Receptor specificity,functional characteristics,and cell-surface phenotype of a highly selected anti-I-Ab-specific,long-term T-cell line

A highly selected alloreactive T-cell line was developed by repeated restimulation of B10.D2/n lymph-node cells with irradiated C57BL/10Sn (BIO) spleen cells in long-term MLC for up to 2 1/2 years. Continuous growth of the line requires restimulation every 2 to 4 weeks with fresh H-2^b stimulator cells. The line proliferates strongly against H-2^b but not againstH-2 ^d ,H-2 ^f ,H-2 ^q ,H-2 ^r , orH-2 ^s stimulators. Analysis of recombinant mouse strains showed that the proliferative response is directed against I-A^b but not K^b or D^b determinants. During the growth period of the line, strong cross-reactivity with H-2^p (B10.P) and weak cross-reactivity with H-2^k strains (e.g., CBA/J and B10.BR) was observed. A clone with exquisite specificity for I-A^b, but with no cross-reactivity with H-2^p or H-2^k was isolated from the line; thus clonal heterogeneity of the line still exists despite the highly selective growth conditions. — The majority of T cells from the line or clone were shown to bind I-A^b but not K^b or D^b determinants either spontaneously during restimulation with fresh B10 stimulator cells or via membrane vesicles expressing I-A^b determinants. No killing activity by the line in either specific or nonspecific cytolytic T-cell assays was observed nor was the T 145 glycoprotein, characteristic of killer T cells, detected.Abbreviations used in this paper B6 C57BL/6J - B10 C57BL/10Sn - Con A Concanavalin A - CTL cytotoxic T lymphocyte - FCS fetal calf serum - FDA fluorescein diacetate - FITC fluorescein isothiocyanate - Ia I-region-associated antigens - LPS lipopolysaccharide fromE. coli - Lyt T-lymphocyte-defined antigen - MLC mixed leukocyte culture - NP-40 nonidet P-40 - PAGE pofyacrylamide gel electrophoresis - PHA phytohemagglutinin fromPhaseolus vulgaris - PM plasma membrane - SDS sodium dodecyl sulfate - TCGF T-cell growth factor(s) - TdR thymidine     

MHC recognition by clones of Mls specific T-lymphocytes

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

Use ofH-2 mutations to quantitate alloreactivity: Alloreactivity is strongest against H-2 antigens which are closest to self

Lymph-node cells fromH-2 allogeneic, intra-H-2 recombinant andH-2 mutant congenic strains were sensitized in limiting dilution cultures to quantitate the cytotoxic T-lymphocyte precursor frequencies (CTL.Pf) against antigens encoded by different regions of theH-2 complex. When fourH-2K ^b mutants of C57BL/6 (B6) were tested, we observed anti-B6 CTL.Pf that were as high or higher than those of recombinant strains which differ from B6 at theK end of theH-2 complex. Relative to strains completelyH–2 allogeneic to B6, the CTL.Pf inH-2 ^bm1,H-2 ^bm3 andH-2 ^bm5 averaged 40–50 percent, andH-2 ^bm8 averaged 140 percent. Recombinant strains B10.A (4R) and B10.D2 (R103), which differ from B6 at theK end of theH-2 complex, averaged 60 percent of the completelyH-2 allogeneic value. Since the mutant and wild-type gene products have no serological and minimal structural differences relative to other alleles atH-2K, these results indicate that the CTL.Pf does not increase with increasing H-2 antigenic disparity between any two strains. Rather, the data suggests that the T-cell receptor repertoire recognizes those H-2 molecules or determinants closest to self.     

