Regulation of the cytotoxic T lymphocyte response against Qa-1 alloantigens

Spleen cells from B6.Tlaa (Qa-1a) mice primed against C57BL/6 (Qa-1b) splenocytes in vivo generate Qa-1-specific CTL when rechallenged with Qa-1b Ag in vitro. The addition of unirradiated Qa-1b splenocytes to these cultures inhibits the generation of Qa-1-specific CTL. By using highly purified cell populations, we demonstrate that the only cell population in resting spleen capable of causing this inhibition is NK1.1+. Although resting CD8 cells lack inhibitory activity, purified CD8 cells precultured with Con A and IL-2 inhibit anti-Qa-1 CTL. This inhibition is specific for the Qa-1b Ag expressed on the inhibitor cells, is not due to cold target competition, and is thus similar to that ascribed to veto cells. Although NK cells from resting spleen inhibit the generation of Qa-1-specific CTL, NK cells precultured in the presence of Con A and IL-2 show an approximate 30-fold increase in veto activity. Thus, NK cells represent the most likely cell population for down-regulating anti-self class I-reactive CTL.     

Qa-2 antigen encoded by Q7b is biochemically indistinguishable from Qa-2 expressed on the surface of C57BL/10 mouse spleen cells

Qa-2 was immunoprecipitated from the surface of 125I-labeled C57BL/10 (B10) mouse spleen cells and compared with Qa-2 immunoprecipitated from the surface of R1.1 thymoma cells transfected with Q7b. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that Qa-2 glycoproteins from both of these sources have a relative molecular mass of approximately 37 kDa. After treatment with endoglycosidase F, the Qa-2 polypeptide chains derived from C57BL/10 spleen and Q7b-transfected R1.1 cells displayed identical mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis because of removal of N-linked oligosaccharide residues. Furthermore, treatment of Qa-2 proteins from both sources with cyanogen bromide or alpha-chymotrypsin resulted in identical peptide fragmentation patterns. These results therefore provide a biochemical correlation between a cloned Qa-region gene produce expressed on the surface of transfected cells, and the Qa-2 glycoprotein on spleen cells that was described a decade ago by serologic methods.     

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

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

Ontogeny of murine T lymphocytes: II. Postnatal appearance and organ distribution of lymphocytes bearing Qa-4 and Qa-5 surface antigens

Cells from the lymphoid organs of C57BL/6 mice (from birth to 20 weeks) were monitored by the cytotoxicity assay for the presence of Qa-4 and Qa-5 surface antigens. Qa-4- and Qa-5-bearing cells are detectable in spleen, lymph nodes, and Peyer's patches, but not in thymus, liver, or bone marrow. Both antigens are present on small fractions of cells in each of these organs during the first week after birth. At 4–6 weeks of age, the fractions of Qa-4- and Qa-5-bearing cells rise to maximal levels which are then maintained throughout the ages studied (4–20 weeks). The relative proportion of these cell populations is greatest in the lymph nodes and smallest in the Peyer's patches, and in all three organs, more Qa-4- than Qa-5-positive cells are detected. The majority of Qa-4- and Qa-5-positive cells are Thy-1 positive, however, not all Thy-1- positive cells are Qa-4, Qa-5 positive. During postnatal development the ratio of Qa-4 or Qa-5-positive cells to Thy-1-positive cells increases in spleen, lymph nodes, and Peyer's patches indicating that cells bearing these antigens become a larger fraction of the T-cell population with age.     

