Specific immune responses restored by alteration in carbohydrate chains of surface molecules on antigen-presenting cells

Two class I major histocompatibility (MHC) mutant mouse strains, H-2bm14 and H-2bm6, differ from the strain of origin C57BL/6 (B6, H-2b) in one and two amino acids of the H-2Db and H-2Kb molecule, respectively. The bm14 Db mutation results in specific failure of female bm14 mice to generate a cytotoxic T lymphocyte (Tc) response to the male-specific antigen H-Y. The allospecific Tc response of CD8+ B6T cells against bm6 Kb mutant spleen cells, in contrast to that against other Kb mutants, is absolutely CD4+ T helper cell dependent. Purified CD8+ T cells completely fail to respond. We now report that the inability to mount these specific immune responses is restored by the use of dendritic cells (DC) as antigen-presenting cells (APC). Comparison of MHC expression on various types of APC by cytofluorimetry and quantitative immunoprecipitation showed very high expression of class I and class II MHC molecules on DC. Strikingly, examination of class I and class II molecules by isoelectric focusing revealed qualitative differences as well. We show that the surface MHC class I molecules of DC are present in greater quantity and carry on average fewer sialic acids than the same molecules isolated from other APC types such as spleen cells, lipopolysaccharide blasts or concanavalin A blasts. That sialic acids on cell surface molecules, including MHC, may play a role in antigen presentation is suggested by our finding that removal of sialic acids, by neuraminidase, can restore specific responses to nonresponder APC as well.     

Two HLA-DQ-specific human-human hybridoma antibodies (TrG6;TrC5) define epitopes also expressed by a transcomplementing hybrid DQ molecule (DQw7 alpha/DQw4 beta)

We have generated two IgG human-human hybridoma Abs, TrG6 and TrC5, that define subsets of HLA-DQ. TrG6 combined selectively with lymphoblastoid cell lines that expressed DQw1 or DQw4. By sequential immunoprecipitation, competition with other mAbs of defined specificity for binding to antigen, and experiments where HLA antigens from cell lysates crosslinked pairs of mAbs, it was established that TrG6 bound a DQ-molecule. mAb TrC5 specifically recognized DQw2+DR3+ and DQw7+DR5+ cells. The reaction pattern of TrC5 with HLA-loss mutants indicated that TrC5 bound to DQw2 of the DQw2+DR3+ haplotype. Antigens in lysate from DQw7+DR5+ cells crosslinked TrC5 to the murine mAbs Tü22 (anti-DQ monomorphic) and IVD-12 (anti-DQw7 + DQw8 + DQw9), demonstrating that on these cells the TrC5 epitope is located on DQw7 molecules. Lysates from DQw7+DR5+/DQw4+DRw8+ heterozygous cells crosslinked TrG6 and TrC5, and available evidence indicated that the epitopes defined by these two mAbs were expressed by the transcomplementing DQ-molecule DQw7 alpha/DQw4 beta, where the DQw7 alpha chain specifies epitope TrC5 and the DQw4 beta chain specifies epitope TrG6. Taken together with published nucleotide sequences of DQ alpha and beta genes, our data are consistent with the conclusion that the amino acids at positions 69 and/or 75 of the DQ alpha chain of DQw2+DR3+ and DQw7+DR5+ haplotypes are critical for epitope TrC5. The previously reported human-human hybridoma Ab TrB12 reacts with DQw6, DQw8, and DQw9. The specificity of the murine mAb IIB3 is similar to that of TrB12, but, unlike TrB12, IIB3 also binds DQw4+ cells.     

Production of epitope specific monoclonal IgG antibodies to HLA class II molecules by combining in vivo and in vitro immunization.

