown to be efficiently lysed by CTL specific for non-H-2 H antigens defined by congenic strains. Therefore, it was concluded that immunodominant antigens detected by B6 anti-BALB.B CTL have a restricted tissue distribution in comparison to non-H-2 H antigens defined by congenic strains. The implications of these results for our understanding of the origin and heterogeneity of non-H-2 cell-surface antigen recognized by effector T cells are discussed. more...

Immunization of mice with multiple non-H-2 histocompatibility antigens results in the generation of cytolytic T lymphocytes that are specific for a limited number of immunodominant antigens. The experiments presented in this communication were designed to reveal immunodominance in pairwise combinations of autosomal and sex-linked non-H-2 histocompatibility (H) antigens. Priming and boosting responders with the male antigen, H-Y, paired with the H-4.2, H-7.1, or H-3.1 antigens, resulted in the generation of cytolytic T cells specific for the autosomal H antigens but not the H-Y antigen. Furthermore, co-immunization and boosting of C57BL/6 female responder spleen cells with BALB.B male cells resulted in the generation of cytolytic T cells specific for the BALB.B immunodominant antigens but not H-Y. No dominance was observed in H-4-plus H-7-incompatible combinations. Co-immunization of three different H-3 congenic strains with H-3.1 plus H-Y demonstrated that an efficient anti-H-3.1 T cell response is required for observing H-3.1 immunodominance over H-Y. Co-expression of H-3.1 and H-Y on the same priming and boosting cells was required for immunodominance. In fact, immunization with H-3.1 and H-Y presented on different cells resulted in normal generation of H-Y-specific cytolytic T cells, but no generation of H-3.1-specific cytolytic T cells resulted unless H-Y-specific cells were stimulated in the mixed lymphocyte cultures. These observations suggest that in vitro T cell responses to paired, non-H-2 H antigens may be independent, competitive, or synergistic, depending on the identity of the antigens and the priming and boosting conditions. more...

The RT1m haplotype of MNR rats has been suggested to be a recombinant RT1 haplotype inheriting RT1.A (class I) alleles from RT1a (DA) and RT1.B (class II) alleles from RT1c (AUG). Additional serologic and biochemical assays, however, have suggested that RT1m and RT1c share a single identical RT1.B molecule, although differing in the expression of the second RT1.B molecule. To resolve this contradiction, RT1.B class II molecules, comparable to I-A and I-E molecules in mice, expressed by the RT1c and RT1m haplotypes were immunoprecipitated by cross-reactive mouse anti-Ia antibodies and were compared by two-dimensional gel electrophoresis and by high pressure liquid chromatographic separation of tryptic peptides. Respective subunits expressed by the two haplotypes co-migrate on two-dimensional gels and have identical tryptic peptide maps. The results at the protein level were confirmed at the DNA level by Southern blot analysis of MNR and AUG genomic DNA. Identical restriction fragments associated with the RT1m and RT1c haplotypes hybridized with each of the DC1 beta, DR alpha, and DR beta cDNA probes. The results at both the protein and DNA levels suggest that the RT1m and RT1c haplotypes share identical expressed alleles at the RT1.Ba, RT1.Bb, RT1.Bc, and RT1.Bd loci. more...

Cytotoxic effector T cells specific for non-H-2 histocompatibility (H) antigens were examined for phenotypic expression of lymphocyte differentiation (Ly) antigens. Virtually all H-Y-specific cytotoxic effectors generated in mixed lymphocyte culture were Ly-1+2+ T cells. H-3-specific effectors comprised both Ly-1+2+ and Ly-1-2+ T cells. However, cytotoxic effectors specific for multiple non-H-2 H antigens were predominantly Ly-1-2+ T cells. The optimal generation of H-Y- and H-3-specific effectors required Ly-1+2+ T cells; optimal generation of multiple non-H-2 H antigen-specific effectors required an interaction between Ly-1+2- and Ly-1-2+ T cells. These observations suggest that the identity of the target H antigen in part determines the Ly type of responsive T cells. Our observations suggest that 2 alternative pathways of T cell response exist for non-H-2 H antigens. The first pathway involves an interaction between Ly-1+2- helper T cells and Ly-1-2+ cytotoxic effector precursors. The 2nd pathway simply involves the response of Ly-1+2+ T cells proliferating and generating H antigen-specific cytotoxic effectors. more...

