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     

Functional requirements of (Phe,G)-A--L-specific T-cell clones of (H-2 b x H-2 kF1 origin

T-cell clones specific for the synthetic polypeptide antigen poly(LPhe, LGlu)-poly(DLAla)--poly(LLys) of (C57BL/6 x C3H/HeJ)F₁ origin were tested for their biological activities. One group of clones was restricted in its proliferative response to the H-2 ^b haplotype, the second to the H-2 ^k haplotype, and the third to the F₁ unique Ia determinants. All the clones which proliferated in response to antigen secreted interleukin-2 (IL-2) following stimulation. The H-2 restriction of the IL-2 secretion was the same as that of the proliferation. Two of the clones tested, C.6 and C.10, could provide help to B cells in antibody production. However, the genetic restriction profile of the helper activity was less stringent than that for the proliferative response. Thus, C.6, which proliferated in the presence of F₁ antigen-presenting cells only, could help B cells and accessory cells of C3H/HeJ. C.10, which was restricted in its proliferative response to the H-2 ^b haplotype, could collaborate with B cells and accessory cells of the H-2 ^k haplotype as well. The antibody response of both clones was restricted to the parental or F₁ strains.Abbreviations used in this paper (T, G)-A-L poly-(LTyr, LGlu)poly(DLAla)--poly(LLys) - (Phe, G)-A--L poly(LPhe, LGlu)-poly(DLAla)--poly(LLys) - APC antigen-presenting cells - Con A concanavalin A - FCS fetal calf serum - IL-2 interleukin-2     

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.     

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.     

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

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

Analysis of haplotype preference in the cytotoxic T-cell response to H-Y

The mechanism determining which parental haplotype is selected in (CBA × 1310) (k × b)F₁ female mice for major histocompatibility complex (H-2) restricted, male-specific (H-Y), immune, cytotoxic T-cell (Tc-cell) responses, was investigated. The data show that haplotype preference is variable, and may be directed towards one, both, or neither of the parental haplotypes. This preference is reflected in the precursor frequency of memory Tc cells as measured by limiting dilution assays. It was further shown that maternal influence, antigen dose, route of immunization, and a feedback mechanism on the stimulator cells in vivo could not influence haplotype preference or its observed variability. Evidence for cross-reactive killing by H-2^k and H-2^b H-Y immune Tc cells on H-2^b and H-2^k allogeneic targets, respectively, (i. e., the independent haplotype of the other parent of the F₁ mice), provide evidence for natural tolerance as a possible mechanism to explain haplotype preference.     

Murine responses to (Tyr-Glu-Ala-Gly)n: II. H-2 and non-H-2 gene effects

Mice of the H-2^b haplotype responded to the sequential polymer poly(Tyr-Glu-Ala-Gly) in the in vitro T-cell proliferative assay, irrespective of whether they were homozygous or heterozygous at the H-2^b locus. The antibody responses of the H-2^b congenic mice to this polymer were variable, with A.BY and BALB.B showing responses better than those of C57BL/6 and C57BL/10 strains. The antibody responses of the F₁ progeny of (responder × nonresponder) strains of mice to this polymer are generally lower than the responder parents. F₁ mice with C57BL/10 background were the poorest responders. Studies with F₂ mice and backcross progenies of selective breeding of high and low antibody responder (C57BL/6 × BALB/c) F₁ to high responder C57BL/6 mice indicated that both non-H-2 genes and H-2 gene dosage effects influenced the magnitude of the humoral antibody responses. Animals having low responder non-H-2 background and only half the dosage of the responder immune response genes has greatly diminished antibody responses.     