Genetic control of the target structures recognized in hybrid resistance

Hybrid resistance of lethally irradiated (C57BL/6 × DBA/2)F₁ and (C57BL/10 × C3H)F₁ hybrid mice to the engraftment of parental C57BL/6 or C57BL/10 bone marrow cells is controlled by the H-2-linked Hh-1 locus. This resistance can be specifically blocked or inhibited by the injection of irradiated spleen cells from lethally irradiated, marrow reconstituted donor mice of certain strains. By testing the ability of regenerating spleen cells from various donor strains to block the resistance, we studied the genetic requirements for the expression of putative cell-surface structures recognized in hybrid resistance to H-2^b marrow cells. Strains of mice bearing informative intra-H-2 or H-2/ Qa-Tla recombinant haplotypes provided evidence that the Hh-1 locus is located telomeric to the H-2S region complement loci and centromeric to the H-2D region class I locus in the H-2 ^b chromosome. Two mutations that affect the class I H-2D ^b gene have no effect on Hh-1 ^b gene expression. The H-2D region of the H-2 ^S haplotype contains an allele of the Hh-1 locus indistinguishable from that of the H-2D ^b region, as judged by the phenotypes of relevant strains and F₁ hybrids. Collectively these data indicate that the Hh-1 locus is distinct from the class I H-2D (L) locus in the H-2 ^b or H-2 ^s genome, and favor the view that the expression or recognition of the relevant determinants is not associated with class I gene products.Abbreviations used in this paper BM(C) bone marrow (cells) - CML cell-mediated lympholysis - CTL cytotoxic T lymphocytes - FBS fetal bovine serum - HBSS Hanks' balanced salt solution - SC spleen cells from irradiated, bone marrow-reconstituted mice Address correspondence to: Dr. I. Najamura, Department of Pathology, School of Medicine, State University of New York at Buffalo, Buffalo, NY 14214, USA     

H-2-linked genes determine the level of the primary in vitro anti-Mls response

The level of cell proliferation and interleukin-2 (IL-2) production observed in an anti-Mls mixed lymphocyte reaction between spleen cells from H-2 compatible, Mls incompatible mouse strains is determined by the H-2 haplotype of the mouse combination. Thus, while AKR (H-2 ^k) spleen cells stimulated strong M1s^a responses in H-2^k responder cells, AKR H-2b spleen cells stimulated no or negligible M1s^a responses in responder cells from H-2 ^bmouse strains. This effect was observed at the levels of IL-2 production and cell proliferation. The magnitude of the response observed using F₁ (H-2 ^k/H-2 ^b) responder cells was found to be a function of stimulator rather than responder cells. The poor stimulatory capacity of AKRH-2 ^bspleen cells was also shown not to be due to the loss of the stimulatory Mls ^aallele during the construction of the congenic strain from AKR and C57BL/6 parental strains. Using stimulator cells from a second series of congenic mice, we found H-2 ^b(strain DLLP) again to represent a poorly Mls^a stimulatory H-2 haplotype. In addition, H-2^q (DBA/1) cells displayed very poor Mls^a stimulatory potential while H-2^d (D1.C) cells were efficient Mls^a stimulators. Again the effect was shown to be at the level of the stimulator cells. In toto, our findings indicate that the H-2 ^kand H-2 ^dhaplotypes encode strong Mls^a stimulatory potential while the H-2 ^band H-2 ^qhaplotypes determine poor Mls^a stimulatory potential in primary in vitro responses, measured as cell proliferation and IL-2 production.Abbreviations used in this paper: CTL cytotoxic T lymphocyte - IL-1 interleukin-1 - IL-2 interleukin-2 - MLR mixed lymphocyte reaction - NMS normal mouse serum     

A genetic analysis of natural resistance to nonsyngeneic cells: The role of H-2

The genetic control of natural resistance in vivo to four natural killer (NK) cell-resistant H-2 homozygous lymphoid tumor cell lines was investigated by following the survival and organ distribution of cells prelabeled with radioactive iododeoxyuridine. Backcross mice derived from DBA/2J and CBA/J parents were injected with H-2 ^dtumor cells and tumor cell elimination was lowest in H-2 ^dhomozygotes. Natural killer cell activity was also reduced in mice with the H-2 ^dhaplotype, but no direct correlation between NK cell levels against YAC-1 or SL2-5 lymphoma cells and natural resistance in vivo was demonstrable. Analysis of 23 BXD recombinant inbred strains indicated that natural resistance to H-2 ^dtumors was restricted to H-2 ^bstrains. There was no direct association of NK cell activity with H-2 type in the BXD strains and NK cell levels did not correlate with tumor survival in vivo. By comparing natural resistance to H-2 ^dand H-2 ^btumors in DBA/2, C57BL/6, B6D2F₁, and B10.D2 mice we found that H-2 nonidentity between the tumor and the host, rather than the host H-2 haplotype, determined whether natural resistance occurred. Again, NK cell activity against YAC-1 cells was not predictive of tumor survival in these strains. These results provide genetic evidence that NK cells alone cannot account for natural resistance to H-2 nonidentical cells of hemopoietic origin.     

