The specificity and significance of the inhibition of Fc receptor binding by anti H-2 sera

The possibility that Ia antigens are unique among H-2 antigens in their relationship to the Fc receptor was investigated in an EA rosette assay. Antibody specific for antigens in various regions of theH-2 complex was incubated with mouse cells, and the ability of the cells to form rosettes with antibody-coated chicken erythrocytes was tested. Antibody raised against the H-2 antigens of Ia-negative tumor cells was highly effective in inhibiting rosette formation. A variety of antisera againstK-, I-, andD-region antigens tested in recombinant mice inhibited EA rosette formation, suggesting that antigens in each of these regions could be detected in rosette inhibition. The F(ab′)₂ fragments of all antisera tested also produced specific EA rosette inhibition. Finally, antibody against Ia antigens failed to inhibit bone marrow RFCs, although antibody against H-2K and H-2D antigens did inhibit. Although H-2 serology is in a state of rapid change at present, it must be concluded that in this assay, antibody against antigens in theK andD regions as well as theI region can inhibit EA rosette formation. Inhibition of these rosettes by anti H-2 sera is therefore not due to a special association of Ia antigens with Fc receptors.     

Cytotoxicity of autosensitized lymphocytes restricted to the H-2K end of targets

Lymphocytes from rodents cultured on syngeneic fibroblasts become cytotoxic against syngeneic but not against allogeneic target cells. We investigated whether known antigens are involved in the phenomenon and the data indicate that H-2 antigens must be shared between sensitizing fibroblasts and responder lymphocytes to generate autocytotoxic cells. Furthermore, the cytotoxicity of autosensitized lymphocytes is restricted to target cells identical with respect to theK and/orI regions. F₁ hybrid lymphocytes cultured on parental fibroblasts develop cytotoxicity towards sensitizing cells. In contrast, parental lymphocytes cultured on F₁ hybrid fibroblasts will not damage the F₁ cells, although they are cytotoxic against both syngeneic and allogeneic parental cells. In addition, parental or F₁ hybrid lymphocytes cultured on parental fibroblasts are not cytotoxic against F₁ hybrid target cells. Fibroblasts heterozygous for theK end only, are also resistant to the cytotoxic action of such lymphocytes. Thus it seems that H-2 antigens, specifically theK end, antigens have a significant role in the phenomenon of autosensitization.     

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

Immune response (Ir) genes mapping in theI region of the mouseH-2 complex appear to regulate specifically the presentation of a number of antigens by macrophages to proliferating T cells. We have investigated the possibility that similarIr genes mapping in theH-2K andH-2D regions specifically regulate the presentation of target antigens to cytotoxic effector T cells. We report that the susceptibility of targets expressing specific non-H-2 H alloantigens to lysis by H-2-compatible, H-antigen-specific cytotoxic effector T cells is controlled by polymorphicH-2K/D genes. This control of susceptibility to lysis is accomplished through what we have defined operationally as antigen-specific regulation of non-H-2 H antigen immunogenicity. High immunogenicity of the H-4.2 alloantigen is determined by a gene mapping in theH-2K region ofH-2 ^b . However, high immunogenicity of H-7.1 is determined by a gene mapping in theH-2D region ofH-2 ^b . High immunogenicity of the H-3.1 alloantigen is determined by genes mapping in both theH-2K andH-2D regions ofH-2 ^b . Therefore, genes mapping in theH-2K andH-2D regions serve a function in presenting antigen to cytotoxic effector T cells. This function is analogous to that played byI-regionIr genes expressed in macrophages which present antigen to proliferating T cells. We present arguments for classification of theseH-2K/D genes as a second system ofIr genes and discuss the implications of twoH-2-linkedIr-gene systems, their possible functions, and their evolution.     

Analysis ofH-2 mutants: Evidence for multiple CML target specificities controlled by theH-2K b gene