A new serologically defined locus,Qa-1, in theTla-region of the mouse

A new cell-surface antigen, specified by a gene betweenH-2D andTla is described. The provisional notationQa-1 is suggested for the locus determining this newly recognized cell surface component. Qa-1 is distinguished from known TL antigens by the following two criteria. Its expression is not confined to thymocytes — it occurs on lymph node cells (LNC) also; and the phenotypes of the new congenic recombinant strains B6.K1 and B6.K2, derived fromH-2D/Tla crossovers, are Qa-1⁺ Qa-2^–TL^– and Qa-l⁺Qa-2⁺TL^–. Qa-1 antigen is defined by reaction of the standard TL typing serum, (B6 × A -Tla ^b)F₁ anti-A strain leukemia ASL1, with lymph node cells (LNC) in the cytotoxicity assay. Qa-1 antigen evidently is expressed, at least, on a subpopulation of T cells as well as on thymocytes. The gene order isH-2D, Qa-1, Qa-2, Tla.Abbreviations used in this paper LNC lymph node cells pooled from inguinal, axillary, brachial, and mesentric nodes - BA⁺ (C57BL/6-Tla^axA)F1 - BA^– (C57BL/6 × A -Tla ^b)F₁ - PBS phosphate buffered saline pH 7.2 - Thy thymocytes - RMIg Rabbit anti-mouse immunoglobulin Please address proofs and communications concerning this paper to Dr. Thomas Stanton, Sloan Kettering Institute, 1275 York Ave., New York, N.Y. 10021     

Comparative ability of Qdm/Qa-1b, kb, and Db to protect class Ilow cells from NK-mediated lysis in vivo

The class Ib molecule Qa-1(b) binds the class Ia leader peptide, Qdm, which reacts with CD94/NKG2R on NK cells. We have generated a gene that encodes the Qdm peptide covalently attached to ss(2)-microglobulin (ss(2)M) by a flexible linker (Qa-1 determinant modifier (Qdm)-ss(2)M). When this construct is expressed in TAP-2(-) or ss(2)M(-) cells, it allows for the expression of a Qdm-ss(2)M protein that associates with Qa-1(b) to generate the Qdm epitope, as detected by Qdm/Qa-1(b)-specific CTL. To test the biological significance of expression of this engineered molecule, we injected TAP-2(-) RMAS-Qdm-ss(2)M cells into C57BL/6 mice and measured their NK cell-mediated clearance from the lungs at 2 h. RMAS cells transfected with Qdm-ss(2)M were resistant to lung clearance, similar to RMA cells or RMAS cells in anti-asialo-GM(1)-treated mice, while untransfected or ss(2)M-transfected RMAS cells were rapidly cleared. Further, pulsing RMAS cells with either Qdm, a K(b)-, or D(b)-binding peptide showed equivalent protection from clearance, indicating that a single class Ia or Ib molecule can afford complete protection from NK cells in this system. In contrast, injection of RMAS cells into DBA/2 animals, which express low levels of receptors for Qdm/Qa-1(b), resulted in protection from lung clearance if pulsed with a K(b)- or D(b)-binding peptide, but not the Qa-1(b)-binding peptide, Qdm.     

Homeostatic proliferation of a Qa-1b-restricted T cell: a distinction between the ligands required for positive selection and for proliferation in lymphopenic hosts

Naive T cells proliferate in response to self MHC molecules after transfer into lymphopenic hosts, a process that has been termed homeostatic proliferation (HP). Previous studies have demonstrated that HP is driven by low level signaling induced by interactions with the same MHC molecules responsible for positive selection in the thymus. Little is known about the homeostatic regulation of T cells specific for class Ib molecules, including Qa-1 and H2-M3, though it has been suggested that their capacity to undergo homeostatic expansion may be inherently limited. In this study, we demonstrate that naive 6C5 TCR transgenic T cells with specificity for Qa-1(b) have a capacity similar to conventional T cells to undergo HP after transfer into sublethally irradiated mice. Proliferation was largely dependent on the expression of beta(2)-microglobulin, and experiments with congenic recipients expressing Qa-1(a) instead of Qa-1(b) demonstrated that HP is specifically driven by Qa-1(b) and not through cross-recognition of classical class I molecules. Thus, the same MHC molecule that mediates positive selection of 6C5 T cells is also required for HP. Homeostatic expansion, like positive selection, occurs in the absence of a Qa-1 determinant modifier, the dominant self-peptide bound to Qa-1 molecules. However, experiments with TAP(-/-) recipients demonstrate a clear distinction between the ligand requirements for thymic selection and HP. Positive selection of 6C5 T cells is dependent on TAP function, thus selection is presumably mediated by TAP-dependent peptides. By contrast, HP occurs in TAP(-/-) recipients, providing an example where the ligand requirements for HP are less stringent than for thymic selection.     