Until recently, the analysis of Class II genes linked to the rat major histocompatibility complex, RT1, has been confined to serologic and electrophoretic analysis of their gene products. To obtain a more definitive estimate of the number and relative polymorphism of RT1 Class II sequences, we performed Southern blot analysis of rat genomic DNA employing human cDNA probes specific for Class II heavy and light chain genes. Southern blots of EcoRI and BamHI digests of genomic DNA from ten inbred strains, expressing eight RT1 haplotypes, were hybridized with the human DQ beta or DR beta cDNA that are homologous to Class II light chain sequences. Four to eight bands were observed to hybridize with the light chain cDNA: band sizes ranged from 2.5 to 28 kb. Restriction fragment patterns were polymorphic; the only identical patterns observed were those associated with RT1 haplotypes with identical RT1.B regions. The number and size of bands hybridizing with DQ beta and DR beta suggested a minimum of four light chain sequences in each haplotype. Southern blots of BamHI and EcoRI digests of genomic DNA from the same strains were hybridized with a DR alpha cDNA that is homologous to Class II heavy chain sequences. All RT1 haplotypes expressed either a 10.0-kb or 13.0-kb band when digested with BamHI, and either a 17-kb or 3.7-kb band when digested with EcoRI. Considerably less polymorphism was detected with the DR alpha probe; this observation is consistent with previously reported limited protein polymorphism of the rat equivalent of the I-E alpha subunit. The size and number of bands hybridizing with the DR alpha probe suggests a minimum of two heavy chain sequences. These observations suggest that the RT1 complex includes more Class II sequences than have been observed in serologic and electrophoretic analyses of Class II gene products. Furthermore, the level of polymorphism of RT1 Class II sequences appears to be comparable with mouse and human Class II sequences. more...

RT1.B Class II molecules, comparable to I-A and I-E molecules in mice, have been characterized by two-dimensional (2D) gel electrophoresis for seven rat strains expressing different RT1 haplotypes. RT1.B molecules were immunoprecipitated from radiolabeled rat lymphocyte preparations with mouse monoclonal antibodies reactive with mouse I-Ab antigens and I-Ek antigens. For each RT1 haplotype, two I-A-like subunits, alpha and beta, and two I-E-like subunits, gamma and delta, have been identified. 2D gel analysis of RT1.B molecules from different strains in paired combinations reveals that, with a few notable exceptions, the alpha, beta, and delta subunits associated with different RT1 haplotypes are characterized by unique 2D gel spot patterns. In contrast, the gamma subunits of all RT1.B haplotypes appear identical. This evidence confirms and extends our previous analysis of RT1.B molecules by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and suggests a high level of polymorphism for genes in the RT1.B region, with the exception of the RT1.Bc gene encoding gamma. Interestingly, three rat strains appear exceptional in the identity of their alpha, gamma, and delta subunits, which suggests that these strains express closely related RT1 haplotypes derived from a common progenitor. more...

Addition of anti-Ia sera to cultures of mouse spleen cells stimulated with oxidative mitogens, neuraminidase/galactose oxidase (NaGO) or sodium periodate (NaIO4), inhibits the subsequent proliferative response 30 to 70%. Anti-H-2K or D sera were not specifically inhibitory. Similar inhibition was seen when cells were pretreated with anti-Ia sera and washed before exposure to the mitogenic enzymes. Treatment with anti-Ia serum and complement depletes greater than 89% of the NaGO and the NaIO4 responses but 2% or less of the phytohemagglutinin (PHA) response. The response to NaGO was sensitive to depletion with anti-Thy-1 serum, rabbit anti-mouse brain serum, anti-Ly-1, or anti-Ly-2 serum. Mixtures of Ly-1 and Ly-2-depleted populations did not restore responsiveness. Thus both an Ia+ cell and an Ly-1+2+ T cell are required for [3H]TdR incorporation in response to NaGO treatment. more...