Paired H-2-specific monoclonal antibodies react differently to red cells and lymphocytes

Two hybrid clones from a fusion of C57BL/6 anti-DBA/2 spleen cells and the myeloma line Sp2/0 secrete antibodies reactive with a product of the murine major histocompatibility complex (MHC). The two antibodies are provisionally designated 513.11 and 513.29. Both react in rabbit-complementmediated cytotoxicity with spleen cells of H-2 ^d , H-2 ^f , H-2 ^r , and H-2 ^p strains. In addition, both antibodies hemagglutinate red blood cells from these strains. S13.11 is also cytotoxic for H-2 ^a , H-2 ^k , H-2 ^u , and H-2 ^v spleen cells but does not hemagglutinate red blood cells from mice bearing these haplotypes. With the exception of H-2 ^v , this strain pattern mimics the public specificity H-2.8. Quantitative absorption of S13.11 shows that H-2 ^d cells are twice as efficient as H-2 ^k cells in their ability to remove the 513.11 antibody. 513.29 reacts weakly in cytotoxicity with H-2 ^kv spleen cells and does not react with cells from H-2 ^u or H-2 ^v . Blocking studies indicate that 513.11 and S13.29 react with the same or a closely related molecule on the cell surface.     

Polymorphic and autoreactive H-2-specific monoclonal antibody isolated after injections of syngeneic sendai virus-coated lymphocytes

An H-2-specific monoclonal antibody (mAb Q-1) was obtained from B10.Q (H-2 ^q) mice injected with syngeneic Sendai virus-coated cells. The IgM monoclonal antibody recognizes the public determinant H-2.25 shared by H-2 ^k (K ^k) and H-2 ^r haplotypes and cross-reacts with H-2^d, H-2^s, H-2^p, and H-2^q cells, the latter being the haplotype of the challenged B-cell donor. The binding of mAb Q-1 to H-2^d, H-2^s, H-2^q, and H-2^p cells was lower than to H-2^k and H-2^r and of decreasing affinity but could be clearly distinguished from the negative reactions with H-2^b and H-2^f cells. MAb Q-1 distinguishes between Sendai virus-coated and uncoated lymphocytes only cells with low-affinity binding. On virus-coated or infected (H-2^p, H-2^q, H-2^d, H-2^s) cells lysis was stronger than on normal lymphocytes. We interpret the enhanced lysis of Sendai virus-positive cells by mAb Q-1 to be due to recognition of a modified exposure of public H-2 determinants induced by Sendai virus.On leave from The Institute of Immunology and Experimental Therapy, Wroclaw, Poland     

Polymorphism of Hh-1, the mouse hemopoietic histocompatibility locus

The major goal of these studies is to more fully assess the polymorphism of the hemopoietic histocompatibility (Hh) genetic system. H-2 homozygosity is required for optimal immunogenicity of bone marrow cell (BMC) grafts, and hybrid resistance to grafts of parental strain BMC by irradiated H-2 heterozygous F₁ hybrid mice suggests that Hh-1 antigens are inherited recessively. The Hh-1 antigens are also expressed on other normal hematopoietic cells and lymphoid tumors, and natural killer cells are the effectors which mediate the elimination of BMC grafts in an Hh-specific manner. Previous studies have demonstrated three different antigens mapping to the Hh-1 locus near H-2D. We test the expression of Hh-1 on BMC of all nonrecombinant H-2 haplotypes of independent origin and H-2 ^j , a presumed natural recombinant. Hh-1 typing is based on the pattern of growth and rejection in a panel of hosts. F₁ hybrids with H-2 ^b , H-2 ^d , and H-2 ^k are produced and used as donors and hosts to confirm the phenotype. Grafts of b-, d-, and j-haplotype marrow serve as prototypical examples of determinants that are provisionally designated as 1, 2, and 3, respectively. We describe a new determinant, 4, in the k haplotype. It is non-codominantly expressed, maps to H-2D, and is also expressed on H-2^b BMC. NZW, H-2^Z grafts exhibit a phenotype similar to k, but express a unique determinant 5 which can be distinguished from determinant 4. This additional determinant is also expressed by the b haplotype. The d, f, and p haplotypes all express determinant 2, and grafts of j-haplotype marrow are found to express determinants 2 and 5 in addition to determinant 3. The q and r haplotypes are null for all known determinants. Finally, we describe a phenotype which is a new combination of previously described determinants: s-haplotype grafts express determinants 1, 2, and 4. The polymorphism of Hh-1 detected thus far consists of seven alleles which are combinations of five distinct determinants.     

