Genetic control of acquired resistance to visceral leishmaniasis in mice

A series of H-2 and non-H-2 congenic resistant (CR) strains on a C57BL/10Sn background were infected with 10(7) amastigotes of Leishmania donovani. Non-H-2 congenic strains B10.LP-H-3b and B10.CE(30NX) and (B10.LP-H-3b x B10)F1 hybrids showed a very rapid decrease in liver-parasite burdens beyond day 21. Parasite counts for these strains at day 35 were significantly lower than for all other strains tested. The rapid decrease in parasite numbers, massive lymphocellular infiltration into the liver and strong delayed hypersensitivity reactions to parasite antigens in strains congenic for a portion of chromosome 2 indicated that acquired immunity to L. donovani was controlled by a dominant gene at or near the Ir-2 locus. In addition, B10.129(10M) mice, which differ from C57BL/10Sn at the H-11 locus, showed highly significant increases in parasite numbers at day 35. Other observations supporting the absence of acquired immunity in B10.129(10M) included negative delayed hypersensitivity tests to parasite antigens and the absence of lymphocellular infiltrate into the liver. Although the differences were not as pronounced, H-2 CR strains with H-2b, H-2a, and H-2k haplotypes also showed significantly greater decreases in parasite numbers by day 35 as compared to other H-2 CR strains.     

An H-11-linked gene has a parallel effect on Leishmania major and L. donovani infections in mice

The courses of visceral infection following intravenous injection of Leishmania donovani amastigotes, or lesion growth following subcutaneous injection of L. major promastigotes, were examined in B10.129(IOM) (H-2 ^b, H-11 ^b) mice and compared with disease profiles observed in congenic C57BL/10ScSn(=B10) (H-2 ^b, H-11 ^a) and B10.D2/n (H-2 ^d, H-11 ^a) mice, and in BALB/mice. Possession of alternative alleles at H-11 and closely linked loci transformed the normal curing/healing phenotype of B 10 mice into a characteristically different noncuring/nonhealing phenotype affecting both visceral and subcutaneous infections in B10.129(10M) mice. In reciprocal radiation bone marrow chimeras made between the congenic B10 and B10.129(10M) strains, both cure and noncure phenotypes were transferable with the donor hematopoietic system. Although it was possible to demonstrate transfer of suppression with T-enriched spleen cells from day 61 L. donovani-infected B10.129(10M) donor mice into 550 rad syngeneic recipients, the pretreatment of mice with sublethal irradiation did not, as in the earlier studies of Scl-controlled L. major nonhealing or H-2-controlled L. donovani noncure phenotypes, have a clear or consistent prophylactic effect. Together with the progressive disease profile observed even for L. donovani at low parasite doses this suggests that, despite their ability to develop initial delayed-type hypersensitivity reactions to parasite antigen early in L. major infection, B10.129(10M) mice possess some inherent defect in ability to mount a cell-mediated response effective at the level of macrophage antileishmanial activity in vivo even when suppressor T cells are not generated. Further elucidation of this characteristically different noncuring/nonhealing phenotype may provide important insight into common events involved in the development of the cell-mediated immune response to both visceral and subcutaneous forms of leishmaniasis.     

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.     

Genetic control of contact sensitivity in mice: Effect ofH-2 and nonH-2 loci

The genetic control of delayed-type hypersensitivity in mice was investigated by contact sensitization with picryl chloride. Distribution patterns of contact sensitivity in 11 inbred strains of mice showed significant differences among strains. Comparison of levels of response between congenic-resistant lines and their inbred partners, at 9 to 11 weeks of age, revealed a clear association betweenH-2 haplotype and the magnitude of response. Testing ofH-2 recombinants further suggested the influence of two genes mapping at either end of theH-2 complex. While theH-2K ^d andH-2D ^k alleles were associated with a high response, theH-2K ^k ,H-2K ^b ,H-2D ^d , andH-2D ^b alleles were associated with a low response. Analysis of the ontogeny of response suggested that theH-2 haplotype manifests its effect through the maturation of contact sensitivity. On both the C57BL/6By and C57BL/10Sn backgrounds, theH-2 ^d haplotype was associated with early maturation of response, while theH-2 ^b haplotype was associated with late maturation. Analysis of the response of congenic lines with different genetic backgrounds and of CXB recombinant-inbred lines further revealed the marked effects of yet other genes on this trait.     

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.     

