A new cytotoxic lymphocyte-defined antigen coded by a gene closely linked to the H-3 locus

F1 complementation results indicate that a new gene, putatively controlling a minor histocompatibility antigen, is closely linked to the minor histocompatibility gene, H-3, in the fifth linkage group of chromosome 2 of the mouse. This gene controls a product that was capable of inducing as well as acting as a target for cytotoxic lymphocytes (CTL). The lytic activity of CTL developed in B10.LP-H-3D mice specific for the product of the new gene of B10 was restricted to target cells possessing H-2Db antigens. This contrasts to the H-2Kb-restricted activity of H-3.1 specific CTL.     

Responses against antigens encoded by theH-3 histocompatibility locus: Antigens stimulating class I MHC- and class II MHC-restricted T cells are encoded by separate genes

The purpose of this work was to study the genetic basis of histocompatibility antigens encoded by the mouse minor histocompatibility (H) locusH-3. Both class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) and class II MHC-restricted helper T cells (TH) specific for antigens encoded by genes within theH-3 locus were isolated and analyzed. Typing a number of mouse strains for expression of antigens recognized by these TH and CTL suggested that there was a different strain distribution pattern of expression of the antigens recognized by TH compared with those recognized by CTL. Separation of the genes whose products stimulate TH from those whose products stimulate CTL was suggested by: (1) analysis of the strain B10.FS(92NX)/Grf that has undergone recombination within theH-3 region; (2) genetic segregation studies of (B10.UW-H-3 ^b/Sn×C57BL/10Sn)F₂ mice; and (3) F₁ complementation studies in which CTL specific for products of the TH-defined gene(s) could not be detected, even in the absence of immune responses to products of the CTL-defined genes. Taken together, these data suggest that in addition to two genes (B2m andCd-1) within theH-3 region whose products typically stimulate class I MHC-restricted CTL, there is at least one additional gene whose product selectively stimulates class II MHC-restricted TH. This new gene is located telomeric from the CTL-defined genes and between the lociwe andun on chromosome 2. These data demonstrate a novel degree of complexity of theH-3 “locus” and suggest selective presentation of minor H gene products in the context of class I or class II MHC proteins.     

Dissociation ofH-2 recognition by antibody and cytotoxic T cells of a cloned murine leukemia cell line

The differential expression of H-2 specificities recognized by antibody and by cytotoxic T lymphocytes (CTL) has been studied using a clone (FY7) of the C57BL/6 leukemia cell line FBL-3 (H-2 ^b /H-2 ^b ). Unlike C57BL/10 spleen cells, EL-4 lymphoma cells and Y57-2C leukemia cells (allH-2 ^b /H-2 ^b ), FY7 failed to induce the primary in vitro generation of anti-H-2^b CTL by (B10.A x A)F₁ (H-2 ^a /H-2 ^a or (B10.D2 x BALB/c)F₁ (H-2 ^d /H-2 ^d ) responder spleen cells. In addition, FY7 was not lysed by, and did not competitively inhibit anti-H-2^b CTL. Quantitative absorption tests with H-2K^b and H-2D^b antisera revealed that FY7 expressed these antigens in quantitatively similar amounts to EL-4. The H-2K^b product of FY7 appeared to be identical with that of C57BL/10 spleen cells both in apparent molecular weight and isoelectric point. Yet FY7 failed to inhibit anti-H-2K^b CTL competitively in a cold target inhibition assay. Possible mechanisms are discussed for the lack of T-lymphocyte recognition of the H-2K^b-gene product expressed by FY7.Abbreviations used in this paper CTL cytotoxic T lymphocytes - MHC major histocompatibility complex - MLC mixed lymphocyte culture - PAGE polyacrylamide gel electrophoresis     