Immunogenetic analysis ofH-2 mutations

Spleen cells carrying theH-2K ^b allele and sensitized against TNP-modified stimulator cells in vitro displayed a cytotoxic effect against TNP-modified target cells carrying a mutation in theH-2K ^b allele (haplotypesH-2 ^ba ,H-2 ^bd , andH-2 ^bf ). Similar crossreactivity in TNP-CML was observed in the reciprocal direction. Spleen cells carrying theH-2K ^k allele and sensitized against TNP-modified stimulators displayed a cytotoxic effect against TNP-modified target cells carrying a mutation in theH-2K ^k allele (haplotypeH-2 ^ka ) and vice versa. The effector cells in these assays were sensitive to anti-T cell serum in the presence of complement, and supernatants from immune cultures did not induce nonimmune cells to display a cytotoxic effect. Titration of effector cells from mutant and wild-type strains of theH-2 ^b haplotype indicated no detectable quantitative differences in their activities. These data demonstrate that crossreactivity in TNP-CML occurs in closely related allogeneic strains that have recently undergone mutation in theH-2 complex.     

Characterization of newly isolated monoclonal antibodies against MHC of a Japanese wild mouse

We have already developed nine B10.MOL congenic strains carrying H-2 haplotypes derived from Japanese wild mice, Mus musculus molossinus, with the C57BL/10 genetic background. To obtain monoclonal antibodies against the H-2 antigen of the Japanese wild mouse, we carried out cell fusion using spleen cells from the animal immunized with one of the B10.MOL strains, B10.MOL-SGR (H-2 ^wm7). As a result, 19 hybridomas producing monoclonal antibodies were produced. Analysis with the intro-H-2 recombinants derived from B10.MOL-SGR indicated that 8 of them reacted with the class I and II with the class II molecule. The class I antibodies were tested for their cross -reactivities on wild mice and on the panels of standard inbred and B10.MOL strains. Most of the antibodies reacted with both the Japanese wild mice and the other subspecies, including standard inbred, while two antibodies highly specific for the donor H-2K region reacted with only three wild-derived mice, two M. m. molossinus from Anj o and Shizuoka, Japan, and one M. m. domesticus from Pigeon, Canada. In addition, all of the other four antibodies reactive with the K antigen of B10.MOL-SGR also reacted with the same three wild mice. The wild mice belonging to different subspecies might share very similar H-2K antigenic determinants in spite of their genetic and geographical remoteness.     

Studies ofH-2K b mutant mice

Three newH-2 ^b mutant strains, B6.C-H-2 ^bm9 , B6.C-H-2 ^bm10 and B6.C-H–2 ^bm11 , are described. The three mutant strains are of the gain and loss type as they reject skin grafts reciprocally with the parental C57BL/6Kh. The mutations, which arose independently, are all allelic at the same locus as 11 other mutant strains already described. By complementation and other studies the mutated gene has been shown to beH-2K ^b . The strains were typed directly and by absorption with antisera specific for H-2K^b and H-2D^b private and public specificities and for Ia^b specificities. Each strain typed differently with these sera. The strain B6.C-H-2 ^bm9 was found to be serologically identical with C57BL/6. The strains B6.C-H-2 ^bm10 and B6.C-H-2 ^bm11 were found to have alterations in the private H-2K^b specificity, H-2.33, and in the public specificity, H-2.5, but to a different extent. B6.C-H- 2^bm10 had a marked decrease in the amount of H-2.33 expressed on the splenic cell surface as compared to C57BL/6 and also has a marked decrease in the expression of H-2.5 on both spleen and red blood cells. In comparison, B6.C-H-2 ^bm11 has a decrease in the expression of H-2.33 but an increase in the expression of H-2.5 on both splenic and red blood cells. The other H-2^b specificities appeared to be unaltered as compared with C57BL/6.     