Congenic and double congenic mice expressing different H-7 alleles with varying H-2 haplotypes on the C57BL/10 (B10) background were tested for their susceptibility to Friend virus (FV) infection. Mice expressing the H-7b allele derived from BALB/c were susceptible to FV infection whereas mice expressing the H-7a allele of B10 were absolutely resistant. Coexpression of H-7b and the H-2a and H-2d haplotypes determined high susceptibility; mice expressing both H-7b and H-2b were relatively resistant compared to H-7b mice expressing H-2a or H-2d. Parallel experiments with BALB/c and BALB.B recipients did not demonstrate differences in susceptibility associated with H-2d and H-2b. Mapping experiments with the H-2h4 and H-2i5 recombinants indicated that the gene determining relative resistance to FV-induced spleen focus formation mapped to the K-end (KbAb) of H-2b. Female recipients expressing H-7b, regardless of their H-2 haplotype, were more susceptible to FV infection than their syngeneic male counterparts. These experiments demonstrate the utility of congenic and double congenic strains that define H-7 and Fv-2 on different H-2 haplotype backgrounds for the analysis of Fv-2:H-7:H-2 interactions in determining susceptibility to FV infection. more...

The effects of a panel of H-2Kb mutants on the presentation of the non-H-2 histocompatibility (H) antigens H-4.2 and H-3.1 to antigen-specific T cells were examined. Kb-restricted cytotoxic effector T cells were presented with H-4.2 and H-3.1 in the context of mutant Kb alleles. H-4.2 was differentially presented by Kb mutants to Kb-restricted cytotoxic effectors. Cold target inhibition analysis demonstrated that Kb-restricted, H-4.2-specific cytotoxic effectors recognized H-4.2 in the context of at least three restriction sites on the Kb molecule. One site is specific for Kb alone, whereas two additional sites are differentially expressed by Kb mutant molecules. The effect of Kb mutations on the presentation of H-4.2 and H-3.1 was H antigen-specific. Identical, respective patterns of presentation of H-4.2 and H-3.1 by Kb mutants were observed with Kb-restricted T cells proliferating in secondary mixed lymphocyte culture. Further, individual mice varied in their preference for H-3.1 presented in the context of different Kb mutant molecules. These observations demonstrate that the presentation of H antigens by mutant Kb molecules is antigen-specific; analysis of presentation of H antigen by mutants to wild-type Kb-restricted effectors allows the identification of multiple restriction sites on the Kb molecule. more...

Lymphocyte populations from B6, C-H-2bml (KbmlDb) mutant mice cannot, after both in vivo and in vitro negative selection for alloreactivity, be induced to recognize vaccinia virus presented in the context of H-2Kb. This finding may mean that the T cell receptor(s) expresses a component that is very specific for a particular "active site" on the self-H-2 molecule. Alternatively, (if recognition is directed at a virus-H-2 complex) the more similar 2 H-2 molecules are, the more likely it may be that precursor thymocytes in the mutant with the capacity to bind H-2Kb + vaccinia virus may be deleted during ontogeny as a result of cross-reaction with H-2Kbml + endogenous antigen. more...

The integration and expression of Moloney-murine leukemia virus (M-MuLV) into the germ line of Mov mouse strains on the C57BL/6 background results in the expression of a cell-surface Ag with characteristics expected from non-H-2 histocompatibility Ag: the ability to stimulate graft rejection and generation of CTL. However, both the previously studied Mov-3 and Mov-14 strains differ from the coisogenic C57BL/6 strain by different length segments of chromosome derived from the ICR strain in addition to the integrated M-MuLV genome. To conclusively demonstrate that an Ag encoded by M-MuLV is solely responsible for rejection of Mov skin grafts by coisogenic recipients, we have studied additional Mov strains that differ from coisogenic 129 or BALB/c backgrounds only by integration of an M-MuLV genome. A total of 129 strain recipients reject skin grafts from two viremic Mov strains, Mov-17 and Mov-18. A total of 129 strain hosts primed with either 1) multiple sets of Mov-17 and Mov-18 skin grafts or 2) single injections of Mov-17 and Mov-18 spleen cells produce M-MuLV-specific CTL that could be boosted in primary mixed lymphocyte culture. Generated CTL were reactive with Con A-stimulated lymphoblasts from all tested viremic Mov strains on the B6 and 129 backgrounds as well as B6 lymphomas. Further, we have observed that 129 strain mice reject Mov-9 skin grafts if these skin grafts are transplanted to virgin 129 recipients which have not received prior skin grafts from non-viremic Mov donors. In addition, skin grafts were transplanted from two viremic Mov strains, Mov-15 and Mov-16, to coisogenic BALB/c recipients; rejection of both sets of grafts was observed. However, BALB/c responders did not generate specific CTL after priming in vivo, with either multiple sets of allogeneic grafts or spleen cell injections, and boosting in vitro. These observations confirm the ability of integrated and expressed M-MuLV genomes to encode what is operationally defined as a non-H-2 histocompatibility Ag. more...