C57BL/6 (H-2 ^b ) mice and two mutants derived from this strain, B6.C-H-2 ^ba (Hz1) andE6-H-2 ^bd (M505), were studied in a number of functional tests, in vitro and in vivo, that assay for differences at theH-2 complex. All three strains give rise to reciprocal mixed lymphocyte reactivity (MLR) and cell-mediated lympholysis (CML) in vitro as well as graft-host reactivity (GVHR) and skin graft rejection in vivo. Analysis for cross-reactivity between these strains in CML revealed that the gained antigens in each mutant do not cross-react, and that Hz1 has lost an antigen shared by C57BL/6 and M505 strains. In addition, spleen cells from B10.A(4R) mice, which differ from theH-2 ^b haplotype only at theK end of theH-2 complex, recognize a common antigen shared by all three strains tested. Provided that the mutations occurred in theH-2K ^b gene, these data indicate that a) there are at least three antigenic specificities coded for by theH-2K ^b gene(s) that serve as targets for receptors on thymus-derived (T) cells in CML; b) since C57BL/6 strain mice and the mutants are serologically indistinguishable on a qualitative basis, the antigens recognized by the receptors on T cells and by humoral H-2 antibody are nonidentical; and c) mutation in theH-2K ^b locus itself can give rise to allogeneic recognition phenomena such as MLR and GVHR.     

A T-cell antigen system of chickens: Ly-4 and Marek's disease

A search was made for lymphocyte antigens associated with resistance or susceptibility to the T-cell lymphoma induced by the herpes virus of Marek's disease (MD), the experimental model for Burkitt's lymphoma of humans. Antisera were produced by reciprocal immunization with whole blood between an MD-resistant and susceptible line of chickens compatible at the major histocompatibility complex (MHC), and were tested against lymphocytes of both lines. The lymphocytes were not agglutinated, immobilized, or lysed, but their ability to evoke graft-versus-host (GVH) splenomegaly was reduced. This inhibitory activity was line-specific, and these sera had a maximum limiting effect on GVH splenomegaly at a dilution of 1/50 and a minimum at 1/800 dilution. A test based on the differential limitation of GVH splenomegaly by a pair of alloantisera was used to identify the antigens in F₁ and F₂ generations. The segregation results established a locus,Ly-4, with two codominant alleles,Ly- 4^a andLy-4 ^b .Ly-4 is distinct from theA, B, orC blood group loci and from theBu-1 locus determining B-cell antigens, but may be linked to theTh-1 locus determining T-cell antigens (recombination frequency of 32 percent). Tentative evidence was obtained from comparisons of homozygous F₂ and F₃ progeny for association of theLy-4 allele characteristic of the susceptible line with increased incidence of MD.     

Cytotoxic T-cell responses to H-Y:Ir genes and associative antigens map inH-2

The secondary cytotoxic responses to the male-specific antigen (H-Y) in mice showH-2 restriction so that the cytotoxic female cell must share the K- and/or D-end antigen with the male target cells. The association with the K and/or D end varies with differentH-2 haplotypes,e.g., H-2 ^b cytotoxic cells require the H-2D^b antigen(s) on the target cells, while cytotoxic cells fromH-2 ^b/H-2 ^d F₁ mice sensitized toH-2 ^d male cells kill only male targets having H-2K^d antigen(s). This association of H-Y with appropriate K/D antigens seems to be needed also in the induction of the cytotoxic response. Of the independent haplotypes, onlyH-2 ^b strains are capable of making secondary anti-H-Y responses and this trait seems to be dominant,i.e., the F₁ strains with oneH-2 ^b parent are able to produce anti-H-Y cytotoxic cells against both theH-2 ^b parent and the nonresponder parent. The mating of the two nonresponder strains may produce F₁ mice which are responders, thus suggestingIr gene complementation. Mapping data indicates that at least one of these complementary genes is located in theI-C region fork/s complementation.     

B lymphocyte alloantigens controlled by theI region of the major histocompatibility complex in mice

An antiserum was produced by reciprocal immunization of congenic resistent inbred strains of mice which differed only with respect to theI andS regions of theH- 2 complex. This antiserum permitted the serological detection of lymphocyte alloantigens, designated Ia (=I region associated antigens). Ia determinants are only present on mature B lymphocytes. They could not be found on thymocytes, splenic or lymph node T cells, or on the majority of bone marrow cells. Absorption studies demonstrated existence of several Ia specificities which are associated with differentI region types. Thus, theI region of theH- 2 complex appears to control not only T-cellexpressed antigen specific immune response genes, but also B-cell-expressed Ia determinants. The relevance of the Ia alloantigen system for cellular interaction in immune reactions is discussed.     