Immune response to the H-X antigen on P815-X2

In a preceding report, the detection of an H-2-linked immune response to the H-X ^d antigen on the P815-X2 mastocytoma was demonstrated by the significantly increased survival of (C57BL/6 × DBA/2)F₁ (B6D2F1) male hybrids (H-X ^b ) compared with female siblings (H-X ^{b/H-X d} ) after injection with the histocompatible tumor (H-X ^d ). This interpretation was supported by the absence of this sex effect in reciprocal D2B6F1 hybrids (H-X ^d and H-X ^{d/H-X b} ). Additional findings presented in this paper support the conclusion that this sex effect is due to a true immunological response to H-X ^d : (a) Reciprocal (DBA/2 × C57BL/6 H-2 mutant)F₁ hybrids, as well as D2B6F1, failed to exhibit the sex effect: (b) the demonstration of the sex effect in (BALB/c × DBA/2)F₁ and (BALB/c-H-2 ^dm2 × DBA/2)F₁ hybrids and in (C57BL/10 × DBA/2)F₁ hybrids was consistent with the known H-X incompatibilities between the strains BALB/c and DBA/2 and C57BL/10 and DBA/2, respectively, previously demonstrated by skin grafting; and (c) the sex effect was not abrogated by castration of male B6D2F1 hybrids. Variability in the presence or absence of the sex effect was observed in various [recombinant inbred (RI) × DBA/2]F₁ hybrids and may be attributed to the influence of a regulatory non-H-2 gene which is closely linked to the gene coding for mouse kidney-androgen-regulated protein (KAP) but androgen-independent, or to variability in inheritance of the H-X ^b allele among the RI lines. It is proposed that the P815-X2 model may be utilized to type RI lines derived from a cross between C57BL/6 and DBA/2 for their H-X genotypes.Abbreviations B C57BL/6 origin allele - B6 C57BL/6 - B10 C57BL/10 - B6D2F1 (C57BL/6 × DBA/2)F₁ - B6 ^m D2F1 (C57BL/6 H-2 mutant × DBA/2)F₁ - bm10 B6.C-H-2 ^bm10 - C BALB/c - D DBA/2 origin allele - D2 DBA/2 - dm2 BALB/c-H-2 ^dm2 - H-X X chromosome-determined histocompatibility antigen of the mouse - Ir gene, immune response gene - KAP kidney androgenregulated protein - MST median survival time - RI recombinant inbred - SDP strain distribution pattern     

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     

B-Cell responses to male-specific antigen(s) in mice

It has been found that B-cell responses to male-specific antigen(s) can be clearly demonstrated by reversed plaque assays. Female mice injected with syngeneic male spleen cells showed significant increases (greater than 100 × in some strains) in the number of immunoglobulin-secreting cells in lymph nodes draining the injection site. There was a variation in B-cell responsiveness between strains and this correlated only partially with previously reported T-cell responsiveness to the H-Y antigen. C57BL (H-2 ^b ) mice were among the most responsive, while CBA (H-2 ^k ), (CBA × C57BL)F₁, and BALB/c (H-2 ^d ) were all much less responsive. These results apparently open up a new approach to the investigation of B-cell responses to male-specific antigen(s).     

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     

H-2 class I antigen expression on mouse teratocarcinoma cell lines

Immunity against PCC3 teratocarcinoma cells (129, H-2 ^b) was induced in allogeneic (C3H, H-2 ^k) mice by preimmunization with L cells (C3H, H-2 ^k) expressing cosmid-introduced K ^b or D ^b genes, but not with nontransfected L cells. In addition, the growth of PCC3 cells in sublethally irradiated (C3H × B6-H-2 ^bm1)F₁ and (C3H × B6-H-2 ^bm13 )F₁ mice bearing the K ^bm1 and D ^bm13 mutations, respectively, was either prevented, stopped, or delayed in comparison with the (C3H × B6)F₁ (k × b) mice, which failed to reject the PCC3 cells. The teratocarcinoma line OC15S was exceptional because it reacted specifically with K^b- and D^b-specific (but not I^b-specific) alloantisera, and because K^b- and D^b-specific antibodies could be absorbed by OC15S cells. The subpopulation of OC15S cells bearing the ECMA-7 antigen characteristic for embryonic carcinoma (EC) cells was isolated by the fluorescence-activated cell sorter and was shown to react specifically with K^b- and D^b-specific antisera. These experiments show that teratocarcinoma cells express antigens similar or identical to the K-and D-region products of differentiated cells. The lack of expression of class I antigens is thus neither a condition nor a consequence of the pluripotentiality of the EC cells. The exact nature of the major histocompatibility complex antigens on EC cells has yet to be established using the methods of molecular biology and biochemistry.     