Addition of anti-Ia sera to cultures of mouse spleen cells stimulated with oxidative mitogens, neuraminidase/galactose oxidase (NaGO) or sodium periodate (NaIO4), inhibits the subsequent proliferative response 30 to 70%. Anti-H-2K or D sera were not specifically inhibitory. Similar inhibition was seen when cells were pretreated with anti-Ia sera and washed before exposure to the mitogenic enzymes. Treatment with anti-Ia serum and complement depletes greater than 89% of the NaGO and the NaIO4 responses but 2% or less of the phytohemagglutinin (PHA) response. The response to NaGO was sensitive to depletion with anti-Thy-1 serum, rabbit anti-mouse brain serum, anti-Ly-1, or anti-Ly-2 serum. Mixtures of Ly-1 and Ly-2-depleted populations did not restore responsiveness. Thus both an Ia+ cell and an Ly-1+2+ T cell are required for [3H]TdR incorporation in response to NaGO treatment. more...

The cytotoxic T lymphocyte (CTL) responses directed towards SV40 tumor-associated specific antigen (TASA) in nine strains of spontaneously arising Kb mutant mice were analyzed. All nine mutants generated normal levels of H-2Db-restricted response, but the K-end-restricted CTL response varied. B6.C-H-2bm1 (bm1) did not produce K-end-restricted SV40 TASA-specific CTL upon immunization, and SV40-transformed bm1 cells were not lysed by intra-H-2 recombinant Kb [B10.A(5R)] CTL. Nonreciprocal cross-reactive lysis was seen between B6-H-2bm8 (bm8) and B10.A(5R). Strain B6-H-2bm8 mice produce highly specific Kbm8-restricted CTL that lyse SV40-transformed bm8 cells (Kbm8SV) but not B10.A(5R) target cells (K5RSV), although Kbm8SV targets can be partially lysed by B10.A(5R) CTL. The other seven Kb mutants cross-react with B10.A(5R). These experiments definitively show that genes mapping to the K and/or D region directly control the H-2-restricted CTL response to SV40 TASA. more...

The RT1m haplotype of MNR rats has been suggested to be a recombinant RT1 haplotype inheriting RT1.A (class I) alleles from RT1a (DA) and RT1.B (class II) alleles from RT1c (AUG). Additional serologic and biochemical assays, however, have suggested that RT1m and RT1c share a single identical RT1.B molecule, although differing in the expression of the second RT1.B molecule. To resolve this contradiction, RT1.B class II molecules, comparable to I-A and I-E molecules in mice, expressed by the RT1c and RT1m haplotypes were immunoprecipitated by cross-reactive mouse anti-Ia antibodies and were compared by two-dimensional gel electrophoresis and by high pressure liquid chromatographic separation of tryptic peptides. Respective subunits expressed by the two haplotypes co-migrate on two-dimensional gels and have identical tryptic peptide maps. The results at the protein level were confirmed at the DNA level by Southern blot analysis of MNR and AUG genomic DNA. Identical restriction fragments associated with the RT1m and RT1c haplotypes hybridized with each of the DC1 beta, DR alpha, and DR beta cDNA probes. The results at both the protein and DNA levels suggest that the RT1m and RT1c haplotypes share identical expressed alleles at the RT1.Ba, RT1.Bb, RT1.Bc, and RT1.Bd loci. more...