To study the expression of HLA-DQ beta chain alleles associated with type 1 diabetes, mAbs were generated from mice immunized with synthetic peptides representing allelic HLA-DQw7 and HLA-DQw8 beta chain sequences. The splenocytes from immunized mice were fused with myeloma cells, either immediately after or following additional in vitro boosting with peptide. Peptide-specific mAbs, predominantly of the IgG isotype, were isolated only from in vitro boosted splenocytes. Immunoblot analysis showed that several of the mAbs cross-reacted with DQ beta chain molecules. One mAb to a peptide representing DQw8 beta position [49-60] specifically recognised the DQw8 beta chain. Three mAbs to a peptide representing DQw8 beta position [39-52] specifically recognised an epitope consisting of Gly-Val-Tyr in position 45-47, i.e., all DQ beta alleles except DQw7 beta (position 45-47: Glu-Val-Tyr) and DQw2 beta (position 45-47; Gly-Glu-Phe). In FACS analysis these mAbs bound lymphocytes with the same specificity as found by immunoblotting analysis. Thus, by combining in vivo and in vitro immunization we have generated a number of epitope specific monoclonal IgG antibodies that distinguish closely related HLA-DQ beta chain alleles in predetermined positions.     

Butyric acid derivative induces allospecific T cell anergy and prevents graft-versus-host disease

Graft-versus-host-disease (GVHD) is a common and potentially fatal complication following bone marrow transplantation. This study was initiated to test whether MEB [n-butyrate 2-(4-morpholinyl) ethyl butyrate hydrochloride], a derivative of the G1 blocker butyric acid, could specifically inactivate the alloantigen-specific T cells that mediate GVHD. MEB was shown to inhibit proliferation in a one-way mixed lymphocyte reaction (MLR) in which spleen cells from C57BL/6 mice (H-2b) were stimulated with spleen cells from DBA/2 mice (H-2d). The addition of MEB to the MLR prevented the expansion of alloantigen-stimulated CD8+ and CD4+ T cells in association with decreased IL-2 production. In addition, MEB inhibited the CTL activity of CD8+ T cells from the MLR. Most importantly, T cells from the MEB-treated MLR, unlike T cells from an untreated MLR, were unable to induce the splenomegaly and increased serum TNF-alpha levels characteristic of acute GVHD when injected into B6D2F1 mice. The splenomegaly found in the B6D2F1 mice injected with T cells from an untreated MLR encompassed the expansion and activation of CD8+ T cells, CD4+ T cells, B cells and macrophages. In contrast, the spleens of mice injected with T cells from MEB-treated MLR looked essentially identical to those of control B6D2F1 mice in terms of the numbers and activation state of the spleen cell populations. Similarly, the increase in IFN-gamma and TNF-alpha production by CD4+ and CD8+ T cells from the spleens of mice undergoing acute GVHD was not observed if the mice were injected with T cells from an MEB-treated MLR instead of an untreated MLR. The use of MEB to inactivate host-specific T cells ex vivo underlines the potential clinical importance of this compound in the prevention and treatment of unwanted immune responses such as GVHD.     

Restricted blocking of cytotoxic T-cell function by anti-H-2K/D antibodies

Antibodies specific for H-2K and H-2D, the murine major histocompatibility complex (MHC)-encoded class I antigens, can block cytotoxic T (Tc)-cell function. Antibodies specific for the Tc cell and not the target cell have been used to map inhibition to the effector cell, suggesting a role for class I antigens in Tc-cell function. These antibody effects have been demonstrated for both alloreactive and MHC-restricted Tc cells, but inhibition has only been revealed by measuring cytotoxicity in a short-term assay. Using the neutral red assay for cytotoxicity, blocking effects evident after a 1.5-hr assay were lost by 2.5 hr. For some Tc-cell responses, only anti-H-2K antibodies have been found to be inhibitory, despite evidence of the expression of both H-2K and H-2D molecules on these cells. Some Tc-cell populations can be blocked by antibodies specific for both the H-2K and H-2D molecules. B10.A(4R) anti-Sendai Tc cells can be inhibited by anti-H-2Kk antibodies, but five different anti-H-2Db antibodies have been ineffective inhibitors. In contrast, B10.A(4R) anti-ectromelia Tc cells can be inhibited very effectively by each of these anti-H-2Db antibodies, as well as by anti-H-2Kk antibodies. Anti-H-2 antibodies also inhibit the function of cloned alloreactive Tc-cell lines such that the inhibitory capacity of antibodies specific for K versus D determinants appears to be consistent and specific for each Tc-cell line. A long-term Tc-cell clone, AR1, has been inhibited specifically by anti-H-2Kb and not anti-H-2Db antibodies, suggesting a clonally 'restricted' phenomenon.     

