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.     

BALB/c-H-2 db : A newH-2 mutant in BALB/cKh that identifies a locus associated with theD region

A newH-2 mutant, BALB/c-H-2 ^db , is described. This mutant originated in BALB/c, is inbred, and is coisogenic with the parental BALB/cKh strain. The mutation is of the loss type since BALB/c-H- ^db rejects BALB/c, but not vice versa. Complementation studies have localized the mutation to theD region of theH-2 complex. A cross between BALB/c-H-2 ^db and B10.D2-H-2 ^da failed to complement for either BALB/c or B10.D2 skin grafts, indicating that these are two separate mutations at the same locus (Z2). Direct serological analysis and absorption studies revealed that, with one exception, theH-2 andIa specificities of BALB/c and BALB/c-H-2 ^db are identical. In particular,H-2.4, the H-2D^d private specificity, is quantitatively and qualitatively identical in the two strains. The exception is that of the specificities detected by antiserum D28b: (k×r)F₁ anti-h, which contains anti-H-2.27, 28, and 29. These specificities appear to be absent from theH-2 ^db mutant since they are not detected directly or by absorption. Other public specificities are present in normal amounts,e.g., the reaction with antisera to H-2.3, 8, 13, 35, and 36. The reaction with antiserum D28 (f×k)F₁ anti-s, which contains antibodies to H-2.28, 36, and 42, is the same in both strains. Antiserum made between the two strains (H-2 ^db anti-H-2 ^d ) reacts like an anti-H-2 serum, in that it reacts with both T and B cells by cytotoxicity, but is not a hemagglutinating antibody. The serum reacts as does the D28b serum in both strain distribution and in cross-absorption studies. We conclude that theH-2 ^db mutation occurred at a locus in theD region, resulting in the loss of the H-2.28 public serological specificity and of a histocompatibility antigen. Whether these are one and the same antigen is not yet known. The data, in view of other evidence, imply that the public and private specificities are coded for by separate genes.Abbreviations used in this paper are as follows CML cell-mediated lysis - MLR mixed lymphocyte reaction - GVHR graft-versus-host reaction - RFC rosette-forming cells - RAM-Ig rabbit anti-mouse IgG     

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.     

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.     

The role ofH-2 regions in overcoming tissue incompatibility

Neonatal transplantation tolerance to the products of theH-2 ^b complex was induced in B10.A (H-2 ^a ) mice. On the basis of the survival of skin allografts it was found that antigens determined by theD region of theH-2 ^b complex (of the B10.A(2R) strain) were most easily overcome and that tolerance to the products of theD end of theH-2 complex (of the B10.A(4R) strain) was also easy to induce. The antigens produced by theK end ofH-2 (of the B10.A(5R) and B10.A(3R) strains) represented a stronger incompatibility barrier and a difference in the entireH-2 ^b complex caused strongest resistance to tolerance induction. When tolerance to the products of the entireH-2 ^b complex was induced in newborn B10.A mice, and the neonatally treated animals were grafted simultaneously with five different grafts, those disparate at theK end ofH-2 and in the entireH-2 region were rejected in some animals, while the grafts disparate at theD end of H-2 remained intact in the same mice. No dependence on theI-J subregion was observed in this system. Furthermore, tolerance was more easily inducible in male than in female B10.A mice.     

Spleen cells from animals neonatally tolerant to H-2Kk antigens recognize trinitrophenyl-modified H-2Kk spleen cells

A.TL mice injected with (A.AL × A.TL)F₁ cells within 24 hours after birth were rendered tolerant to H-2K^k antigens, as evidenced by acceptance of A.TL skin grafts. When spleen cells from these tolerant animals were cocultured with A.AL stimulator cells, no cytotoxic effector cells were generated in a cell-mediated lympholysis assay. However, when the A.AL stimulator cells were derivatized with trinitrophenol, effector cells that displayed a cytotoxic effect against trinitrophenyl-modified H-2K^k target cells were generated. These data indicate that animals tolerant to H-2 determinants but chimeric to only a minor extent possess cytotoxic precursor cells in sufficient frequency to mount a primary in vitro response against trinitrophenol in the context of an allogeneicH-2K region.     