Variable effect of anH-21 barrier on response to skin allografts in the mouse

(AQR×B10)F₁ mice were grafted with skin from donors differing in theK, I, KI, andISD regions of theH-2 complex. A dichotomy was observed in the fate of theH-2I-disparate grafts: either they were rejected acutely within the second week or were accepted indefinitely. Acceptances were much more common among male than female hosts. Acceptor status was limited to the I group, was unpredictable in occurrence, was not well-correlated with positive serum anti-Ia titers, and did not confer protection of grafts that were alike atH-2I but different atH-2K orH-2D. Since theH-2I barrier studied here elicited such divergent responses in genetically identical hosts, it is unlikely that any histocompatibility typing test could predict graft fate.Abbreviations used in this paper are MST median survival time - MHC major histocompatibility complex - CTL cytotoxic T lymphocyte - B10 C57 BL/10 - 6R BIO.T(6R) - B10.A BIO. ASn - H-2-Ia serologically detected antigens coded in theI region ofH-2 This term is used in preference toIa, since it has recently been shown that Ia-like alloantigens may be coded outside the MHC (Dickleret al. 1975).     

Immune response of mice to the Thy-1.1 antigen: Effect of theIr-5 alleles studied in 129/J and B10.129 (6M) mice

The primary immune response to the Thy-1.1 antigen was measured by a plaque assay that detected cells producing antibodies lytic for AKR thymocytes. B10.129(6M) mice carrying theH-2 complex of an intermediate responder (129) on a low-responder (B10) background, were low responders. Studies employing different F₁ hybrids and segregating generations of 129/J and 6M mice indicated that differences in responsiveness of those two strains depend on alleles at a single locus, loosely linked to theH-2 complex. These results lend further support to the previously advanced concept that the expression of theIr-Thy-1 allcles controlling the response to the Thy-1.1 antigen is influenced by the alleles at theIr-5 locus. In addition, studies employing F₁ hybrids produced through matings of 129/J, 6M, C3H.B10 and C57BL/6J mice to a panel of inbred strains suggested that in regard to the responsiveness to the Thy-1.1 antigen, 129/J and 6M mice are phenotypically, and presumably genotypically, similar to C3H.B10 and C57BL/6J mice, respectively.     

Effect of mouse genotype on interferon production II. Distribution ofIf-1 alleles among inbred strains and transfer of phenotype by grafting bone marrow cells

TheIf-1 alleles of many inbred strains, some of common parentage with either BALB/c Gif or C57BL/Lac, were determined. Of the 23 inbred lines examined, all were eitherIf-1 ^h orIf-1 ^l , except the C57BR/cdJ strain, which gave intermediate results; the latter will have to be explored more thoroughly before the existence of a third allele can be established. Survey of the 23 lines showed no correlation betweenH-2 haplotypes andIf-1 alleles. The absence of linkage betweenH-2 andIf-1 was confirmed by typing (BALB/c×C57BL)F₁×C57BL backcross progeny for bothH-2 andIf-1. The fact that some high and some low producers were of sameH-2 type made it possible to study the production of interferon in radiation chimeras. Mice of a low-producer strain, C3H/Lac (If-1 ^l , were lethally irradiated and their hemopoietic function restored by grafting bone marrow from eitherIf-1 ^l orIf-1 ^h mice, all donors of the sameH-2 haplotype as that of the recipients (H-2 ^k . NDV-induced serum interferon production was measured 31 days after irradiation and restoration. The production of interferon in all mice restored with marrow fromIf-1 ^l donors was equal or lower than that of syngeneic chimeras (alsoIf-1 ^l ). In contrast, titers of interferon in all four groups restored withIf-1 ^h marrow were higher than those of control chimeras, three being higher than titers in unirradiatedIf-1 ^l controls. These data indicate that theIf-1 locus is expressed through cells derived from hemopoietic stem cells, possibly the interferon-producing cells themselves.     

Evidence for a third,Ir-associated histocompatibility region in theH-2 complex of the mouse

Skin grafts transplanted from B10.HTT donors onto (A.TL × B10)F₁ recipients are rapidly rejected despite the fact that the B10.HTT and A.TL strains should be carrying the sameH-2 chromosomes and that both the donor and the recipient contain the B10 genome. The rejection is accompanied by a production of cytotoxic antibodies against antigens controlled by theIr region of theH-2 complex. These unexpected findings are interpreted as evidence for a third histocompatibility locus in theH-2 complex,H-2I, located in theIr region close toH-2K. The B10.HTT and A.TL strains are postulated to differ at this hypothetical locus, and the difference between the two strains is explained as resulting from a crossing over between theH-2 ^t1 andH-2 ^s chromosomes in the early history of the B10.HTT strain. TheH-2 genotypes of the B10.HTT and A.TL strains are assumed to beH-2K ^s Ir ^s / ^k Ss ^k H-2D ^d andH-2K ^s Ir ^k Ss ^k H-2D ^d , respectively. Thus, theH-2 chromosomes of the two strains differ only in a portion of theIr region, including theH-2I locus. The B10.HTT(H-2 ^tt) and B10.S(7R)(H-2 ^th) strains differ in a relatively minor histocompatibility locus, possibly residing in theTla region outside of theH-2 complex.     