Polymorphism of minor histocompatibility genes in wild mice

H-2^b-restricted cytolytic T lymphocytes (CTL) were generated against H-1, H-3, and H-4 antigens and tested against target cells of F₁ hybrids between wild mice and inbred H-2 ^b mice. The congenic strain combinations for the CTL production were such that they tested one allele each at the H-1 and H-4 loci and four alleles at the H-3 locus. Most of the wild mice tested came from Southern Germany, but a few mice came from other European countries and Egypt and Israel. Virtually all wild mice typed as positive with CTL directed against H-3^b and H-4^b antigens; 32% of the F₁ hybrids tested reacted with anti-H-1^cCTL and 9% reacted with anti-H-3^d CTL. The positive results were not caused by cross-reaction with allogeneic H-2 antigens controlled by the major histocompatibility complex (Mhc) genes of the wild mice. At least some of the H-3 and H-4 antigens detected by the CTL in the F₁ hybrid were not identical with antigens of the immunizing strains. These results suggest a relatively low degree of polymorphism of the tested minor H loci in wild mice and further support the notion that minor H loci are unrelated to the Mhc.     

Use of hybridoma-resistant variant cell lines to study H-2D b/FMR-specific cytotoxic T cells

An H-2D ^b b heterozygous tumor cell line and a variant subclone bearing a mutant gene product were used to analyze the H-2D^b specificity of cytotoxic T lymphocytes (CTL) generated during a Moloney murine sarcoma virus (MSV) infection. When the mutant cells were used as targets for MSV-specific CTL, the amount of cell lysis, compared with that seen with the nonmutant parental cells, was drastically decreased. However, cells of the mutant clones remained susceptible to allogeneic CTL specific for the nonmutant H-2D^b molecule. The mutant cells also did not differ from the parent cells in their level of viral antigen expression. Biochemically the parental and mutant molecules were similar but not identical. The data indicate that minor alterations of the H-2 antigens caused by somatic mutation may prevent virus-infected cells from being recognized as targets by CTL.     

A third class I major histocompatibility complex antigen encoded by a gene in the D region of the H-2 d haplotype recognized by cytotoxic T lymphocytes

The D region of the H-2 ^d haplotype contains five class I genes: H-2D ^d , D2 ^d , D3 ^d , D4 ^d and H-2L ^d . Although previous studies have suggested the presence of D-end encoded class I molecules in addition to H-2D^d and H-2L^d, segregation of genes encoding such molecules has not been demonstrated. In this report we have used cãtotoxic T lymphocytes (CTL) to examine the D region of the H-2 ^d haplotype for the presence of additional class I molecules. CTL generated in (C3H × B6.K1)F₁ (K ^k D ^k , K ^b D ^b ) mice against the hybrid class I gene product Q10^d/L^d expressed on L cells cross-react with H-2L^d but not H-2D^d molecules, as determined by lysis of transfected cells expressing H-2L^d but not H-2D^d. Although H-2L^d-specific monoclonal antibodies (mAb) completely inhibit H-2L^d-specific CTL from killing B10.A(3R) (K ^b D ^d L ^d ) target cells, only partial inhibition of anti-Q10 CTL-mediated lysis was observed, suggesting the presence of an additional D-end molecule as a target for these latter CTL. To identify the region containing the gene encoding the Q10 cross-reactive molecule, we show that anti-Q10 CTL lyse target cells from a D-region recombinant strain B10.RQDB, which has H-2D ^d , D2 ^d , D3 ^d , D4 ^d , and H-2D ^b but not the H-2L ^d H-2 ^d , and H-2L ^d (including D2 ^d , D3 ^d , and D4 ^d , lacks this anti-Q10 CTL target molecule. Together, these data demonstrate that a class I gene mapping between H-2D ^d and H-2L ^d encodes an antigen recognozed by anti-Q10 CTL. A likely candidate for this gene is D2 ^d , D3 ^d or D4 ^d .     