The production,testing, and utility of double congenic mouse strains

Two new double congenic strains, B10-H-2 ^a H-7 ^b /Wts and B10-H-2 ^d H-7 ^b /Wts, were selected to differ from B10.A and B10.D2/o, respectively, at theH-7 locus. The survival time ofH-7-incompatible skin grafts is dependent upon theH-2 haplotype of recipient and donor.     

CML typing of serologically identicalH-2 mutants

Cell-mediated lympholysis (CML) reactions were studied among four strains of C57BL/6 (B6) mice carrying mutant alleles (H-2 ^ba ,H-2 ^bd ,H-2 ^bg , andH-2 ^bh ) at thez1 locus in theK end ofH-2 ^b and the original B6 (H-2 ^b ) strain. Cross killing of target cells from lines that had not participated in the mixed lymphocyte reaction (MLR) was extensive, but usually less intense than that of target cells of stimulator cell genotype. The extent of CML crossreactivity could be limited by using cells from F₁ hybrid mice as responders in MLR. In a comprehensive analysis of the cytotoxicity exerted by 20 MLR combinations with homozygous, and 10 MLR combinations with F₁ hybrid responder cells, 19 different CML cytotoxicity patterns were identified, corresponding to at least 19 different CML target specificites. When the number of CML mismatches of each mutant with the originalH-2 ^b was calculated,H-2 ^ba was found to be most distinct fromH-2 ^b ,H-2 ^bs andH-2 ^bd were closest toH-2 ^b , andH-2 ^bh occupied an intermediate position. The validity of this sequence of relatedness is supported by published reports on skin graft survival times and on the interaction of T lymphocytes with virus-infected target cells using cells fromz1 locus mutants.     

Do histocompatibility antigens recognize themselves?

In the Simonsen spleen weight assay, theH-2K ^ba mutant does not respond against theH-2K ^bd mutant orH-2K ^bd /H-2K ^b hybrid, while the parentalH-2K ^b haplotype does respond. TheH-2K ^ba /H-2K ^b hybrid reacts strongly to bothH-2K ^bd andH-2K ^bd /H-2K ^b , indicating that the donor genotype could influence the reactivity against the same antigenic difference. The response of theH-2 ^ba mutant against a number of unrelated H-2 antigens does not differ from that of the parental haplotype. TheH-2K ^bd mutant reacts againstH-2K ^b andH-2K ^ba , and theH-2K ^b parent reacts against both theH-2K ^ba andH-2K ^bd mutants. The specific defect of reactivity in theH-2K ^ba mutant is effectively complemented by crossing with a number of unrelatedH-2 haplotypes. TheH-2 ^ka andH-2 ^fa mutants complement poorly compared to corresponding parental strains CBA and A.CA, while the B10.M (H-2 ^f ) strain does not complement at all (which is probably attributable to an undetectedH-2 mutation in the last strain). The data strongly suggest that the product of theH-2K locus-which is known to function as a transplantation antigen, lymphocyte activating determinant, and serologically defined antigen-also influences the immune response capacity against a mutant histocompatibility determinant.     

Cardiac allografts in mice congenic at non-H-2 histocompatibility loci

Neonatal mouse heart fragments were grafted under the ear skin of adult recipients. Cardiac allograft survival was evaluated by visual observation of pulsation, electrocardiography, and histology. Employing a series of congenic resistant strains differing from C57BL/10Sn at theH-1, H-3,H-4, H-7, H-8, H-9, H-10, H-11, andH-12 loci, the median survival times of the heart grafts to and from C57BL/10Sn were obtained. The various interallelic combinations resulted in a wide variation of graft survival. Reciprocal transplants frequently showed different survival times.H-1 ^c grafts were rejected by B10.129(5M)/nSn female mice with a median survival time of 90 days.H-1 ^b grafts were not rejected by C57BL/10Sn mice for the experiment's duration of 200 days. The weaker the histocompatibility barrier, the more variable the survival times and the smaller the ratio of rejected to total grafted heart fragments. Female recipients were observed to reject their grafts more rapidly and to reject a higher proportion than males of the same strain. Although the strength of the different non-H-2 barriers generally paralleled that determined by skin transplants, the rankings of the strongest minor barriers were not the same for both tissues.     