TheH-2 class II genes and the susceptibility to the development of pulmonary adenoma in mice

To determine the locus in theH-2 complex that affects susceptibility to the development of pulmonary adenomas in mice,H-2 congenic and recombinant strains of mice with A/Wy, BALB/c, C3H, and B10 backgrounds were subjected to treatment with urethane. The average number and the incidence of adenoma foci were recorded five months after the treatment. InH-2 congenic strains on the A/Wy background, the average number of adenoma foci per mouse was significantly higher in mice of the A/Wy, A/J, and A-Tla ^b (H-2 ^a ) strains than in A.BY (H-2 ^b ) mice. In BALB/c and C3H congenic strains, the strains carrying theH-2 ^k haplotype were more susceptible than those carrying theH-2 ^b haplotype. InH-2 congenic strains on the B 10 background, the average number and incidence of foci was also higher in haplotypesa, h2, k, andj than in haplotypesb, s, f, d, r, h4, i3, i5, and4. The average numbers of adenoma foci in (A/J × A.BY)F₁ (H-2 ^a /H-2 ^b ) and (B10 × B10.A)F₁ (H-2 ^b /H-2 ^a ) were intermediate between the numbers in the parental strains. In [B10.A (4R) × B10.A (3R)]F₁ (H-2 ^h4 /H-2 ⁱ³ ) and [B10.A (4R) × B10.A (5R)]F₁ (H-2 ^h4 /H-2 ⁱ⁵ ), the numbers of adenoma foci were higher than in resistant parental recombinants. These patterns of response to urethane matched the patterns of the immune response to lactate dehydrogenase-B (LDH-B) and immunoglobulin gamma 2a (IgG2a) proteins. These differences between mice in their susceptibility to the development of pulmonary adenomas is probably due to the polymorphism of the class II genes in theH-2 complex.     

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.     

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.     

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.     

H-2 haplotype and the embryotoxicity of serum from nonpregnant congenic mice

Previous studies revealed a significant association between genes at or near the H-2 complex and fetal loss. Reasoning that the maternal serum might contain one or more unknown factors that are harmful to early embryonic or fetal development, or both, we performed an embryotoxicity screen using chick embryos and serum from nonpregnant C57BL/10Sn(H-2 ^b) and B10.A/SnSg (H-2 ^a) congenic mice. Serum from the strain with the higher frequency of fetal loss (C57BL/10 Sn) yielded a significantly greater frequency of chick abnormality, specifically neural tube malformation and death, than the serum from the strain with the lower frequency of fetal loss (B10.A/SnSg). Further, the C57BL/10 Sn serum demonstrated a highly significant dose-response. These results suggest that analogous studies may be profitable with women who have a history of chronic fetal wastage and/or offspring with neural tube defects.     

Single gene control of resistance to cutaneous leishmaniasis in mice

A series of inbred, congenic resistant, and hybrid strains of mice were intradermally inoculated with 10⁶ promastigotes of Leishmania tropica. These mice were divided into susceptible and resistant groups using the criteria of lesion size, development of metastatic foci and skin-test reactivity. At 16 weeks of infection, resistant strains A/J, DBA/1J, AKR/J, CBA/J, C3H/HeJ, NZB/BINJ, C57BL/6J, C57BL/10Sn, B10.D2, B10.129(10M), and B10.CE(30NX) had completely resolved their lesions, while susceptible SWR/J and BALB/cJ mice demonstrated large, nonhealing cutaneous lesions. In addition, BALB/cJ developed metastatic lesions on the extremities which progressively increased in size. All BALB/cJ and SWR/J mice died by 7 1/2 months of infection. The BALB/cJ x C57BL/6JF₁ hybrid behaved in an intermediate fashion showing a slower expansion of cutaneous ulcers and a delayed development of metastatic foci, however, the infection ultimately proved fatal. The F₂ generation could be separated into three distinct groups: resistant, intermediate, and susceptible mice with a lesion size distribution pattern in conformity with a 1:2:1 ratio. Male/female susceptibility differences were not noted. These data indicated that development of acquired resistance may be under the control of a single, autosomal gene. The gene did not appear to be H-2-, Ir-2-, or H-11-linked as is seen with Leishmania donovani infections.     

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.     

The association of H-2 haplotype with implantation,survival, and growth of murine embryos

Using three congenic strains, C57BL/10Sn (H-2 ^b), B10.A/SnSg (H-2 ^a), and B10.D2/nSn (H-2 ^d), we sought to investigate the possible association of H-2 haplotype with the number of implants, fetal survival, and fetal weight, as well as to analyze the possible effects of hybrid vigor and maternal-fetal histoincompatibility in primigravidae mice. The results of this study indicate a significant association between genes at or near the H-2 complex and both fetal loss and fetal weight, but not the number of implants. Haplotype variation accounted for 14 percent of the variation in fetal loss and 20 percent of the variation in fetal weight. With the exception of fetal loss, there was no evidence of a maternal effect. There was also no clear evidence of hybrid vigor or histoincompatibility effects for any of the three variables studied. In summary, the data suggests that particular allelic variants at or near the H-2 complex confer some selective advantage as measured by differential fetal survival and fetal growth.     

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     

Genetic analysis of theT-H-2 region in non-t Chromosomes

A general breeding protocol useful in the construction of congenic lines of mice disparate in the 15 cMT-H-2 region of chromosome 17 in non-t chromosomes is described. Two such congenic lines, B6.TC2/Rn and B6.TC3/Rn, were derived from the C57BL/6J and B6.C-H-2 ^d/By strains using this protocol. Both B6.TC2 and B6.TC3 dissociate the quantitative activity locus for glyoxalase I (Qglo-1) from theH-2 complex, and hence possess BALB/cBy DNA centromeric toH-2. However, neither new strain is able to map anyH-2-associated restriction fragment length polymorphism with anH-2 cDNA probe even though both strains are recombinant in the 2 cMQglo-1-H-2K interval.     