Visualization, fate, and pathogenicity of antigen-specific CD8+ T cells in the graft-versus-host reaction.

To follow the fate of alloreactive T cell effectors in graft-vs-host disease, Ld-specific CD8+ T cells from C57BL/6 2C TCR-transgenic donors were transplanted into sublethally irradiated (750 cGy) Ld+ or Ld- recipients. In Ld- C57BL/6 or (BALB/c-dm2 x C57BL/6)F1 recipients, naive 2C T cells engrafted and survived long term, but did not acquire effector function. In Ld+ (BALB/c x C57BL/6)F1 recipients, 2C T cells engrafted, expanded, became cytolytic, destroyed host B cells and double-positive thymocytes, and later disappeared. Despite marked damage to lymphoid and hemopoietic cells by 2C T cells, no significant pathology was detected in other organs, and recipients survived. Ld+ (BALB/c x C57BL/6)F1 recipients died when LPS/endotoxin was administered on day 7 after cell transfer, while Ld- (BALB/c-dm2 x C57BL/6)F1 recipients survived. Our findings show that under certain conditions, a CD8+ T cell population recognizing an extremely limited repertoire of Ags can initiate graft-vs-host disease.     

Molecular basis of Qa-11 antigen and paradoxical Qa-gene expression in an H-2 recombinant

The Qa-11 Ag expressed in certain strains with the B2-microglobulin-b allele, apparently maps into the Tla region as well as into the Qa-2 region. Moreover Qa-11 has been shown to be biochemically indistinguishable from Qa-2. Genetic complementation studies combining the right Qa and Tla regions failed to lead to Qa-11 expression. To elucidate the molecular basis of this apparent paradox, we examined the expression of Qa-11 on products of transfected Q-region class I genes. Immunochemical analysis has shown that the Qa-11 Ag is expressed on class I molecules encoded by the Q7 gene from both C57BL/10 (Q7b) and BALB/c (Q7d), but not on the protein product of the Q9 gene isolated from the C57BL/10 strain (Q9b). Inasmuch as the predicted protein products of the Q7b and Q9b genes would differ at a single amino acid, a residue critical for Qa-11 expression has been identified. Based on these results it is proposed that among the beta-2-mb strains, the Qa-11+/Qa-2+ mice are likely to express at least the Q7 gene, whereas Qa-11-/Qa-2+ mice express only Q9. In support of this model, the Qa-2+/Q-11- recombinant B6.K2, essential for the apparent mapping of Qa-11 into the Tla region, expresses only Q9 but not Q7 encoded molecules on the cell surface, and only Q9 and no processed Q7 mRNA is detected in the cytoplasm. This expression pattern in B6.K2 cannot be explained on the basis of a single crossing-over event.     

Activation of murine T cells by toxic shock syndrome toxin-1. The toxin-binding structures expressed on murine accessory cells are MHC class II molecules