Study of anH-2K d mutant strain: C.B6-H-2 dm4

A new-H-2 mutant involving theH-2 ^d haplotype is described — C.B6-H- 2^dm4 (dm4). This mutant strain carries a gain and loss mutation which maps to theK ^d gene of theH-2 complex. Serological testing comparing the mutant and the parental BALB/cKh strain failed to detect any difference between the two strains and no antibodies could be produced, although a reciprocal mixed lymphocyte reaction was observed between mutant and parent.     

Liver tissue graft rejection in murine major histocompatibility complex mutants

Liver tissue grafts between seven H-2 mutants and their parental strains have been studied. Each of these mutants was originally identified by reciprocal mutant—parental strain skin graft rejection. However, liver grafts among mutants and parental standard strains are not uniformly rejected. Liver graft rejection also fails to correlate with mutant—parental stimulation in CML and MLC. In addition, the immune reaction pattern of female mutant animals against grafts of male liver differs from the reaction pattern found in parental standard strains. Several explanations for the differences between immune response to liver and skin grafts are proposed, including different T cell subsets involved in recognition, availability of antigenic sites to immunocompetent cells, and structural differences between mutant and parental H-2 antigens. Abbreviations used in this paper: bml, 2, 3, 4,14; dml; fm2=mutants of strains C57BL/6, B10.D2 and B10.M respectively; B6=C57BL/6     

Generation of cytotoxic lymphocytes in mixed lymphocyte reactions II. Importance of private and publicH-2 alloantigens on the expression of cytotoxicity

MLC were established to test for the generation of specific cytotoxic effector cells in CML. The target cell used to assay for CML in the five combinations tested was of a differentH-2 haplotype from the stimulating cell population. Cytotoxicity was observed against this target only when it shared private alloantigens (antigens that are specific for theH-2D andH-2K region of differentH-2 haplotypes) with the stimulating cell population. Very weak or no Cytotoxicity was found when such alloantigens were not shared, although cross-reactive publicH-2 specificities were. These findings indicate that T cells display a cytotoxic potential against privateH-2 antigens in a primary response in vitro and are not capable of responding to publicH-2 specificities to the same level.BSS balanced salt solution - CML cell-mediated lympholysis - GPC guinea pig complement - ¹²⁵IUdR ¹²⁵I-iodo-deoxyuridine - MLC mixed lymphocyte culture - SE standard error     