Study of H-2 mutations in mice. IV. A comparison of the mutants M505 and Hz1 by skin grafting and serological techniques

The line B6.M505 is congenic with C57BL/6JY and carries a mutant form of theH-2 ^b haplotype designatedH-2 ^bd . The mutant site 505 was located by the F₁ tests in theK end of theH-2 gene complex. The M505 mice are histoincompatible with the B6.C(Hz1) line (haplotypeH-2 ^ba ) carrying another mutation in theK end ofH-2 ^b . Inability of M505 to complement Hz1 in tests with B6 skin grafting is considered as an evidence that the same gene was altered by both mutations. The gained H antigens of two mutants can cross-react in vivo as revealed by accelerated rejection of Hz1 skin grafts by B6 recipients presensitized with M505 spleen cells. The lost antigenic determinants are not identical as shown by accelerated rejection of B6 skin grafts by Hz1 hosts preimmunized with M505 spleen cells. Absorptions of the antiserum ASY-015, (d×a) anti-i, anti-H-2.33 with M505 spleen cells did not clear forH-2 ⁱ ,H-2 ^b andH-2 ^ba , and absorptions with Hz1 did not clear forH-2 ⁱ ,H-2 ^b , andH-2 ^bd . These results show that changes of histocompatibility determinants may be accompanied by loss of some haptenic determinants in the Hz1 and M505 mutations.     

Correlation of Qa-1 determinants defined by antisera and by cytotoxic T lymphocytes

Cytotoxic T lymphocytes (CTL) activated in H-2 identical, Qa-1 disparate mixed leukocyte cultures recognize H-2-nonrestricted target antigens indistinguishable by strain or tissue distribution from serologically defined Qa-1 antigens. Cloned Qa-l-specific CTL define determinants encoded by four Qa-1 genotypes; we used anti-Qa-1 sera in antibody blocking experiments to determine if these determinants reside on molecules recognized by Qa-1-specific antibodies. Antisera containing Qa-1.1-specific and TL-specific antibodies blocked recognition of two CTL-defined determinants associated with Qa-1 ^a . Although both Qa-1 and TL molecules are expressed on activated T cells from appropriate strains, our studies indicated that the CTL recognized Qa-1, not TL. In addition, anti-Qa-1.2 serum inhibited CTL recognition of Qa-1^b- and Qa-1^c-encoded determinants. Qa-1 ^d target cells are unique in that they express determinants recognized by anti-Qa-1^a CTL and by anti-Qa-1^b CTL. Killing of Qa-1 ^d targets by anti-Qa-1^a CTL was not inhibited by anti-Qa-1.1 serum, but was partially inhibited by anti-Qa-1.2 serum. Cytotoxicity of Qa-1 ^d cells by one anti-Qa-1^b CTL clone was inhibited by both anti-Qa-1.2 and anti-Qa-1.1 sera, indicating close association of both serological determinants with the determinants recognized by the CTL. Thus, all of the CTL-defined Qa-1 determinants resided on molecules recognized by Qa-1-specific antibodies, but anti-Qa-1^a CTL and Qa-1.1-specific antibodies did not have identical specificities.Abbreviations used in this paper B6 C57BL/6J - CAB concanavalin A stimulated lymphoblasts - CML cell-mediated lympholysis - CTL cytotoxic T lymphocyte - NMS normal mouse serum - MHC major histocompatibility complex - MLC mixed leukocyte culture - MR maximum release - SMDM supplemented Mishell-Dutton medium - SR spontaneous release     

Spontaneous loss and subsequent stimulation ofH-2 expression in clones of a heterozygous lymphoma cell line