A co-stimulatory role for CD28 in the activation of CD4+ T lymphocytes by staphylococcal enterotoxin B.

In this study we investigated the differential effect of the co-stimulatory receptor ligand molecules CD2/LFA-3, LFA-1/ICAM-1, and CD28/B7 on microbial superantigen mediated activation of CD4+ T cells. Highly purified CD4+ T cells, depleted of antigen presenting cells (APCs), do not proliferate in response to the superantigen, staphylococcal enterotoxin B (SEB). However, CD4+ T cells do respond to SEB in the presence of the LFA-3, ICAM-1, and B7 positive erythroleukemic cell line K562, murine L cells, human B7 transfected L cells or CD28 mAb. The K562 plus SEB induced response can be inhibited by combinations of mAbs to CD2 and LFA-1, and to LFA-3, ICAM-1, and B7. Addition of CD28 mAb to the CD2 and LFA-1 inhibited cultures could restore the response. Furthermore, soluble CD28 mAb alone is able to synergize with SEB to induce a proliferative CD4+ T cell response. CD4+ T cells depleted of APCs could also be activated by a pool of four mAbs directed to the V beta 5, V beta 6, V beta 8, and V beta 12 region of the TCR when a co-stimulatory signal was provided by the CD28 mAb, while the V beta mAbs alone or in combination are unable to activate CD4+ T cells in the absence of APCs. In contrast, addition of soluble mAbs to CD2 and LFA-1 molecules failed to co-stimulate SEB activated CD4+ T lymphocytes. The kinetics of the different modes of activation are distinct. SEB induced proliferation is most efficient in the presence of autologous APCs with maximal proliferation at a log4 lower SEB concentration than when CD28 mAbs were used. SEB plus K562 activation peaks on day 7, while SEB plus CD28 mAb induced proliferative responses do not peak until day 9. Thus, superantigen mediated activation of CD4+ T cells requires co-stimulatory signals, among which CD28 has distinct and unique effects.     

Preferential positive selection of V alpha 2+ CD8+ T cells in mouse strains expressing both H-2k and T cell receptor V alpha a haplotypes: determination with a V alpha 2-specific monoclonal antibody.

A monoclonal antibody, B20.1, was generated by fusing spleen cells from a Lou rat immunized with a soluble alpha/beta T cell receptor (TcR; V alpha 2/V beta 2) to mouse myeloma cells. Analysis of a panel of V alpha 2 mRNA-expressing T cell lines, hybridomas and transfectants revealed that the B20.1 antibody was specific for murine TcR V alpha 2 chains. The V alpha 2+ T cell population was examined in various inbred strains by two-color immunofluorescence using B20.1 and CD4- and CD8-specific antibodies with the following results: (a) the B20.1 antibody detected most members of the TcR V alpha 2 subfamily in the four TcR V alpha haplotypes tested; (b) in most strains examined, TcR V alpha 2 expression was biased to the CD4 subset (7.4%-17.4% V alpha 2+ T cells) as compared to the CD8 compartment (3.8%-13.3%); (c) TcR V alpha 2 expression was not influenced by Mls gene products and (d) increased positive selection of V alpha 2+ CD8+ T cells by H-2k major histocompatibility complex molecules occurred in all murine strains tested of the TcR V alpha a, but not in those bearing the TcR V alpha b haplotype.     

Attenuation of lpr-graft-versus-host disease (GVHD) in MRL/lpr spleen cell-injected SCID mice by in vivo treatment with anti-V beta 8.1,2 monoclonal antibody.