Mechanism of hybrid resistance. The role of a natural antibody in parental bone marrow cell rejection

Hybrid resistance (HR) to parental bone marrow growth is specifically directed against hemopoietic histocompatibility (Hh-1) Ag that are present in parental bone marrow cells (bmc). The mechanism of HR seems to be a multistep process. According to a model we proposed earlier, a T cell recognizes the Hh-1 Ag and stimulates a macrophage to secrete IFN-alpha/beta (recognition phase). IFN-alpha/beta activates a NK-like cell that specifically kills the parental bmc (effector phase). We have also described in a previous paper that serum from resistant F1 hybrids contains a humoral factor that seems to be involved in the effector phase of HR. In the present work, we study the role and the nature of this humoral factor. Our results show that this humoral factor: 1) is present in all resistant H-2Db heterozygous F1 hybrids we have tested but not in nonresistant H-2Db homozygous mice; 2) seems to recognize the Hh-1b Ag because it is absorbed on bmc from Hh-1b mice but not on bmc from Hh-1d and Hh-1- mice; and 3) is an IgG1 Ig (natural antibody). These results could help us to explain the specificity of HR at the effector phase by supposing that this natural antibody recognize the Hh-1 Ag and enable NK-like cells to kill parental bmc cells in Hh-1 specific manner.     

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.     

Immunological and genetic requirements for the parental hemopoietic takeover reaction in adult,H-2-incompatible parent-F1 hybrid parabiosis

Parabiosis of adult DBA/2J (H-2 ^d ) mice with adult (DBA/2J× CSH/HeJ)F₁ (H-2 ^d /H-2 ^k ) mice results in survival beyond 100 days in 44% of such pairs, induction ofin situ unresponsiveness to C3H/HeJ skin, and the complete takeover of the erythroid system of the F₁ by parental cells. However, in vitro responsiveness to C3H/HeJ cells remains. Dye exclusion cytotoxicity assays establish the absence ofF ₁ lymphoid cells in the spleens and bone marrow of both partners. The parental takeover of the erythroid system of the F₁ partner requires immune recognition of the hybrid's alloantigens, because this takeover is not seen with tolerant parental cells. PartialH-2 differences (on the C3H background) influence both survival and the takeover reaction when incorporated into parabioses with DBA/2J partners. When only theK andI subregions ofH-2 were targets of the parental response, 58% of parabionts survived, with complete parental hemopoietic takeover. When onlyH-2D was the target, 83% of parabionts survived, with incomplete hemopoietic takeover. Changing the non-H-2 background of the F₁ target did not significantly affect survival or takeover, while substituting a differentH-2 ^d parental strain (BALB/c) eliminated survival altogether.Thus the parabiont takeover reaction encompasses the lymphoid and hemopoietic systems, requires immunorecognition of the target alloantigens, and seems to require a strongH-2 difference for its induction.     

Xid-defective male (CBA/N x C57BL/6)F1 accessory cells present bovine insulin to long-term cultured F1-restricted T-cells

The reactivity of H-2^b-restricted murine T cells towards bovine insulin was reported to depend on the expression of Ia.W39, a private specificity of I-A^b, on antigen-presenting cells. Cells of male (CBA/N x B6)F₁ mice carrying the mutation xid on the X chromosome lack Ia.W39 on the cell surface. These cells are unable to present bovine insulin to primed T cells derived from female (CBA/N x B6)F₁ mice. We show here that spleen cells of male (CBA/N x B6)F₁ hybrids served perfectly as accessory cells for the insulin-dependent induction of a proliferative response of long-term cultured T cells with (B10 x B10.BR)F₁ genotype, restricted to recognizing insulin in the context of F₁-unique I-A determinants. The epitope on the insulin molecule essential for stimulation was determined to depend on the glutamic acid residue in position 4 of the A chain of insulin. This contrasts with the H-2^b-restricted response of B6 mice to bovine insulin, which appears to be directed at the A chain loop determinant (amino acids A8 and A10). These data suggest that distinct I-A^b-encoded structures, the expression of which is regulated independently, may serve as components of restriction elements for H-2^b and (H-2^b x H-2^k)F₁ restricted T cells, which are specific for different epitopes of bovine insulin.     

Production of anti-self-H-2 antibodies by C3D2F1 mice hyperimmune to L cell/L1210 hybrids and L1210 leukemia cells

During the course of immunization of (C3H × DBA/2)F₁ mice (genotype H-2^k/b) with L cell (H-2^k/k)/L1210 leukemia cell (H-2^d/d) hybrids and L1210 leukemia cells, some of them produced a good titer of anti-self-H-2 (H-2^d) antibodies. Antigens recognized by this anti-self-H-2 antiserum were shown to be controlled by the H-2K-IA-IB-IJ-IE subregions of the H-2^d but not H-2^k nor H-2^b haplotypes of parental as well as F₁ origins and to have a tissue distribution identical to that of class 1 H-2 (H-2K/D) antigens.     