Detection of two allotype-(Ig-1)-linked minor histocompatibility loci by the use ofH-2-restricted cytotoxic lymphocytes in congenic mice

Cytotoxic lymphocytes (CTL) were generated betweenIg-1-congenic strains BALB/c(H-2^d,Ig-1^a) andC.B-17(H-2^d,Ig-1^b) by cross-immunization in both directions and rechallenge in vitro. The effector cell populations specifically lysed target cells sharing both theH-2 haplotype and theIg-1 allele of the sensitizing strain. B- and T-cell blasts were equally good targets, suggesting thatH-2-restricted cytotoxic lymphocytes are not directed against serologically defined conventional allotypic determinants, but probably against minor histocompatibility antigens controlled by genes linked to theIg-1 complex. Competition experiments using cold target cells from a series ofIg-1^b-congenic strains of the BALB/c background (BAB-14, C.B-17, C.B-26) revealed two not yet described minor histocompatibility loci linked to theIg-1 complex: We could demonstrate that BALB/c anti-C.B-17 effector cells recognize at least two distinct antigenic determinants on C.B-17 target cells, but only one on target cells from BAB-14, which carries a recombinantIg-1 complex. From these results we conclude that one of the minor histocompatibility antigens, designated as H(C_H), is encoded by a gene linked to the heavy-chain constant-region (C_H) genes, whereas the second minor histocompatibility antigen, designated as H(V_H), is coded for by a gene linked to the heavy-chain variable-region (V_H) genes. These two new genetic markers may be useful for further analysis of the mouseIg-1 complex because the analysis of the H(C_H) and H(V_H) genes may facilitate the search for recombinants in that chromosomal region.     

Diversity of alpha and beta subunits of T-cell receptors specific for the H4 minor histocompatibility antigen

 The mouse H4 minor histocompatibility antigen (HA) includes a single H2K^b-bound peptide that stimulates rejection of skin allografts and generation of cytolytic T lymphocytes (CTL). We evaluated the diversity of the CTL response to this single minor HA peptide by sequencing alpha and beta chains of T-cell receptors (Tcr) from H4-specific CTLs as a first step toward understanding the diversity of Tcrs specific for single minor HA. We selected H4-specific CTL clones from short-term lines that were restricted by K^b (19 clones), K^bm5 (7 clones), and K^bm11 (10 clones). Whereas multiple V_α and V_β family members were identified in the panel of K^b-restricted CTLs, five VB genes and one VA subfamily were predominant in CTLs derived from multiple individuals. Similar distributions were observed with K^bm5- and K^bm11-restricted CTLs, suggesting that these two mutants did not alter Tcr gene usage observable at the VA and/or VB gene levels. Negatively charged residues were present in the CDR3 regions of 12/13 unique K^b-restricted beta chains. A comparable observation was made with K^bm5-restricted CTLs, and the distributions of these residues among CDR3 positions were similar in the two CTL panels. However, the K^bm11 mutation dramatically altered the distribution of these residues resulting in their presence in positions 10–12 of CDR3 regions in all CTLs. These results indicate that the Tcrs expressed by CTLs specific for this single minor HA peptide are oligoclonal and characterized by the presence of negatively charged residues in beta CDR3 regions, the distribution of which is profoundly altered by the K^bm11 mutation.     

Mutations in H-2K b influence the specificity of alloreactive effector cells included in the repertoire of H-2D b -restricted cytotoxic T lymphocytes against Moloney leukemia virus

Moloney leukemia virus-specific cytotoxic T lymphocytes (CTL), generated by secondary in vitro stimulation of spleen cells with syngeneic virus-infected cells, frequently lysed not only syngeneic virus-infected cells, but also noninfected allogeneic target cells. This phenomenon was studied with B6(H-2 ^b ) responder cells and a series of H-2K ^b -mutant responder cells. Thus, B6 Moloney-specific CTL lysed noninfected K ^b -mutant cells, but not B6 cells, whereas K ^b -mutant Moloney-specific CTL lysed noninfected B6 cells and not noninfected cells of the same mutant. Cold-target-inhibition studies showed that the CTL reactions against different allogeneic cells were mediated by different subpopulations of virus-specific CTL: lysis of allogeneic target cells was fully inhibited only by the same allogeneic and by syngeneic virus-infected cells, but not by another allogeneic cell, also lysed by the same effector-cell population. Lysis of syngeneic virus-infected cells could not be inhibited by allogeneic target cells. These data imply that a minority of virus-specific CTL shows cross-reactivity with a given allogeneic target cell. It is concluded that limited amino acid substitutions in the K^b molecule alter the repertoire of Moloney virus-specific CTL, as reflected in alloreactive CTL populations, even though the virus-specific CTL response. of B6 and all K ^b mutants is mainly D^b-restricted. Thus, the development of tolerance to self class-I major histocompatibility complex (MHC) molecules affects the repertoire of self-restricted cytotoxic T cells.     