When MRL/lpr (H-2k) spleen cells were intraperitoneally injected into C.B-17-scid/scid (severe combined immunodeficient (SCID)) (H-2d) mice, the SCID (SCID-MRL/lpr) mice manifested a severe wasting syndrome with weight loss, splenic atrophy, and lymphoid cell infiltration in the liver and lung, as seen in lpr-GVHD. In contrast, MRL/+ spleen cell-injected SCID (SCID-MRL/+) mice did not show lpr-GVHD. The spleens of SCID-MRL/lpr mice showed progressive increases in donor CD4+ and CD8+ T cells from 4 to 12 weeks after injection and a decrease in B cells at 12 weeks. SCID-MRL/+ mice showed a stable engraftment of CD4+ and CD8+ T cells and a progressive increase in B cells. Analyses of T cell receptor (TCR) repertoires (V beta 6, V beta 8.1,2 and V beta 11) revealed that the V beta 8.1,2+ T cells were found more frequently in SCID-MRL/lpr mice than in SCID-MRL/+ mice. When SCID-MRL/lpr mice were treated with intraperitoneal injection of an anti-V beta 8.1,2 (KJ16) MoAb, V beta 8.1,2+ T cells were markedly depleted, and the severity of lpr-GVHD was attenuated at 4 and 8 weeks after treatment, in contrast to normal rat IgG-injected SCID-MRL/lpr mice. However, the KJ16 MoAb-treated SCID-MRL/lpr mice suffered from severe lpr-GVHD 12 weeks after treatment, although V beta 8.1,2+ T cells were still maintained at a low level. These findings suggest that V beta 8.1,2+ T cells are a major T cell population that mediates lpr-GVHD in the early stage of lpr-GVHD, but that in the later stage, the other T cell populations may proliferate naturally or in accordance with the depletion of V beta 8.1,2+ T cells, and contribute to the development of lpr-GVHD.     

Expression of CD4 can confer major histocompatibility complex class II-associated superantigen reactivity upon a T cell receptor derived from a CD8-dependent cytotoxic T lymphocyte clone

It has been shown that reactivity against major histocompatibility complex (MHC) class II-associated Mlsa determinants is mainly mediated by CD4+ V beta 6+ T cells. 3F9 is a CD8+ CTL clone which is specific for the alloantigen H-2Db. While 3F9 is V beta 6+, it is not Mlsa reactive, presumably because it does not express CD4. 3F9 utilizes the same T cell receptor (TcR) V alpha V beta combination as LB2, a CD4+ T helper clone specific for chicken red blood cells (cRBC)/I-Ab and yet differs from LB2 in the junctional sequences in both TcR chains. CD4+ CD8- and CD4-CD8- hybridomas expressing the 3F9 TcR were tested for reactivity against Mlsa and cRBC/I-Ab. Only the CD4+CD8- hybridomas were Mlsa reactive, and antibody inhibition studies revealed that this reactivity was both CD4 and MHC class II dependent. Therefore the expression of the CD4 molecule can make an MHC class I-restricted TcR Mlsa reactive. Neither type of hybridoma reacted against cRBC, thus the main difference in the antigen reactivity between 3F9 and LB2 lies in the TcR junctional regions.     

Impaired H-2 expression in B16 melanoma variants

We studied the expression of H-2b alloantigens in three different B16 melanoma lines cultures in vitro. Cell lines were B16-F1 and two cell cultures (named B16-A and B16-B) newly derived from two different in vivo sublines of B16 melanoma. The assays used were in vivo tumour growth in allogeneic (BALB/c and B10.BR) as compared to syngeneic mice, in vitro cell-mediated cytotoxicity by anti-H-2b immune lymphocytes and absorption of anti-H-2b antisera activity. The B16-F1 line was able to efficiently kill allogeneic hosts, could not be lysed by anti-H-2b cytotoxic effectors and did not express any serologically detectable amount of H-2b alloantigens. The B16-A line was H-2 positive during the early in vitro passages, then, at the 8th-10th passages, it acquired the capacity to kill allogeneic hosts, lost the sensitivity to anti-H-2b cytotoxic effectors and the H-2Kb antigens became undetectable The expression of H-2Db was reduced, although at a lower degree. Similar data were obtained with B16-B cells, which after 10 in vitro passages grew and killed allogeneic hosts, showed a decreased sensitivity to cytotoxic anti-H-2b effectors and a very low expression of the K region antigens. The results indicate that H-2 expression is altered in B16 melanoma lines and this may influence the different metastatic capacity of such cells.     