Toxic shock syndrome toxin-1 (TSST-1)-binding structures present on murine lymphoid tissues were investigated by using 125I-TSST-1. T-depleted C57BL/6 spleen cells incubated with TSST-1 for 3 h at 0 degree C were mitogenic to splenic T cells, indicating that the former cells bind and present TSST-1 to T cells. TSST-1-binding activity was observed in C57BL/6 splenic B cells and L cells transfected with I-Ab genes, but not in splenic T cells and control L cells. Scatchard plot analysis showed that these B cells and transfectants bound TSST-1 with similar binding affinity. SDS-PAGE analysis showed that lysates of C57BL/6 spleen cells and the I-Ab-positive transfectants contain a single band which bound TSST-1 and comigrated with I-Ab heterodimers. TSST-1-binding activity observed clearly in C57BL/6. BALB/c, and C3H/HeN spleen cells and L cells transfected with I-Ab or I-Ak genes was not reduced by paraformaldehyde fixation. Binding of 125I-TSST-1 to the three spleen cells was markedly reduced by anti-I-A antibodies, but not by anti-I-E antibodies. C57BL/6, C3H/HeN, and (C3H/HeN x C57BL/6) F1 T cells were activated by TSST-1 to proliferate and produce IL-2 in the presence of FT6.2 cells, LT1-30-3 cells and either of them, respectively, but not in the presence of control L cells. These results indicate that I-A molecules function as the structures via that accessory cells directly bind TSST-1 on the cell surface and present a triggering signal of TSST-1 to T cells.     

Activation of murine T cells by staphylococcal enterotoxin E: requirement of MHC class II molecules expressed on accessory cells and identification of V beta sequence of T cell receptors in T cells reactive to the toxin

We investigated a mechanism leading to activation of murine T cells by staphylococcal enterotoxin E (SEE). L cells transfected with I-Ab genes but not control L cells supported IL-2 production by SEE-induced C57BL/6 T lymphoblasts upon restimulation with SEE. mAb to I-Ab markedly inhibited the above response. Flow cytometric analyses showed that SEE-induced C57BL/6 T lymphoblasts are composed of both CD4+ T cells and CD8+ T cells, and that larger parts of them bore V beta 11 (40-75%). mAb to V beta 11 markedly inhibited the SEE-induced proliferative response and IL-2 production by T cells. Analysis of SEE-induced IL-2 production in spleen cells from various mouse strains showed that C57BL/6 and B10.A(4R) mice (I-E, not expressed; V beta 11+ T cells, normally generated) are highly responsive to SEE. In contrast, BALB/c, C3H/HeN, (C57BL/6 x BALB/c or C3H/HeN) F1 mice (I-E, normally expressed and V beta 11+ T cells, deleted), and SJL and C57L mice (V beta 11 genes, deleted) are weakly responsive to SEE. The results indicate that SEE activates mainly T cells bearing V beta 11 in physical association with MHC class II molecules expressed on AC. In addition, the results indicate that SEE activates both CD4+ T cells and CD8+ T cells.     

V gamma 1+ T cells suppress and V gamma 4+ T cells promote susceptibility to coxsackievirus B3-induced myocarditis in mice

Coxsackievirus B3 infections of C57BL/6 mice, which express the MHC class II IA but not IE Ag, results in virus replication in the heart but minimal myocarditis. In contrast, Bl.Tg.Ealpha mice, which are C57BL/6 mice transgenically induced to express IE Ag, develop significant myocarditis upon Coxsackievirus B3 infection. Despite this difference in inflammatory damage, cardiac virus titers are similar between C57BL/6 and Bl.Tg.Ealpha mice. Removing gammadelta T cells from either strain by genetic manipulation (gammadelta knockout(ko)) changes the disease phenotype. C57BL/6 gammadelta ko mice show increased myocarditis. In contrast, Bl.Tg.Ealpha gammadelta ko mice show decreased cardiac inflammation. Flow cytometry revealed a difference in the gammadelta cell subsets in the two strains, with Vgamma1 dominating in C57BL/6 mice, and Vgamma4 predominating Bl.Tg.Ealpha mice. This suggests that these two Vgamma-defined subsets might have different functions. To test this possibility, we used mAb injection to deplete each subset. Mice depleted of Vgamma1 cells showed enhanced myocarditis, whereas those depleted of Vgamma4 cells suppressed myocarditis. Adoptively transfusing enriched Vgamma4(+) cells to the C57BL/6 and Bl.Tg. Ealpha gammadelta ko strains confirmed that the Vgamma4 subset promoted myocarditis. Th subset analysis suggests that Vgamma1(+) cells biased the CD4(+) T cells to a dominant Th2 cell response, whereas Vgamma4(+) cells biased CD4(+) T cells toward a dominant Th1 cell response.     