The nature of hemopoietic histocompatibility determinants. Differential sensitivity of Hh-1b and H-2b determinants to tunicamycin

NK cell-dependent resistance of F1 hybrid mice to parental H-2b hemopoietic allografts is directed to cell surface structures controlled by the Hh-1 locus in or near the H-2D region. Crucial to an understanding of this enigmatic phenomenon is the information on the biochemical nature of the Hh-1 locus-controlled structures. Therefore, we examined the effect of tunicamycin (TM), an inhibitor of asparagine-linked glycosylation and ganglioside biosynthesis, on the expression of Hh-1 determinants in H-2b/Hh-1b lymphomas. The Hh-1b determinants on EL-4 and RBL-5 cells were no longer detectable after TM treatment, as demonstrated by the failure of the treated cells to inhibit hybrid resistance to parental H-2b bone marrow cells in vivo. This interpretation was supported by the unaltered ability of the TM-treated cells to localize in the spleens of irradiated F1 hybrid recipients. In contrast, TM caused only moderate reduction in H-2Kb and H-2Db expression as measured by binding of specific antibodies. This was accompanied by reduced susceptibility to alloimmune anti-H-2Db CTL, but not to anti-H-2Kb CTL. No decrease was found in the susceptibility to NK cell cytotoxicity in vitro. These data indicate that N-linked glycosylation or ganglioside synthesis is crucial for the expression of the Hh-1 locus-controlled target structures, but not for the H-2 class I molecules. The data also show that the Hh-1b determinants are substantially different from those which confer the susceptibility to NK cell-mediated in vitro cytotoxicity.     

Differential effect of hybrid resistance on the localization of virus-immune effector T cells to spleen and brain

The hybrid resistance (Hr) effect operates in the lymphocytic choriomeningitis (LCM) in vivo transfer model to inhibit both the level of cytotoxicity T lymphocyte (CTL) generation in spleen and the induction of inflammation in cerebrospinal fluid (CSF). The effect is seen when LCM virus-immune T cells that are homozygous for H-2D ^b are injected into virus-infected, immunosuppressed recipients that are heterozygous for this allele, or into radiation chimeras that express an appropriate F₁ phenotype. Evidence that Hr to T -cell transfer is cell-dose-dependent and tends to diminish with age was found in both chimeric and normal F₁ mice. Inhibition of the capacity of injected T cells to cause meningitis is a more sensitive measure of Hr than is the further stimulation of CTL effectors in recipient lymphoid tissue. The injection of large numbers of H-2^b virus-immune T cells into (H-2 ^k X H-2 ^bF₁H-2 ^k) virus-infected recipients did not induce any cellular extravasation into CSF, though potent H-2^b-restricted CTL effectors were generated in recipient spleen. Evidence of minimal inflammatory process was found in one experiment where these chimeras were given a comparable dose of (H-2 ^b X H-2 ^d)F₁ immune spleen cells. Development of this Tcell-mediated immunopathological process depends essentially on the expression of the appropriate H-2 restriction element on radiation-resistant host cells which, in this case, presumably constitute part of the physiological barrier between blood and CSF.     

Variable spread of X inactivation affecting the expression of different epitopes of the Hya gene product in mouse B-cell clones

Cloned B-cell lines from a female T16H/XSxr mouse in which Tdy expression was suppressed due to X inactivation and from a male X/XSxr mouse, both of the (kxb)F₁ haplotype, were examined for H-Y expression. This was determined both by their ability to act as targets for H-2^k and H-2^b-restricted H-Y-specific cytotoxic T cells and by their ability to stimulate the proliferation of H-2K^k, H-2D^b (class I) and A^b (class II)-restricted T-cell clones. In B-cell clones from the T16H/XSxr mouse, expression of H-Y/D^b exhibited partial X inactivation and only a proportion ( 30%) of the cells were targets for or stimulated H-2D^b-restricted H-Y-specific T cells. In contrast, H-Y eiptopes restricted by H-2^k (H-Y/K^k, H-Y/D^k) and A^b (H-Y/A^b) exhibited no X inactivation. Furthermore, no inactivation of H-Y/D^b, H-Y/A^b, or H-Y^k was observed in the male X/XSxr mouse. These results indicate that the T16H/XSxr female is a mosaic, as a result of the variable spread of X inactivation into the Sxr region. They further suggest that the H-Y antigen recognized in association with H-2^k and H-2D^b class I molecules and A^b class II molecules may be the product of more than one gene.