Two unusual murine lymphomas, designated CH1 and CH2, were produced in the newly developed double congenic strain of mice, B10 H-2a H-4b p/Wts. Both tumors lack the T cell-specific antigen (thy-1), but express cell surface immunoglobulin and the H-2K, H-2D, and Ia specificities determined by the H-2a haplotype. Further studies have demonstrated that these tumors represent "early" B cells in that they express surface IgM (mu heavy and lambda light chains), but do not bear surface delta, gamma, or alpha heavy chains. CH1 and CH2 lack surface C3 receptors and results from assays for Fc receptors have proven variable. A competition radioimmunoassay directed against the gp71 group-specific antigen of Friend leukemia virus has shown that there is a murine leukemia virus associated with these tumors, however, we have been unable to establish a causal relationship between the virus and this malignancy. A comparison of the surface characteristics of these tumors with other mammalian B cell lymphomas is presented. more...

Limit dilution cultures were used to test for influenza immune T cell populations from bm1 and bm3 mutant mice that were not lytic for virus-infected targets expressing the Kb and Db major histocompatibility complex glycoproteins. Both Kbm3- and Kbm1-restricted cytotoxic T cells were detected. Such effectors showed minimal cross-recognition of influenza on other mutant targets, except for the case of bm1 and bm10 targets. This is dissimilar to previous findings concerning vaccinia presentation in which bm3+bm11, bm1+bm9, and bm3+bm9 pairs each showed high cross-reactivity. These differences illustrate the role of the H-2K glycoprotein in immune responsiveness. Not only are multiple determinants on each H-2K glycoprotein involved in antigen presentation, they appear to play differential roles in the presentation of different viral antigens. more...

Cytotoxic effector T cells specific for non-H-2 histocompatibility (H) antigens were examined for phenotypic expression of lymphocyte differentiation (Ly) antigens. Virtually all H-Y-specific cytotoxic effectors generated in mixed lymphocyte culture were Ly-1+2+ T cells. H-3-specific effectors comprised both Ly-1+2+ and Ly-1-2+ T cells. However, cytotoxic effectors specific for multiple non-H-2 H antigens were predominantly Ly-1-2+ T cells. The optimal generation of H-Y- and H-3-specific effectors required Ly-1+2+ T cells; optimal generation of multiple non-H-2 H antigen-specific effectors required an interaction between Ly-1+2- and Ly-1-2+ T cells. These observations suggest that the identity of the target H antigen in part determines the Ly type of responsive T cells. Our observations suggest that 2 alternative pathways of T cell response exist for non-H-2 H antigens. The first pathway involves an interaction between Ly-1+2- helper T cells and Ly-1-2+ cytotoxic effector precursors. The 2nd pathway simply involves the response of Ly-1+2+ T cells proliferating and generating H antigen-specific cytotoxic effectors. more...

Lymphocyte populations from B6, C-H-2bml (KbmlDb) mutant mice cannot, after both in vivo and in vitro negative selection for alloreactivity, be induced to recognize vaccinia virus presented in the context of H-2Kb. This finding may mean that the T cell receptor(s) expresses a component that is very specific for a particular "active site" on the self-H-2 molecule. Alternatively, (if recognition is directed at a virus-H-2 complex) the more similar 2 H-2 molecules are, the more likely it may be that precursor thymocytes in the mutant with the capacity to bind H-2Kb + vaccinia virus may be deleted during ontogeny as a result of cross-reaction with H-2Kbml + endogenous antigen. more...

RT1.B Class II molecules, comparable to I-A and I-E molecules in mice, have been characterized by two-dimensional (2D) gel electrophoresis for seven rat strains expressing different RT1 haplotypes. RT1.B molecules were immunoprecipitated from radiolabeled rat lymphocyte preparations with mouse monoclonal antibodies reactive with mouse I-Ab antigens and I-Ek antigens. For each RT1 haplotype, two I-A-like subunits, alpha and beta, and two I-E-like subunits, gamma and delta, have been identified. 2D gel analysis of RT1.B molecules from different strains in paired combinations reveals that, with a few notable exceptions, the alpha, beta, and delta subunits associated with different RT1 haplotypes are characterized by unique 2D gel spot patterns. In contrast, the gamma subunits of all RT1.B haplotypes appear identical. This evidence confirms and extends our previous analysis of RT1.B molecules by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and suggests a high level of polymorphism for genes in the RT1.B region, with the exception of the RT1.Bc gene encoding gamma. Interestingly, three rat strains appear exceptional in the identity of their alpha, gamma, and delta subunits, which suggests that these strains express closely related RT1 haplotypes derived from a common progenitor. more...