Suppressor T cells arising in mice undergoing a graft-vs-host response.

We investigated the ability of mice to generate antibody-forming cells when undergoing a graft-vs-host reaction. (C57BL/6 X DBA/2)F1 mice (BDF1) injected with C57BL/6 spleen cells generated suppressor T cells which inhibit antibody synthesis by BDF1 spleen cells in vitro. These T cells arose from the donor inoculum. They differ from helper T cells in size and they act directly on antigen reactive B cells. The suppressor T cells were specifically directed against components of the H-2 region of the reciprocal parental strain (DBA/2 = H-2d) in the hybrid F1 mouse.     

Two roles for CD4 cells in the control of the generation of cytotoxic T lymphocytes.

The generation of CTL against Qa-1 Ag in C57BL/6 (B6) (Qa-1b) and B6.Tlaa (Qa-1a) congenic strains requires in vivo priming with the Qa-1 alloantigen together with a helper Ag, such as H-Y. The primed precursors obtained from these female mice generate Qa-1-specific CTL activity upon culture in vitro. Although the presence of the H-Y helper Ag is not required for the in vitro sensitization, no response occurs in the absence of CD4 cells. Addition of unprimed B6.Tlaa CD4 cells from Qa-1 incompatible radiation bone marrow chimeras (B6.Tlaa----B6), that are presumably tolerant to Qa-1b, provide helper activity for Qa-1b-specific CTL. This indicates that although CD4 cells are obligatory for the Qa-1 response, they need not be specific for alloantigens on the APC to generate helper activity in in vitro cultures. Addition of unirradiated B6 CD8-depleted spleen cells to CD4-depleted B6.Tlaa anti-B6 cultures in the presence of either B6.Tlaa CD4 cells or rIL-2 prevents the generation of Qa-1 specific CTL. This inhibition is not due to an anti-idiotypic Ts cell since B6.Tlaa----B6 chimeric cells do not suppress an anti-Qa-1b response. Rather, this finding is consistent with that of a veto cell mechanism. To determine whether CD4 cells themselves exhibit veto activity, highly purified CD4 populations were tested for their ability to inhibit the generation of Qa-1-specific CTL. CD4 cells precultured for 2 to 3 days with Con A and rIL-2 specifically inhibit CTL activity whereas resting cells do not, similar to that noted for CD8 veto cells. The relative efficiency of activated CD4 cells is greater than that of resting NK cells but is less than that of activated CD8 or NK cells. Thus, CD4 cells not only provide helper activity for CTL precursors, but also act as veto cells to prevent the generation of CTL activity.     

Oligosaccharide-dependent and independent Qa-1 determinants

A contribution of N-linked oligosaccharides to determinants recognized by alloreactive cytotoxic T lymphocytes has not been demonstrated. Employing cloned CTL and tunicamycin, an inhibitor of protein glycosylation, we found that carbohydrate addition was required for the formation of two of six Qa-1 determinants. The other determinants were detectable on nonglycosylated Qa-1 molecules, similar to observations in most reports that allodeterminants on class I molecules are not dependent on glycosylation for serologic detection. Examination of TM-treated, Con A-activated lymphoblasts revealed a direct correlation between the determinants defined by the reactivity of CTL clones with target cells from four Qa-1 genotypes and their dependence on carbohydrate side chains for expression. Most anti-Qa-1b CTL clones recognized either a glycosylation-dependent determinant found only on Qa-1b cells or glycosylation-independent determinants on both Qa-1b and Qa-1c cells. Similarly, clones that killed only Qa-1a cells recognized a glycosylation-independent determinant. However, clones reactive with both Qa-1a and Qa-1d cells recognized a glycosylation-dependent determinant on Qa-1a molecules and a glycosylation-independent determinant on Qa-1d molecules. This result indicates that such clones recognize cross-reactive conformational determinants, not carbohydrate itself. Thus, N-linked oligosaccharides serve to stabilize the conformation of some Qa-1 determinants, but others remain intact on nonglycosylated molecules. The absence of similar data for H-2K/D/L molecules suggest that a reexamination of other class I antigens with cloned CTL is in order to determine whether Qa-1 molecules are unique.     