Bone-marrow-cell grafts involving theH-2D andH-2L mutant haplotypes

TwoH-2 ^d mutants,H-2 ^dm2 (H-2L loss mutation) andH-2dm1 (gainplus-loss mutation involving bothH-2L andH-2D) were evaluated for any change in the immunogenicity of marrow stem cells. Grafts of 2 or 4 × 10⁶ BALB/c(C) or BALB/c-H-2^dm2 (C-H-2 ^dm 2) marrow cells were accepted by lethally irradiated B10.D2(H-2 ^d ) recipients and were rejected by irradiated B10(H-2 ^b ) recipients. Moreover, both (B6 × C)F₁ and (B6 × C-H-2 ^dm 2)F₁ mice, as irradiated recipients, resisted the growth of parental-strain B6(H-2 ^b ) marrow cells but accepted grafts from C or C-H-2 ^dm 2 parental-strain donors. Thus, theH-2 mutation involvingH-2L but notH-2D did not affect the expression ofH-2 ^d -associated Hemopoietic or Hybrid(Hh) antigens of marrow stem cells. Grafts of 2 to 8 × 10⁶ B10.D2 or B10.D2-H-2 ^dm 1 marrow cells were rejected by B10.BR(H-2 ^k ) and B6 hosts and were accepted by B10.D2 hosts. However, B10.D2-H-2 ^dm 1 marrow cells grew to a much greater extent than B10.D2 cells in irradiated (B6 × B10.D2)F₁ or (B6 × B10.D2-H-2 ^dm 1)F₁ host mice. Therefore, theH-2 ^dm 1 mutation has altered the expression of Hh antigens at least quantitatively, resulting in a relative loss of hybrid resistance with the retention of Hh determinants recognized by allogeneic recipient mice which are notH-2 ^d . Since the Hh determinants of B10.D2 marrow cells have been mapped 16 cM to the right ofH-2, this mutation atH-2D/H-2L may have affected a regulatory gene.     

Study ofH-2 mutations in mice VI. M523, a newK end mutant

A newH-2 mutation was found in a mouse belonging to CBA/CaLacSto (H-2 ^k ) strain and designated 523, the proposed haplotype symbol for which isH-2 ^ka . The line CBA.M523 carries this mutation and is fully congenic with the parental strain, except for the mutant site 523. The mutation 523 is located within theK- end of theH-2 gene complex. Phenotypically, it causes prompt skin graft rejection and pronounced graft-versus-host activity in strain combination CBA/Sto⇄C-BA.M523. Attempts to produce active alloantisera in the same strain combination have so far been unsuccessful.     

H-2-associated specificity of virus-immune cytotoxic T cells fromH-2 mutant and wild-type mice: M523 (H-2K ka ) and M505(H-2K bd ) do,M504 (H-2D da ) and M506(H-2K fa ) do not crossreact with wild-typeH-2K orH-2D

B10.A(3R) (H-2K ^b ) mice infected with lymphocytic choriomeningitis virus (LCMV) or vaccinia virus generate cytotoxic T cells capable of specifically lysing virus-infected macrophage target cells fromH-2K ^b mutant mice M505 (H-2K ^bd ), and vice versa. Similarly, virus-immune B10.A(4R) (H-2K ^k ) T cells specifically lyse infected targets from M523 (H-2K ^ka ), and vice versa. In contrast, virus-specific cytotoxic T cells from neither M504 (H-2D ^da ) and B10.A(5R) (H-2D ^d ) nor M506 (H-2K ^fa ) and B10.M(11R) (H-2K ^f ) mutually crossreact at the cytotoxic effector-cell level. As far as tested, the crossreactivity patterns between wild-type and mutantK orD specificities are identical for LCMV- and vaccinia virus-immune spleen cells. Although this finding is no proof for either the altered self nor the dual recognition concept of T-cell recognition, it may be compatible with the latter model.     

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.     

Natural resistance of irradiated 129-strain mice to bone marrow allografts: Genetic control by theH-2K region

A major genetic determinant of natural resistance to bone marrow allografts, designated asHh-3, was mapped to theH-2K region. This gene may code for or regulate the expression of cell surface structures selectively expressed on donor hemopoietic cells and recognized by naturally occurring cytotoxic effectors. Resistance was observed as failure of donor cell growth in the spleen of irradiated 129-strain (H-2 ^bc ) recipients of H-2^k bone marrow cells. The mapping was accomplished by substituting donor cells bearingk alleles throughout theH-2 complex with cells of recombinant mouse lines bearingk alleles at definedH-2 regions. The host antigraft reaction underlying resistance was abrogated by pretreating 129-strain mice with either rabbit antimouse lymphocyte serum or the antimacrophage agent silica. Grafting of H-2K^k cells into mice ancestrally unrelated to 129 but sharing theH-2 ^bc or the similarH-2 ^b haplotype, and intoH-2 ^b/k ,H-2 ^k/bc , andH-2 ^k/d F₁ hybrids revealed that resistance was unique to 129 mice, since mice of the other strains, including F₁ hybrids, were susceptible to the grafts. Thus,Hh-3 incompatibility was a necessary but insufficient condition for the manifestation of allogeneic resistance; other genetic factors not associated withH-2 conferred responder status to 129-strain mice and nonresponder status to D1.LP, B10.129(6M), B10, B6, and possibly to F₁ hybrid mice. The possible relationships between allogeneic resistance to H-2^k marrow grafts, hybrid resistance to H-2^k lymphomas, and F₁ hybrid antiparental H-2^k cytotoxicity induced in vitro are discussed.     