The expression of H-2K^k antigens in a (C3H × DBA/2)F₁ lymphoma cell line growing in vitro was investigated with monoclonal antibodies specific for a public antigen of theH-2K ^k region (H-2.m3) in fluorescence analysis and microcytotoxicity assays and in cell-mediated cytotoxicity with allogeneically stimulated effector cells. Estimates of relative levels of H-2K^k-antigen expression obtained by the different methods were highly correlated. The uncloned, unselected population gradually lost H-2K^k surface antigen expression under culture conditions. This was due to the appearance of H-2K^k negative variants. Fifteen cloned sublines of a population enriched for cells expressing antigen H-2.m3 in the fluorescence activated cell sorter contained either two distinct populations, one consisting of H-2.m3 negative and one of H-2.m3 positive cells, or consisted of H-2.m3 negative cells only. The expression of the H-2.m3 determinant of H-2K^k paralleled that of other serological H-2K^k determinants and of H-2K^k target determinants for cell-mediated cytotoxicity. In nearly all clones where two populations could be detected, the proportion of H-2.m3 negative cells increased with time in culture. The amounts of H-2K^k antigen expressed by the clones appeared not to be correlated to the amounts of H-2D^k antigens on the cell surface as judged by cell-mediated cytotoxicity.In at least one clone and in the uncloned population, H-2K^k-antigen expression detectable by fluorescence analysis could be stimulated by growing the cells in the peritoneal cavities of (C3H × DBA/2)F₁ mice or by adding mouse interferon preparations to the cell cultures. The increase in susceptibility to cell-mediated lympholysis of cells grown in vivo paralleled the increase inH-2 expression detected by fluorescence. In contrast, cells growing in the presence of interferon in vitro showed reduced sensitivity to lysis by alloreactive lymphocytes, although H-2 antigens were strongly expressed as measured by fluorescence.     

Serological and complementation studies in four C57BL/6H-2 mutants

C57BL/6 (H-2 ^b ) mice, and four mutants (B6.C-H-2 ^ba , B6-H-2 ^bg1 , B6-H-2 ^bg2 , B6-H-2 ^bh ) derived from this strain after separate mutations had occurred at the same locus within theH-2 complex, were analyzed to determine whether the mutations had led to anyH-2 (or Ia) difference which could be detected serologically. The strains were typed directly with antisera specific for H-2K and H-2D public and private specificities and for the Ia specificities; quantitative absorption studies were also performed for the relevant H-2K^b, H-2D^d and Ia^b specificities. In no case was any quantitative or qualitative difference detected serologically between any of the strains. In addition, by using a variety of techniques to produce and assay for antibody, we failed to produce any antisera between the parental strains and the four mutants. TheH-2 mutations therefore appear to give rise to a type of antigenic specificity which is recognized byT cells and which generateT, but notB cell responses; nor are they recognized by H-2 or Ia alloantisera. The location of the mutating locus within theH-2 complex was shown by the complementation method to be within theK orIA region and not in theIB region, since crosses of the mutant strains with B10.A(4R) or D2.GD failed to complement for a subsequent C57BL/6 skin graft.     

Allostimulatory analysis of a newly-defined and widely-distributed Mls superantigen

We previously noted that Mls^a,c C58/J responder cells proliferated unexpectedly to H-2^k-compatible Mls^a or Mls^c prototypic stimulator cells in a primary mixed lymphocyte reaction. The present investigation was performed to evaluate whether the response of C58/J T cells to these H-2- and Mls-compatible stimulator cells could functionally identify a newly-defined member of the Mls superantigen family through its allostimulatory ability. We observed that C58/J responder cells also proliferated when cultured with H-2-compatible prototypic Mls^null, Mls^b (nonstimulatory), or Mls^a,c splenic stimulator cells. The widely distributed nature of the non-MHC ligand recognized by C58/J T cells is indicated by the finding that 11 of 12 H-2^k inbred mouse strains clearly expressed this specificity. A gradient of stimulatory capacity from low to high across this newly-defined non-MHC difference was detected with splenocytes from these different inbred mouse strains. The Mls^a,c genetic composition of C58/J was confirmed by the observation that crossing C58/J with parental B10.BR (responsive to both Mls^a and Mls^c determinants) generated F₁ progeny that were unresponsive to H-2^k-compatible Mls^a, Mls^c, or Mls^a,c stimulator cells. Like prototypic Mls^a and Mls^c, the non-MHC specificity recognized by C58/J responder cells, termed Mls^f, was particularly sensitive to radiation (versus mitomycin C) treatment of the stimulator cells, was greatly augmented after anti-IgD activation of splenic stimulator cells, was blocked with anti-MHC class II antibody, and was effectively presented by phenotypically normal female but not B cell-defective xid⁺ male CBA/N F₁ stimulator cells. Address correspondence and offprint requests to: J. J. Ryan     