H2-M3-restricted T cells participate in the priming of antigen-specific CD4+ T cells

H2-M3-restricted CD8+ T cells provide early protection against bacterial infections. In this study, we demonstrate that activated H2-M3-restricted T cells provide early signals for efficient CD4+ T cell priming. C57BL/6 mice immunized with dendritic cells coated with the MHC class II-restricted listeriolysin O peptide LLO(190-201) (LLO) generated CD4+ T cells capable of responding to Listeria monocytogenes (LM) infection. Inclusion of a H2-M3-restricted formylated peptide fMIGWII (fMIG), but not MHC class Ia-restricted peptides, during immunization with LLO significantly increased IFN-gamma-producing CD4+ T cell numbers, which was associated with increased protection against LM infection. Studies with a CD4+ T cell-depleting mAb indicate that the reduction in bacterial load in fMIG plus LLO immunized mice is likely due to augmented numbers of LLO-specific CD4+ T cells, generated with the help of H2-M3-restricted CD8+ T cells. We also found that augmentation of LLO-specific CD4+ T lymphocytes with H2-M3-restricted T cells requires presentation of LLO and fMIG by the same dendritic cells. Interestingly, the augmented CD4+ T cell response generated with fMIG also increased primary LM-specific responses by MHC class Ia-restricted CD8 T cells. Coimmunization with LLO and fMIG also increases the number of memory Ag-specific CD4+ T cells. We also demonstrate that CD8 T cells restricted to another MHC class Ib molecule, Qa-1, whose human equivalent is HLA-E, are also able to enhance Ag-specific CD4+ T cell responses. These results reveal a novel function for H2-M3- and Qa-1-restricted T cells; provision of help to CD4+ Th cells during the primary response.     

Antigenic Heterogeneity of Qa-2 Antigens in C57BL/6

The Qa-2 antigens are class I-like molecules encoded by genes mapped telomeric to the H-2D region on chromosome 17 in the mouse. A panel of 8 new monoclonal anti-Qa-2 antibodies derived from a C3H.KBR anti-C3H. SW immunization was studied. Immunoprecipitation of¹²⁵I-labeled C57BL/6 splenocyte antigens showed that all of these antibodies precipitated 40 kDa molecules which could be completely precleared by the monoclonal antibody 20-8-4, which had previously been shown to crossreact with Qa-2. One of the monoclonal antibodies (1-12-1), however, was found not to completely preclear Qa-2 antigens precipitable by the other 7 antibodies or by 20-8-4, suggesting the existence of at least two different species of Qa-2 molecules. Cell lines transfected with Q7 or Q9 genes were reactive with all 9 antibodies and the Qa-2 antigens expressed on surface membranes of these cells were completely precleared by both 20-8-4 and 1-12-1. Therefore, the observed heterogeneity of these molecules cannot be explained by an antigenic difference between the Q7 and Q9 gene products. 2D gel analyses showed identical pI spectra between Qa-2 molecules precipitated with 20-8-4 and 1-12-1. In addition, all of the monoclonal antibodies reacted with labeled antigen preparations following treatment with Endo F or neuraminidase, indicating that carbohydrate moieties are probably not responsible for the antigenic difference between the two species of Qa-2 antigen.     