CD4+T cells restricted by DQ not by DR support CD8+T cells to grow

Much attention has been paid whether there are any differences in regulating the human immune response between HLA-DR and -DQ molecules encoded by the genes within the HLA class II multigene family. Previous studies have suggested that HLA DQ molecules control low responsiveness through activating CD4 T cells which generate CD8 positive T cells, whereas HLA -DR molecules control high responsiveness through activating CD4 helper T cells. To examine this model we investigated the streptococcal cell wall antigen (SCW) specific T cell lines restricted by either DR or DQ molecule. To identify the restricting molecules, L cell transfectants expressing DQw1, DR2AB1 or DR2AB5 from Dw12 haplotype or DQw4, DR4 or DRw53 from DW15 haplotype were used. 1. From individuals with Dw12 which is a low responder haplotype to SCW, T cell clones specific to SCW and restricted by HLA-DQw1 or DR2 were identified, whereas from individuals with Dw15 which is a high responder haplotype, only DR4 or DRw53 restricted T cell clones were identified and DQw4 restricted T cells were never observed. 2. SCW specific CD4 T cells restricted by DQw1 were able to support the growth of CD8 positive cells, whereas those restricted by DR4 could not do so. 3. The CD8 T cells also required autologous antigen presenting cells and SCW to grow, and they completely blocked the immune response to SCW in vitro. These observations clearly demonstrated the distinct function of HLA-DQ and -DR molecules in regulating the human immune response to SCW.     

CD4 regulates the efficiency of an endogenous superantigen-induced clonal deletion of TCRV beta 11+ cells in the periphery.

Peripheral T-cell antigen receptor V beta (TCRV beta) repertoire is influenced by clonal deletion both in the thymus and periphery. Developing thymocytes expressing certain TCRV beta are deleted by endogenous superantigens presented on major histocompatibility complex (MHC) molecules in the thymus. Likewise, mature T cells bearing particular TCRV beta chains can be clonally deleted by superantigens in the periphery. The efficiency with which T cells expressing particular V beta subunits are deleted differs depending upon which coreceptor is expressed. Indeed, while deletion of V beta 11+ splenic T cells in CBA/J (Mls-1, a I-E, + MTV 9+) mice is quite efficient for CD4+ spleen T cells, it is much less efficient for CD8+ splenic T cells. If the difference in the efficiency of deletion is due solely to the coreceptor expressed, then a transgene encoding CD4 should increase the efficiency with which CD8+ cells are deleted. To address this question, we have produced CD4 transgenic (TG) mice that express physiologic levels of CD4 on all thymocytes and peripheral CD8 T cells. CD4 molecules expressed on CD8+ splenic T cells were associated with P56lck tyrosine kinase, and were functional as evidenced by their ability to facilitate class II alloreactivity. Furthermore, we found that ectopic expression of TG CD4 molecules on CD8+ cells was able to affect the efficiency of deletion in response to superantigen stimulation. In particular, deletion of TCRV beta 11+ T cells was much less efficient for CD8+ than for CD4+ T-cell subpopulations in (CBA/J x B6) F1 mice. However, expression of the CD4 transgene on CD8+ splenic T cells from these mice increased the efficiency of deletion in the CD8+ V beta 11 T cells. Interestingly, this effect was not observed in a mature CD8+ thymocyte subpopulation. The results in this report demonstrate that CD4 molecules are involved in peripheral deletion of TCRV beta 11+ T cells in (CBA/J x B6) F1 mice, and that the TCRV beta repertoire can be altered by ectopic expression of CD4 on all T-lineage cells.     