Properties of reticulum cell sarcomas in SJL/J mice

While T cells from SJL and from F₁ hybrids of SJL that do not express I-E antigens give strong proliferative responses to RCS, T cells from F₁ hybrids expressing surface I-E do not. The nature of the stimulating antigen on the RCS cell surface was examined using monoclonal antibodies. Complete inhibition of the T-cell proliferative response was obtained with antibodies to I-A antigens, whereas antibodies to I-E antigens did not inhibit at all. This inhibition was mediated via an effect of the antibodies on the stimulating cells. Biochemical characterization of immunoprecipitated ¹²⁵I- and 's S-labeled RCS antigens was performed using two-dimensional gel electrophoresis. Using this technique, I-A antigens were readily detected. However, neither Ia.7-specific antibodies nor antibodies specific for E_α : E _β complexes precipitated any E alpha or E beta chains. Comparison of I-A antigens from RCS and normal SJL spleen cells revealed minor mobility differences in the gels, possibly due to differences in glycosylation, the significance of which needs to be further evaluated. Examination of RNA extracted from RCS, using E alpha and A alpha cDNA probes showed that RCS cells do not transcribe the E alpha gene as has been shown previously for normal H-2 ^s cells. Furthermore, DNA from RCS cells showed a defect in the E alpha gene similar to that known to exist in normal H-2 ^s cells. Our findings exclude the presence of E alpha on RCS cells and suggest a major role for I-A, either alone or in conjunction with another as yet unidentified cell surface antigen, in the stimulation of T cells.     

Immune response of mice to Thy-1.1 antigen: Studies on congenic lines

The primary response to Thy-1.1 antigen was measured by a plaque assay that detected cells producing antibodies lytic for AKR thymocytes (PFC). TheH-2 congenic mice (B10.K and B10.BR) carryingH-2 complexes of high responders (CBA and C57BR) on the low-responder background (B10) were found to produce significantly fewer PFC than the corresponding donor of theH-2 complex. On the other hand, C3H.B10 mice carrying theH-2 complex of a low responder on the high-responder background produced significantly more PFC than the donor of theH-2 complex. These findings were interpreted as evidence that alleles at previously described loci believed to be components of theI region of theH-2 complex and controlling immune response to Thy-1.1 are influenced by alleles at another locus. Studies of segregating populations of theH-2 congenic lines supplied evidence that this locus, tentatively calledIr-5, is in chromosome 17 (linkage group IX).     

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.     

Histocompatibility-2 system in wild mice

Ninety-six wild mice trapped at 13 localities in the state of Texas were tested in the dye-exclusion cytotoxic test with a battery of 49 oligospecific H-2 antisera. The antisera detected 36 class I (K and D) and 10 class II (Ia) antigens. The phenotypic frequencies of private class I antigens ranged from 1 to 20%, the majority of them being in the range between 1 and 5%. At least some of the higher frequencies resulted from the presence of more than one antibody in the typing reagents, and from other factors complicating the typing. We estimate that the frequencies of most of the class I alleles among Texas wild mice are 1% or less. This estimate leads to the prediction that at least 200 alleles exist in Texas mice at theH-2K locus, and another 200 alleles exist at theH-2D locus. Frequencies of most of the class I public antigens were in excess of 20%. In the sample of 96 mice, 46 different phenotypic combinations of private class I antigens were found, and the frequency of blanks (mice unreactive with any of the antibodies to private class I antigens) was 27%. The frequencies of private class II antigens ranged from 5 to 15%. Some of the public class II antigens, in particular those controlled by theE region, occurred with frequencies of 80% or higher. The class II antigens were found in 26 phenotypic combinations. No striking linkage disequilibrium was found either between K and D antigens, or between class I and class II antigens. The polymorphism of theK, A, andD region appears to be higher than that of the corresponding regions of the human or rat major histocompatibility complex. The polymorphism of theE region is significantly lower than that of theA, K, andD regions. The polymorphism of theA region is extensive.     