Selective reversal of H-2 linked genetic unresponsiveness to lysozymes

Genes outside of the mouse major histocompatibility complex (H-2) were found to be capable of specifically reversing the previously described nonresponsiveness to hen egg-white lysozyme (HEL) owing to H-2 ^b immune response (Ir) genes. C3H.SW, BALB.B, and C57L, all of the H-2 ^b haplotype, showed responsiveness to HEL, but not to human lysozyme (H UL). Mapping of the reversing gene(s) was attempted by testing H-2 ^b recombinant inbred (RI) strains of mice carrying C3H, BALB, and C57L non-H-2 ^b genes. Analysis of the strain distribution pattern of responsiveness with both CXB and BXH RI strains was consistent with the location of the responsible site within the H-3 region on chromosome 2. The anti-HEL proliferative responsiveness in two H-3 congenic strains of mice, B10.C(28NX) ^SN and B10.C-H-3 ^{cH-3 a} , that have BALB/c genes within the H-3 region confirmed the mapping, as well as localized the reversing gene(s) near the Ir-2 gene. The data are discussed with regard to the site of expression of the reversing gene(s) and its mechanism of action.Abbreviations used in this paper MHC major histocompatibility complex - HEL hen egg-white lysozyme - Ir immune response gene - HUL human lysozyme - SDP strain distribution pattern - PFC plaque-forming cells; ₂ m, ₂-microglobulin - CFA complete Freund's adjuvant - PT-LN parathymic lymph nodes - RI recombinant inbred mice     

Transfer of long-lasting tumor immunity by immune T cells from MHC congenic mice: Migration,survival and tumor-protectivity of cytotoxic donor cells

Immunocompetent B10.D2 (H-2^d) mice are able to reject the highly malignant lymphoma ESb of DBA/2 (H-2^d) origin very effectively. Seven days after intravenous injection of the ESb tumor cells, B10.D2 mice developed a strong tumor-rejection response which was associated with the generation of anti-tumor T cells in their spleens with direct cytotoxic activity. Most of the cytotoxic potential was directed against the minor histocompatibility differences as demonstrated by the lysis of unrelated DBA/2 derived Eb tumor cells and normal DBA/2 but no B10.D2 derived ConA lymphoblasts. A previously performed clonal analysis, however, revealed a minority population of CTL clones which specifically recognized the ESb specific transplantation antigen (ESb-TATA). When transferred systemically into DBA/2 mice, the B10.D2 anti-ESb immune spleen cells could delay the outgrowth of s.c. transplanted ESb tumor cells. When the ESb tumor cells were experimentally distributed in a s.c. implanted sponge-matrix, the i.v. injected B10.D2 immune cells could confer complete protective immunity against the metastatic tumor, provided the recipients were pre-treated with 5 Gy to allow a better take of the allogeneic cells. The distribution of intravenously injected B 10D2 donor spleen cells was assessed in the recipients up to 50 days by cytotoxicity testing and assaying for the expression of the ₂ microglobulin allelic form b ( ₂m^b). These tests revealed a high propensity of donor cells to populate the spleen and lymph nodes of the DBA/2 recipients. Again this was particularly marked in sublethally irradiated mice where a long-lasting lymphoid chimerism was established.     