Alloreactive T-cell clones raised in an HLA-B/D crossing-over family dissect HLA-DR5 and HLA-DQw3 subtypes.

This study was undertaken to resolve a positive mixed lymphocyte reaction between HLA-ABC identical, HLA-D different siblings. Three CD3+ CD4+ CD8- alloreactive T-lymphocyte clones, called 2/6, 7/1, and 7/2, were generated and extensively studied. Proliferation of 2/6 cells and 7/2 cells was blocked by anti-DQ monoclonal antibodies (mAbs), whereas anti-DR and DP were not effective. Stimulation of 7/1 cells was inhibited by anti-DR, but not by anti-DQ and DP mAbs. Testing on a well-characterized panel of reference B-lymphoblastoid cell lines showed that the DQ-specific clones 2/6 and 7/2 were able to proliferate upon stimulation by cells carrying the DQw7 and DQw8 but not the DQw9 subtype of DQw3. Clone 7/1 was proliferative towards cells expressing DRw11.1 but not towards DRw11.2- or DRw12-positive cells. Moreover, this clone detected determinants present on some DRw8 cells. Correlation of the reactivity of clone 7/1 with available sequence data suggests that amino acids 67, 71, and 86 of DR beta 1 molecules played a crucial role in forming the epitope recognized by this clone. In contrast, sharing of T-cell epitopes between DQw7 and DQw8 subtypes was not inferable from specific amino acid residues. The implication of these findings for T-cell allorecognition is discussed.     

The requirement of intrathymic mixed chimerism and clonal deletion for a long-lasting skin allograft tolerance in cyclophosphamide-induced tolerance

Mechanisms of cyclophosphamide (CY)-induced tolerance were studied. When C3H/He Slc (C3H; H-2k, Mls-1b) mice were primed i.v. with 1 x 10(8) viable spleen cells from H-2-identical AKR/J Sea (AKR; H-2k, Mls-1a) mice and treated with 200 mg/kg of CY 2 days later, a long-lasting skin allograft tolerance to AKR was established. When [C57BL/6 Sea (B6; H-2b, Mls-1b) x AKR]F1 (B6AKF1) cells were used as the tolerogen, however, only a moderate, but not long-lasting, skin tolerance to AKR was observed. In the C3H mice treated with AKR cells and CY, the intrathymic clonal deletion of V beta 6+ T cells, which are strongly correlated with reactivity to Mls-1a antigens, was observed in the chimeric thymus on day 35, although neither the clonal deletion of V beta 6-bearing T cells nor the mixed chimerism was observed in the thymus on day 14. In the C3H mice treated with B6AFKF1 cells followed by CY, however, neither the clonal deletion of V beta 6+ T cells nor the mixed chimerism was observed in the thymus throughout the test period. In the lymph nodes of the C3H mice treated with AKR cells and CY, only CD4+ V beta 6+ T cells, bur not CD8+V beta 6+ T cells, had selectively decreased by day 14, and they were hardly detectable on day 35. The selective decrease of CD4+V beta 6+ T cells in the lymph nodes was also observed by day 14 when B6AKF1 cells were used as the tolerogen, although CD4+V beta 6+ T cells gradually increased on day 35, at which time almost all skin grafts from AKR had already been rejected. These results strongly support the necessity of the intrathymic mixed chimerism and clonal deletion of donor-reactive T cells for a long-lasting skin allograft tolerance in CY-induced tolerance.     

Alloreactive T-cell clones identify multiple HLA-DQw3 variants.