Antigen-presenting cells that induce anti-H-2K T-cell responses: Differences in stimulator-cell requirements for induction of proliferation and cell-mediated lympholysis

Splenic T or B cells, which have been depleted of adherent cells by passage through Sephadex G10 columns, fail to stimulate allogeneic lymph-node cells (LN) in primary mixed lymphocyte reactions (MLR) both when the stimulating antigens are H-2 plus Ia and H-2K only. This failure cannot be ascribed to lack of viability of G10-passed cells, since by dye exclusion they are 95 percent viable and can be induced to proliferate in vitro by exposure to LPS or allogeneic cells. Stimulation of MLR activity could be restored by addition of small numbers of plastic-adherent spleen cells (SAC) which had to be syngeneic with the G10-passed stimulator cells. Further, SAC alone without G10-passed cells induced MLR activity which was, on a cell-for-cell basis, 40 times more effective than that induced by unfractionated spleen cells. If the SAC were first depleted of Ia⁺ cells, no stimulation was obtained. This result was observed both in cases where responder and stimulator strains differed across the entireH-2-gene complex and in a mutant-wild type combination (CBA and H-2^km1) in which the difference between the two strains has been mapped to theK region only. These results indicate that Ia⁺ SAC contain a subset(s) of cells which are responsible for stimulation in MLR, regardless of whether the alloantigenic differences involve either Ia or H-2K. In contrast to the inability of G10-passed splenic cells to stimulate MLR activity, these cells were able to stimulate CTL from cytotoxic T lymphocyte precursors (CTL.P) in combinations where the antigenic differences between responder and stimulator were at the entireH-2 complex or atH-2K only. However, SAC were more potent stimulators of cell-mediated lympholysis (CML) activity on a cell-for-cell basis. Thus, either CTL.P can be stimulated by nonadherent spleen cells or they are specifically sensitive to a small subpopulation of contaminating cells that cannot readily be removed by G10 passage.     

Murine antigen H-2.7: Its genetics,tissue expression,and strain distribution

Reinvestigation of alloantisera containing antibodies to murine antigen H-2.7 revealed that the crucial recombinant, A.TFR1 (H-2 ^an1 ), which was reported to separate theH-2G locus from theSs-Slp loci, has ak-like instead off-like H-2.7 antigen. Therefore, the crossover position inH-2 ^an1 and the position of the G locus in theH-2 map are now uncertain. By using the hemagglutination-serum inhibition test, anti-H-2.7 reactive substance was found to be present in normal mouse serum in a strain-specific manner. Tissue distribution study by absorption analysis indicated that H-2.7 antigen is present, in addition to RBCs, on spleen and lymph node cells, but is absent on thymus cells. Thirty B10.W congenic lines were analysed for the presence of the H-2.7 antigen. Two lines (B 10.CHA2 and B 10.KPA44) were found to be H-2.7 positive by both direct hemagglutination and absorption tests.Abbreviations used in this paper ACT Ammonium chloride Tris-buffer - BSA Bovine serum albumin - HA Hemagglutination - HASI Hemagglutination serum inhibition - HBSS Hanks balanced salt solution - PBS Phosphate buffered saline - PVP Polyvinylpyrrolidone - RBCs Red blood cells