Immunogenetic analysis ofH-2 mutations

A.BY, B10.LPa, and B10.129(5M) mice were presensitized in vivo against B10.A(5R) cells and then restimulated in vitro by the same cells in the standard CML assay. The effector cells thus generated lysed not only B10.A(5R), but also C57BL/6 targets, indicating that, in addition to anti-H-2D_d response [measured on the B10.A(5R) targets], response to minor histocompatibility (H) antigens (measured on the C57BL/6 targets) also occurred. The latter response was directed against multiple minor H antigens in the case of the A.BY effectors, and against H-1 and H-3 antigens in the case of B10.129(5M) and B10.LPa effectors, respectively. The sensitization against minor H antigens occurred in the context of H-2K^b H-2D^d antigens, but by testing the response on C57BL/6 targets, only cells reacting with minor H antigens in the context of H-2K^b were assayed. The same effector cells were then tested against H-2^b mutant strains, in which theH-2K ^b allele was replaced by a mutant one. All three effector types [A.BY, B10.LPa, and B10.129(5M)] behaved in a similar way: they all reacted with theH-2 ^bg1 mutant to the same degree as withH-2 ^b, they did not react at all or reacted only weakly with theH-2 ^bd andH-2 ^bh mutants, and they reacted moderately or strongly with theH-2 ^ba mutant. The degree of crossreactivity with the mutants reflects, with one exception, the degree of relatedness of these mutants toH-2 ^b, as established by other methods. The one exception is theH-2 ^ba mutant, which is the most unrelated toH-2 ^b, and yet it crossreacted strongly. Further testing, however, suggested that in this instance the crossreactivity was probably directed against H-2 antigens: the anti-H-2D^d effectors apparently crossreacted with the H-2K^ba antigens. This finding is an example of cell-mediated crossreactivity between the products of two differentH-2 genes (H-2K andH-2D). It is also an example of anH-2 mutation generating an antigenic determinant known to be present in another strain.     

Somatic cell variants of H-2k: b: A point mutation in the first extracellular domain results in altered immune recognition

A cell-surface-associated variant H-2K product was expressed by an Abelson virus-induced pre-B-cell line after chemical mutagenesis with ethyl methane sulfonate. The variant cell line (R8.313) was previously demonstrated to have altered allodeterminants in K^b as demonstrated by both K^b-specific monoclonal antibody binding and alloreactive cytotoxic T lymphocyte (CTL) cytolysis. The mutant H-2K ^b gene from R8.313 was cloned and characterized in detail. DNA sequence analysis of the region of the gene corresponding to the three extracellular domains identified a single point mutation resulting in a leucine-to-phenylalanine substitution at amino acid residue 82. The site of mutation within the 1 domain was confirmed by oligonucleotide hybridization analysis. Mouse L-cell fibroblasts transfected with the mutant gene were recognized with the same monoclonal antibody binding and CTL lytic pattern as the R8.313 cell line, confirming that the altered phenotype of the mutant cell line was due to a point mutation in the H-2K ^b gene. These data further extend the hypothesis that the region of amino acid residues 70–90 in the 1 domain is important in the formation of both antibody and CTL-defined recognition structures on major histocompatibility complex class I molecules.     

The Mouse H-2A Region Influences the Envelope Gene Structure of Tumor-Associated Murine Leukemia Viruses

C57BL/10 (B10) strains congenic at the mouse major histocompatibility locus (H-2) were injected with a modified ecotropic SL3-3 murine leukemia virus (MuLV) to determine the effect of the H-2 genes on the envelope gene structure of recombinant MuLVs. All tested strains rapidly developed T-cell lymphomas, and recombinant proviruses were detected in the tumor DNAs by Southern blot. The B10.D2 (H-2^d), B10.Br (H-2^k), B10.Q (H-2^q), and B10.RIII (H-2^r) strains exhibited a TI phenotype in which almost all tumors contained type I recombinants. These recombinants characteristically acquire envelope gene sequences from the endogenous polytropic viruses but retain the 5′ p15E (TM) gene sequences from the ecotropic virus. The parental B10 (H-2^b) strain, however, had a novel phenotype that was designated NS for nonselective. Only 30% of the B10 tumors had detectable type I recombinants, whereas a proportion of the others appeared to contain type II recombinants that lacked the type I-specific ecotropic p15E gene sequences. Studies of other B10 congenic strains with hybrid H-2 loci and selected F₁ animals revealed that the NS phenotype was regulated by a dominant gene(s) that mapped to the A region of H-2^b. These results demonstrate that a host gene within the major histocompatibility complex can influence the genetic evolution of pathogenic retroviruses in vivo.     