Asialo-GM1-positive T killer cells are generated in F1 mice injected with parental spleen cells

(C57BL/6 x DBA/2)F1 mice transplanted with parental C57BL/6 spleen cells become splenic chimeras, show donor antihost cytotoxic T cell activity, and lose their T cell-mediated, humoral, and natural immunity. Injection of anti-asialo-GM1 (ASGM1) into transplanted mice strongly suppresses splenic cytotoxic activity and causes a significant reduction of spleen cells expressing ASGM1, Thy-1, and Lyt-2. In vitro treatment of spleen cells from transplanted mice with antibody and complement shows that the cytotoxic effector cells are ASGM1+, Thy-1+, Lyt-2+, L3T4-, NK1.1-, and H-2d-, hence of donor origin. The cytotoxic effector cells are specific for H-2d targets and lack NK activity. In an attempt to explore whether in vivo elimination of the cytotoxic effector cells has any influence on splenic chimerism or humoral immunity, F1 mice injected with parental splenocytes were treated with anti-ASGM 1. Results show that this treatment eliminates a substantial proportion of cytotoxic effector cells but has no effect on splenic chimerism or restoration of humoral immunity. It therefore appears that cytotoxic effector cells are not primarily responsible for induction of chimerism or suppression of humoral immunity. In support of this injection of parental spleen cells with the nu/nu mutation induces killer cells in F1 mice but fails to induce splenic chimerism or immunosuppression. In contrast, injection of parental spleen cells with the bg/bg mutation generates both splenic chimerism and suppression of humoral immunity although their ability to generate cytotoxic effector cells in F1 hosts is seriously impaired and comparable to the cytotoxic potential of C57BL/6 nu/nu cells. It is concluded that the ASGM1 + cytotoxic T cells are not primarily responsible for splenic chimerism and suppression of humoral immunity and that the two effects are likely caused by parental cells with a different phenotype and function.     

A new Tla region antigen Qa-11, similar to Qa-2 and associated with B-type beta 2-microglobulin

A new antigen, Qa-11, is detected as a 40,000 dalton band in the SDS-PAGE of immunoprecipitates of radiolabeled lymphocyte membrane preparations. In C57BL H-2 congenic strains, its presence is controlled by a gene in the Tla region. In strains with genetic background other than C57BL it is not expressed. Tests with recombinant inbred strains and with H-3 congenic strains show that, in addition to the Tla region, a gene linked to or identical with the beta 2-microglobulin-b-allele is required for the expression of Qa-11 as well. The mobility of the Qa-11 antigen in SDS-PAGE and in isoelectrofocusing is the same as that of Qa-2 antigen. The Cleveland peptide maps of Qa-2 and Qa-11 are identical as well. This finding, that the Tla region controlled Qa-11 antigen is structurally very similar to the Qa-2 antigen, contrasts with the fact that Tla region products do not react with anti-Qa-2 sera. This paradox could be explained by a separate Qa-11 region between Qa-2 and Tla. Alternatively, it is possible that the Qa-11 antigen is the result of the action of a modifying gene in the Tla region upon a Qa-2 gene product, or that the structural gene for Qa-11 is located in the Qa-2 region and a Tla region gene controls its expression.     

Further biochemical data on Qa-2

The Qa-2 differentiation alloantigen is coded by a gene situated between the D and Tla loci of the murine major histocompatibility complex (H-2). Qa-2-bearing protein was isolated by immunoprecipitation and found to be composed of subunits of 40 000 and 12 000 daltons by SDS polyacrylamide gel electrophoresis (PAGE). The 12 000 dalton material was identified as 2-microglobulin (2M) by its molecular weight (SDS PAGE), charge (isoelectric focusing), antigenicity (reactivity with xenogenic anti- 2M), and genetics. The 40 000 dalton mol. wt. of Qa-2 heavy chain is 5 000 daltons less than that of D and K molecules (45 000 daltons). The quantity of Qa-2 isolated by immunoprecipitation was found to vary in a strain-specific fashion and as much as a 15-fold difference was observed.Abbreviations used in this paper B6 C57BL/6 strain mice - B10 C57BL/10 mice - 2M beta 2-microglobulin - IEF isoelectric focusing - K 1000 daltons - MHC major histocompatibility complex - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - TL thymusleukemia antigen