The effects of a panel of H-2Kb mutants on the presentation of the non-H-2 histocompatibility (H) antigens H-4.2 and H-3.1 to antigen-specific T cells were examined. Kb-restricted cytotoxic effector T cells were presented with H-4.2 and H-3.1 in the context of mutant Kb alleles. H-4.2 was differentially presented by Kb mutants to Kb-restricted cytotoxic effectors. Cold target inhibition analysis demonstrated that Kb-restricted, H-4.2-specific cytotoxic effectors recognized H-4.2 in the context of at least three restriction sites on the Kb molecule. One site is specific for Kb alone, whereas two additional sites are differentially expressed by Kb mutant molecules. The effect of Kb mutations on the presentation of H-4.2 and H-3.1 was H antigen-specific. Identical, respective patterns of presentation of H-4.2 and H-3.1 by Kb mutants were observed with Kb-restricted T cells proliferating in secondary mixed lymphocyte culture. Further, individual mice varied in their preference for H-3.1 presented in the context of different Kb mutant molecules. These observations demonstrate that the presentation of H antigens by mutant Kb molecules is antigen-specific; analysis of presentation of H antigen by mutants to wild-type Kb-restricted effectors allows the identification of multiple restriction sites on the Kb molecule. more...

The immunization of C57BL/6 responder mice with spleen cells from H-2-matched BALB.B donors, which differ by multiple non-H-2 histocompatibility (H) antigens, results in the generation of cytotoxic T lymphocytes (CTL) that are specific for only a limited number of immunodominant antigens. Previous analysis of the genes encoding these dominant antigens has not mapped these genes to any of the non-H-2 H loci defined by congenic strains. It would have been expected that the histogenetic techniques employed for congenic strain selection would have preferentially identified the "strongest" H antigens. Therefore, we have investigated the possibility that immunodominant antigens do not belong to the class of non-H-2 H antigens encoded by genes mapping to H loci defined and mapped by congenic strains. The first experiments were aimed at identifying antigens that were expressed by independently derived inbred strains and were cross-reactive with the immunodominant cytotoxic T cell target (CTT-1) antigen of BALB.B. Strong cross-reaction with the C3H.SW (H-2b) strain was observed; the C3H gene encoding this antigen was mapped with BXH recombinant inbred strains. Contrary to the mapping of the CTT-1 gene to chromosome 1 in BALB.B, the C3H gene was shown to map to either chromosome 4 or chromosome 7. This result indicates that identical, or at least extensively cross-reactive, non-H-2 antigens may be encoded by genes mapping to independently segregating loci in different inbred strains. The tissue distribution of immunodominant antigens was approached by determining the reactivity of CTL specific for these antigens with either lymphoid-derived or fibroblast-derived targets. These CTL effectively lysed lymphoblast and lymphoid tumor targets but did not lyse an SV40-transformed fibroblast line that was shown to be efficiently lysed by CTL specific for non-H-2 H antigens defined by congenic strains. Therefore, it was concluded that immunodominant antigens detected by B6 anti-BALB.B CTL have a restricted tissue distribution in comparison to non-H-2 H antigens defined by congenic strains. The implications of these results for our understanding of the origin and heterogeneity of non-H-2 cell-surface antigen recognized by effector T cells are discussed. more...

The immunological surveillance hypothesis was formulated prior to the realization of the fact that an individual's effector T cells generally only see neoantigen if it is appropriately presented in the context of self MHC glycoproteins. The biological consequence of this mechanism is that T lymphocytes are focused onto modified cell-surface rather than onto free antigen. The discovery of MHC-restricted T cell recognition, and the realization that T cell-mediated immunity is of prime importance in promoting recovery from infectious processes, has thus changed the whole emphasis of the surveillance argument. Though the immunological surveillance hypothesis generated considerable discussion and many good experiments, there is no point in continuing the debate in the intellectual context that seemed reasonable in 1970. It is now much more sensible to think of "natural surveillance" and "T cell surveillance," without excluding the probability that these two systems have elements in common. We can now see that T cell surveillance probably operates well in some situations, but is quite ineffective in many others. Part of the reason for this may be that the host response selects tumor clones that are modified so as to be no longer recognized by cytotoxic T cells. The possibility that this reflects changes in MHC phenotype has been investigated, and found to be the case, for some experimental tumors. In this regard, it is worth remembering that many "mutations" in MHC genes that completely change the spectrum of T cell recognition are serologically silent. The availability of molecular probes for investigating the status of MHC genes in tumor cells, together with the capacity to develop cloned T cell lines, monoclonal antibodies to putative tumor antigens, and cell lines transfected with genes coding for these molecules, indicates how T cell surveillance may profitably be explored further in both experimental and human situations. more...