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.     

H-2 restriction of cytolysis after immunization of minorH congenic pairs of mice

Mice of three congenic resistant lines differing from C57BL/10 at theH-3, H-13, H-7, andH-8 minor histocompatibility loci were used to immunize, and were immunized with, C57BL/10. Cytotoxic cells which were capable of causing rapid lysis of cells from the immunizing strain were generated at least one-way in all combinations tested. In order for a target to be susceptible to cytolysis, it had to carry both the sameH-2 ^b haplotype and the same differential minor histocompatibility alleles as the immunizing strain. That is, B10.C(47N) (H-2 ^b ,H-7 ^b ) anti-C57BL/10 (H-2 ^b ,H-7 ^a ) cytotoxic cells lysed C57BL/10 targets but not B10.BR (H-2 ^k ,H-7 ^a ) targets, nor BALB.B (H-2 ^b ,H-7 ^b ) targets. Crossreactions in the cytotoxic assay suggest that theH-3, H-13 congenic pair —B10.LP and C57BL/10 —may differ in at least two more minor histocompatibility loci which have not yet been defined. Although 6 x 10⁶6 C57BL/10 lymphoid cells primed B10.D2(57N) (H-8 ^b ) mice for a secondary in vitro cytotoxic response, a tenfold higher dose apparently made them tolerant. It is concluded that all minor histocompatibility differences can generate cytotoxic T cells which show specificity both for the minor and major histocompatibility alleles.     

Interaction between MHC-encoded products and cloned T cells

The interaction between class I major histocompatibility complex (MHC) products and T cells was studied using H-2K^b-specific alloreactive T-cell lines and clones obtained by repeated in vitro stimulation with allogeneic cells. Induction of proliferation of these T cells appeared to involve two signals: the H-2K^b alloantigen and interleukins. Immunopurified liposome-inserted H-2K^b, which stimulates specific secondary in vitro cytotoxic T lymphocyte (CTL) responses, could not replace cell-associated H-2K^b in the stimulation of these T-cell lines, even in the presence of feeder cells and interleukins. When T-cell lines were initiated in vitro and repeatedly stimulated with H-2K^b liposomes and feeder cells, it was possible to obtain T cells that could proliferate in response to H-2K^b liposomes in the presence of feeder cells and interleukin-2-containing supernatants or on H-2K ^b -expressing cells. Only stimulation with cells permitted maintenance of these T cells in culture for more than 12 weeks. Analyses of cell surface markers and of patterns of inhibition of proliferation by monoclonal antibodies (mAb) of T-cell lines induced in vitro with cell- or liposome-associated H-2K^b indicated that T-cell stimulation by class I antigen can occur in at least two ways. In the first, the H-2K^b-induced proliferation of Lyt-1^- Lyt-2⁺ T4^- T cells is inhibited by H-2K^b- and by Lyt-2-specific mAb, but not by Ia or T4-specific mAb. In the second, both Lyt-2⁺ and T4⁺ T cells are involved and the H-2K^b-induced proliferation is inhibited by H-2K^b- and Lyt-2-specific mAb and by Ia- and T4-specific mAb.Abbreviations used in this paper Ab antibody - mAb monoclonal antibody - C complement - i.p. intraperitoneally - PBS phosphate-buffered saline - PBS-B-N PBS containing bovine serum albumin and NaN₃ - CTL cytotoxic T lymphocyte - Th T helper cell - MHC major histocompatibility complex - PMA 4-phorbol 12-myristate 13-acetate - SCA concanavalin A-stimulated rat spleen cell supernatant - SC16 EL4 clone 16 supernatant - IL-1 interleukin-1 - IL-2 interleukin-2 (T-cell growth factor) - FCS fetal calf serum - H-2K^b-lip. H-2K^b inserted in liposomes - C. E. cell equivalents     