Lymphokine production in mouse mixed lymphocyte reaction (MLR)

We have investigated alloantigen differences which stimulate lymphokine release and³H-TdR uptake in primary ‘one-way’ MLC among allogeneic mice. When mice differing at the wholeH-2 region were tested, MIF and immune IF release was observed, along with a marked³H-TdR uptake. Differences atK, D, orI-S-G regions stimulate both lymphokine release and³H-TdR uptake, though stronger immune IF and³H-TdR responses were observed with differences atI-S-G regions. On the other hand, when mice differing in their minor histocompatibility antigens, and notably at theMls locus, were tested, lymphokine release took place even in the absence of proliferation. Lastly, in MLC between mice differing at multiple minor loci, butH-2 andMls matched, MIF release only, and not immune IF and³H-TdR responses were observed in a few combinations. These findings show that T lymphocytes can recognize alloantigens by releasing lymphokines even without going through proliferation. Moreover, different levels of T-lymphocyte activation exist, depending on the kind of stimulating alloantigens present.     

Idiotypic regulation of mouse anti-H-2 antibody responses

Mouse-immunoglobulin (MIg) tolerant rabbits immunized with mouse H-2 antibodies produced anti-idiotype antisera, which were reactive towards specific B- and T-cell receptors. One such rabbit antiserum (from rabbit 5936) defines a family of idiotypes (Id) designated 5936-idiotypes (Rubin et al. 1979). The present experiments were performed in order to establish (1) the nature of 5936-Id⁺ serum molecules, (2) the specificity of 5936-Id⁺ serum molecules, (3) the association of the 5936-Id genes to allotype and/orH-2 genes and (4) the immunological role of 5936-Id⁺ serum molecules. A sensitive, radioimmunoassay employing¹²⁵I-labelled-F(ab)₂ fragments of B6 anti-B10.BR MIg pool, 5936 antiserum, and a sheep anti-rabbit immunoglobulin antiserum, was used.—The results suggested that 5936-Id⁺ serum molecules were exclusively MIg, and that they were mainly of the IgG₁ class. Such molecules were induced in B6 mice (H-2 ^b /Ig-1 ^b ) upon immunization with H-2^k but not with H-2^q alloantigen or conventional antigens. The 5936-Id were found to be associated with Ig-1^b allotypes and theH-2 ^b complex may contain immune response (Ir) genes which, in comparison withIr genes inH-2 ^d andH-2 ^s , favor the expression of 5936-Id.—Adsorption of 5936-Id⁺ B6 anti-CBA MIg preparations on CBA (IA^k) spleen cells demonstrated that CBA antibodies were 5936-Id^?. It is dicussed whether 5936-Id⁺, IgG₁ molecules in B6 anti-CBA sera are anti-(anti-CBA) antibodies or nonspecific antibodies, the production of which is augmented by immunization with IA^k alloantigen.     

Genetic analysis of anH-2 mutant,B6.C-H-2 ba,using cell-mediated lympholysis: T- and B-cell dictionaries for histocompatibility determinants are different

B6.C-H-2 ^ba [H (z1)] is a mutant derived from C57BL/6. The two strains mutually reject their skingrafts and are incompatible in the mixed leucocyte reaction (MLR) and in cell-mediated lympholysis (CML) assays. They are serologically indistinguishable. This report shows that H(z1) carries a new, privateK end CML specificity clearly distinguishable from that of B6 by a third party strain, HTG. Antisera directed against the private H-2K specificity of B6 present on H(z1) cells) can block CML between the two strains in either direction. The new CML specificities of H(z1) cross-react with (public) CML specificities controlled by bothK andD regions of other unrelated haplotypes. The results suggest that H(z1) carries a mutation in theH-2K locus itself or in a closely linked gene, the product of which is also physically associated with the H-2K molecule corresponding to the cis-configuration of the alleles in both loci. These findings indicate that T- and B-cell dictionaries for histocompatibility determinants are different.     

Genetics of histocompatibility in mice

Twenty-five new congenic lines with distinctive BALB/cBy-strain histocompatibility alleles introduced onto the C57BL/6By-strain background by a regimen of backcrossing and tailskin grafting have been established. Twenty-one of the histocompatibility loci represented by these lines are new, while four duplicate theH-1, H-2, H-7, andH-8 loci identified by Snell.