Thirteen new chromosome-7 congenic lines

Thirteen new congenic lines have been produced which have chromosome-7 segments introduced from different strains onto the C57BL/10Sn background. Sublines B10.P(61NX)C,D, and E received chromosome-7 segments from P/J, B10.CE(62NX) from CE/J, B10.SEC(64NX)A,C,E, and F from SEC/1Re, B10.SM(65NX) from SM/J, B10.WB(66NX) from WB/Re, B10.A(67NX) from A/SnGrf, B10.AKR(68NX) from AKR/SnGrf, and B10.K(69NX) from C3H.K. Isograft testing indicated that three sublines, B10.P(61NX)D, B10.CE(62NX)B, and B10.WB(66NX)B are histoisogenic, i.e., histocompatible within each line. With the exception of B10.A(67NX), B10.AK(68NX), and B10.K(69NX), which have not been isografted, the remaining sublines showed residual heterozygosity on isografting. The three histoisogenic lines have undergone F₁ testing and have been found to possess theH-4 ^a allele and new and distinct alleles at theH-1 locus. They have been designated B10.P(61NX)-H-4^a H-1 ^d , B10.WB(66NX)-H-4^a H-1 ^e , and B10.CE(62NX)-H-4^a H-1 ^f . Direct exchange of grafts has indicated the following genotypes: B10.A(67NX)-H-4^a H-1 ^b , B10.AK(68NX)-H-4^a H-1 ^b , and B10.K(69NX)-F-4^a H-1 ^b . The B10.SEC(64NX) and B10.SM(65NX) sublines have not been typed completely forH-4 andH-1. F ₁ testing or direct exchange of skin grafts indicated that B10.P(61NX)-H-4^a H-1 ^d , B10.WB(66NX)-H-4^a H-1 ^e , B10.A(67NX)-H-4^a H-1 ^b B10.AK(68NX)-H-4^a H-1 ^b and B10.K(69NX)-H-4^a H1 ^b possess nonon-H-1 histocompatibility differences from the G57BL/10 background.     

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.     

Histocompatibility genes (theH-2 complex) and susceptibility to spontaneous lung tumors in mice

The incidence of spontaneous lung tumors in relation toH-2, the major histocompatibility complex, was studied in congenic strains of mice on the B10-, A-, and C3H-backgrounds.The most relevant results were obtained with congenic strains on the B10-background. The strains could be divided into two groups: one with a low frequency of spontaneous lung tumors carrying the haplotypesH-2 ^b ,H-2 ^h4 ,H-2 ^d ,H-2 ⁱ H-2 ^r and one with a higher incidence of lung tumors carrying the haplotypesH-2 ^f ,H-2 ^m ,H-2 ^h2 ,H-2 ^a . The differences between these two groups were highly significant.Analysis of the results obtained with the recombinant strains indicated that genes in theIB region determined the susceptibility to the development of spontaneous lung tumors.The comparison of the results in the B10, B10.A and A strain has shown that the incidence in the B10.A strain carrying the haplotypeH-2 ^a derived from the highly susceptible strain A (H-2 ^a ) on the resistant background strain B10 (H-2 ^b ) is intermediate between these two strains. This shows, that other genes of the background are also involved.The lung tumor incidence in (B10.A × B10)F₁ hybrids was intermediate between the two parental strains.The results obtained in the strains C3H with the haplotypeH-2 ^k , C3H.B10 with the haplotypeH-2 ^b and C3H.NB with the haplotypeH-2 ^p , were inconclusive because of the early mortality which occurred among the animals of these strains. The strains A (H-2^a) and A.SW (H-H-2 ^s ) were both equally susceptible.     

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.     

Studies of twoH-2D b mutants: B6.C-H-2 bm13 and B6.C-H-2 bm14

Two new C57BL/6H-2 mutants,B6.C-H- 2^bm13 and B6.C-H- 2^bm14 are described. They arose independently in C57BL/6 as spontaneous mutations of the gain and loss type. Complementation studies map the mutations in both bm13 and bm14 to theH-2D ^b gene. How ever, these two mutant strains are not identical, but occurred as independent mutations at the same locus, as shown by reciprocal graft rejection and by the inability of the (bm13 × bm114)F₁ hybrid to accept C57BL/6 grafts. Serological studies by direct testing (cytotoxicity and hemagglutination) and by quantitative absorption demonstrated a decrease in the H-2D^b private specificity H-2.2 in both bm13 and bm14 when compared to C57BL/6. This was confirmed by SDS-PAGE analysis using antisera detecting the H-2.2 specificity. Attempts to produce antibodies to either the gained or lost specificities of the two mutant strains failed.