Glyoxalase I activity inH-2 b andH-2 d cells

Erythrocytes and thymocytes of theH-2 ^d genotype express between 1.6 and 2.6 times as much glyoxalase I activity per milligram of protein as those of theH-2 ^b genotype; the activity in theF ₁ (H-2^b/H-2^d) cells is intermediate between these two values. Activity of glyoxalase II is not affected by the H-2 genotype. The H-2^d:H-2^b ratio of glyoxalase I activity is higher in the BALB background where it is 2.6:1 than in the B10 background where it is 1.6:1. Thermal denaturation kinetics of enzymes from the H-2^b and H-2^d genotype cells are the same.     

Thy.1lowCD3− Cells Sorted from Nylon Wool-Passed Bone Marrow Cells Can Augment the H-2 Identical but Not Non-Identical Cytotoxic T Lymphocyte Precursors in Mixed Lymphocyte Cultures

Thy. 1^lowCD3^– cells obtained from nylon wool-passed murine bone marrow (NW-BM) cells by cell sorting did not express CD4, CD8, or T cell receptor-α/β and -γ/δ on their cell surfaces. An extremely limited number of B10.BR (H-2^k) responder lymph node (LN) cells were stimulated with B10. D2 (H-2^d) stimulator spleen cells in cultures containing the minimum required dose of rat T cell growth factor (TCGF). In these cultures, the generation of cytotoxic T lymphocytes (CTL) was very low. B10.BR Thy.1^lowCD3^– NW-BM cells, added to these cultures, could augment the CTL generation vigorously, but neither B10 (H-2^b) nor B10.D2 cells could. When B10 LN cells were used as responder cells in these cultures, B10 Thy. 1^lowCD3^– NW-BM cells could augment the CTL generation, but neither B10.BR nor B10.D2 cells could. Similar findings were obtained when Lyt-2⁺ cells or Thy.1⁺ L3T4^– (CTL precursor) cells sorted from spleen cells were used as responder cells. Both elements, rat-TCGF and Thy.1^low CD3^– NW-BM cells, were essential for this augmentation of the CTL generation in this culture system because neither one alone could augment generation, and rat-TCGF could be replaced by Thy.1⁺ Lyt-2^– helper T (Th) cells sorted from spleen cells. These findings showed that NW-BM cells could augment CTL precursors in a self-major histocompatibility complex (self-MHC)-antigen restricted manner, and further that both NW-BM cells and Th cells had different and independent functions to induce CTL.     

Antigen-specific helper T cells recognize determinants encoded in the H-2D region of the H-2 gene complex

Observations have frequently been interpreted as showing that the helper T cells which collaborate with alloantigen-specific cytotoxic T-cell precursors can only recognize antigens encoded in the I region of the H-2 gene complex. An experimental system is described here that allows analysis of the recognition repertoire of these helper cells. CBA helper T-cell precursors can be primed in vitro to antigens encoded in the H-2 ^b gene complex. These helpers can then be tested for the existence of a subset of helper cells which recognize antigens encoded in the D region of H-2 ^b haplotype. CBA thymocytes were used as a source of cytotoxic T-cell precursors that respond poorly in the absence of exogeneous helper activity. The source of alloantigen was varied by using irradiated spleen cells from various (BALB/c × recombinant)F₁ hybrid mice as stimulator cells. When the stimulator cell bears BALB/c determinants recognized by the cytotoxic T-cell precursor and also bears only the D region antigens of the H-2 ^b haplotype, an anti-BALB/c cytotoxic response is generated only if the anti-H-2^b helper population contains cells able to recognize H-2D^b. A positive cytotoxic response was obtained, indicating that helper cells are not limited to recognition of I region antigens and can efficiently recognize antigens encoded in the D region of the H-2 gene complex. This was confirmed by the demonstration of helpers specific for H-2D^d. We were unable to detect any evidence for Ia-restricted recognition of the H-2D alloantigens, suggesting that, as for cytotoxic T lymphocytes (CTL), helper cell recognition of class I alloantigens is an unrestricted event.