Trinitrophenylated syngeneic spleen cells (TNP-SC) are potent tolerogens of the anti-TNP plaque-forming cell (PFC) response in vivo and in vitro. This unresponsive state requires T cells for both its induction and maintenance. Because H-2K/D-restricted cytotoxic T cells are also induced by exposure to TNP-SC, we determined the role(s) of histocompatibility antigens (K, I, and D) in the suppression of the PFC response by TNP-SC. We treated syngeneic TNP-modified stimulator cells with antiserum directed at K-, I-, or D-region determinants and found that blocking of H-2K or D antigens on TNP-SC transformed these tolerogens into immunogens capable of eliciting an anti-TNP PFC response in the absence of extrinsic immunogens like TNP-polymerized flagellin. In H-2k or H-2a(k/d) mice, only H-2Kk needs to be blocked on the stimulator cells, whereas H-2K or D recognition was apparent in B10.A(4R) mice. These observations indicate that suppression of the PFC response by TNP-SC shows the same restriction in recognition as does the cytotoxic T-cell response. Furthermore, our results suggest that TNP-I-A is recognized by the helper cells in this system as the intrinsic antigen. When both TNP-K and TNP-I-A are present and available on the same stimulator cell, suppression (via modified K recognition) is dominant over help. more...

Until recently, the analysis of Class II genes linked to the rat major histocompatibility complex, RT1, has been confined to serologic and electrophoretic analysis of their gene products. To obtain a more definitive estimate of the number and relative polymorphism of RT1 Class II sequences, we performed Southern blot analysis of rat genomic DNA employing human cDNA probes specific for Class II heavy and light chain genes. Southern blots of EcoRI and BamHI digests of genomic DNA from ten inbred strains, expressing eight RT1 haplotypes, were hybridized with the human DQ beta or DR beta cDNA that are homologous to Class II light chain sequences. Four to eight bands were observed to hybridize with the light chain cDNA: band sizes ranged from 2.5 to 28 kb. Restriction fragment patterns were polymorphic; the only identical patterns observed were those associated with RT1 haplotypes with identical RT1.B regions. The number and size of bands hybridizing with DQ beta and DR beta suggested a minimum of four light chain sequences in each haplotype. Southern blots of BamHI and EcoRI digests of genomic DNA from the same strains were hybridized with a DR alpha cDNA that is homologous to Class II heavy chain sequences. All RT1 haplotypes expressed either a 10.0-kb or 13.0-kb band when digested with BamHI, and either a 17-kb or 3.7-kb band when digested with EcoRI. Considerably less polymorphism was detected with the DR alpha probe; this observation is consistent with previously reported limited protein polymorphism of the rat equivalent of the I-E alpha subunit. The size and number of bands hybridizing with the DR alpha probe suggests a minimum of two heavy chain sequences. These observations suggest that the RT1 complex includes more Class II sequences than have been observed in serologic and electrophoretic analyses of Class II gene products. Furthermore, the level of polymorphism of RT1 Class II sequences appears to be comparable with mouse and human Class II sequences. more...

Provirus modifications which can occur in tumors may lead to tumor antigenic variants. To investigate this possibility we compared the restriction fragment length patterns of mouse mammary tumor virus (MMTV), murine leukemia virus and xenotropic and mink cell-focus inducing (MCF)-related sequences, using Southern blot analysis, in a panel of Balb/c and C57BL/6 lymphomas, some of which were known to express novel histocompatibility (H) antigens. The results indicate differences in amplification and novel integration sites of retroviral sequences in all tumors analyzed, the number of new sequences depending on the specific retroviral sequence. A relationship has been found between integration of new MMTV sequences and a known susceptibility to lysis by syngeneic alloactivated lymphocytes in the same group of tumors. The possibility that retroviruses can induce the expression of novel histocompatibility antigens on tumors is discussed. more...