H-2 effects on cell-cell interactions in the response to single non-H-2 alloantigens

Individual mice were tested for their proliferative T-cell response to H-Y- and H-3-incompatible stimulator cells in secondary mixed lymphocyte culture. Responders expressing the H-2 ^bhaplotype were restricted in their response to stimulators presenting H-Y and H-3 in the context of H-2 ^b. Lymphocytes from individual B10 females proliferated in response to H-Y presented with I-A ^band D ^b. The ratio of I-A ^b/D ^b-restricted responses varied between individual responders, indicating significant qualitative variation between genetically identical responders. The majority of the proliferative response in all tested mice was restricted to the entire H-2 ^bhaplotype suggesting complementation of I-A ^b- and D ^b-region genes in presenting the H-Y antigen. Similar observations were made in the response of individual B10.LP mice to the H-3 antigen. H-3-specific, proliferating T cells were restricted to H-3 antigen presented with K ^bA^band D ^bwith significant variation between individuals in their preference for H-3 plus K ^bA^band D ^b. In contrast to the response to H-Y, the proliferative response to H-3 plus H-2 ^bcould be accounted for by the summation of the proliferative responses to H-3. plus K ^bA^band D ^b. These observations demonstrate that the proliferative response to non-H-2 H antigens in the context of I-region determinants is not a sine qua non for the T-cell response to these antigens. Further, the individual qualitative and quantitative variation observed with individual genetically identical mice has strong implications for our knowledge of intrastrain variation in immune responsiveness and the characterization of inbred strains for immune responsiveness.     

Genetics of graft-versus-host reactions (GVHR)

Injection of 20×10⁶ donor lymph node cells (LNCs) into newborn allogeneic recipients incompatible with donors at theIC subregion of the mouseH-2 complex evoked both GVH splenomegaly and GVH mortality. The strength or severity of the allogeneic reactions induced varied as a function of the interallelic strain combination and was influenced particularly by properties of the recipientIC determinants. Thus,IC ^s determinants on recipient cells led to strong GVHR, whileIC ^d determinants induced a moderate GVHR, even when donors carrying differentIC alleles were used. However, responder donor genes also affected the degree of GVHR in some combinations. The effect on donor GVH potential of pre-exposing B10 donors to antirecipient antiserum (B10 anti-B10.A) was also studied. Spleen cells from B10 donors pre-exposed to alloantiserum for two to seven days exhibited a markedly reduced ability to cause GVH splenomegaly and GVH disease in newborn B10. A or (B10. A × B10) F₁ recipients. Inhibition of donor lymphocyte GVH potential waned eight to 14 days after antiserum pretreatment. Inhibition was shown to be specific for variousH-2 determinants and to be caused by antirecipient alloantibodies. Pre-exposure of donors to alloantiserum reduced the GVH potential of spleen cells but did not affect LNC reactivity.Ia antibodies and, to a lesser extent, anti-H-2D serum were shown to be able to inhibit GVHR. The results suggest that the observed reduction in donor GVH reactivity is caused by antibody-mediated central feedback inhibition. Anti-H-2 alloantibodies evidently play an important role in the network regulating allogeneic responses.     

Immune response to the Slp alloantigen in the mouse:H-2-linked qualitative and quantitative control

Immunization of inbred mouse strains lacking the Slp allotype results in the production of Slp antibodies in some strains but elicits no detectable response in other strains. Analysis of standard inbred and congenic resistant strains reveals that both the qualitative and quantitative ability to respond to the Slp allotype is associated with theH-2 haplotype of the recipient. Three different response phenotypes can be identified utilizing complement fixation and quantitative immunodiffusion tests. Strains which carry theH-2 ^q haplotype are high responders,H-2 ^k strains are intermediate in response, whileH-2 ^b andH-2 ^v strains produce no detectable antibody. The characteristic response patterns of high and intermediate responders were manifest by day 35 of immunization and continued as discrete response types after a second booster. Quantitative data in the immune response of the intra-H-2 recombinant B10.A(4R) suggest that the recombination event which established theH-2 ^h4 chromosome disturbs the proper function of the genetic determinant controlling response to Slp.