HLA-DQw3 is a broadly defined alloantigen that has been subdivided by serological, biochemical, and molecular methods into three distinct specificities: DQw7, DQw8, and DQw9. In order to characterize functionally relevant structural polymorphisms within this family of alloantigens, we generated a series of DQw3-reactive T-cell clones that together recognize six different variants of DQw3. T-cell clones IG11 and IG9 were found to recognize three distinct functional variants associated with a majority of DQw3+ cells, while clones 21J, IE6, 64B, and IC3 recognized four more narrowly distributed functional variants associated with unique DQw7, DQw8, and DQw9 subsets. Comparison of known DQB gene sequences suggested candidate recognition sites for clones IG11 and 64B in the region of amino acid residues 66 to 71 and residue 57 of the DQ beta chain. In contrast, no unique DQB or DQA sequences were found that individually corresponded to the reactivity patterns of clones 21J, IE6, IG9, or IC3, suggesting that an interaction between DQ alpha and DQ beta chains determines allo-recognition. These data are consistent with the hypothesis that T cells recognize specific alloepitopes on HLA class II molecules, either as distinct structural elements that trigger an alloresponse or, more indirectly, as contact elements that influence alloreactivity by governing the binding of foreign peptide. The results illustrate the diversity of possible T cell responses directed toward HLA-DQ molecules and suggest that T cell recognition of the DQ heterodimer alone, or a peptide antigen bound to the DQ heterodimer, can be affected either by the individual DQ alpha and beta chains, or by a more complex interaction between the two.     

Immunohistology of T cell differentiation in the thymus of H-Y-specific T cell receptor alpha/beta transgenic mice

We examined the immunohistological aspects of the H-Y specific T cell receptor (TcR) alpha/beta transgene expression in the thymus of male and female transgenic (Tg) mice. Virtually all thymocytes expressed the beta transgene in both the male and female thymus. Expression of accessory molecules (co-receptors) in Tg mice deviated from control mice. In the male Tg thymus, CD8 expression was either low or absent on both cortical and medullary thymocytes. In contrast, in the thymus of female mice, CD8+ cells were found both in the cortex and in the medulla. The majority of medullary thymocytes was bright CD8+. This is in clear contrast to the CD8 distribution in control B6 mice, where only a few percent of medullary cells are CD8+. Similarly, the proportion of cells expressing CD4 antigens was reduced in the cortex and medulla of the thymus from male Tg mice, as compared to the thymus of female Tg mice and B6 control mice. Comparative analysis of the stromal cell types of the thymic microenvironments in the three groups of mice revealed that the cortical thymic microenvironment of male Tg mice differed, compared to that of female Tg mice. In particular, the deep cortex showed a closely packed meshwork of epithelial reticular cells. Moreover, H-2Db molecules (which are the restricting elements for the Tg TcR alpha/beta) were abnormally expressed in the thymic cortex of male mice. The cortical microenvironment in female mice, on the other hand, appeared normal. Together, the data indicate that TcR alpha/beta transgene expression in male mice leads to an aberrant co-receptor expression in both cortical and medullary lymphoid cells as well as an abnormal composition of the cortical microenvironment. Both phenomena may be the consequence of "negative selection" of developing H-Y-specific T cells, as it occurs only in the male Tg thymus. The absence of the H-Y antigen, but presence of the restricting element H-2Db in the thymic cortex of female mice, leads to accumulation of CD8+ in the medulla, a phenomenon interpreted as "positive selection".     

CD3+ T cells in severe combined immunodeficiency (scid) mice. IV. Graft-vs.-host resistance of H-2d scid mice to intravenous injection of allogeneic H-2b (C57BL/6) spleen cells.

Intraperitoneal injection of 2 x 10(7) nonfractionated spleen cells (SC) from C57BL/6 (B6, H-2b) mice into completely allogeneic immunodeficient H-2d scid mice induced clinical and histological signs of acute graft-vs.-host disease (GVHD), with all transplanted severe combined immunodeficiency (scid) mice dying in the 3rd week post-transfer. In contrast four out of five scid mice survived for greater than 7 weeks after intravenous (i.v.) injections of equal numbers of B6 SC. Intravenously allotransplanted scid mice analyzed in the 8th week post-transfer had engrafted donor-type CD4+ and CD8+ T cells in the spleens but showed no clinical or histological evidence of GVHD. i.v. injection of 10(7) or 10(6)O B6 SC engrafted allogeneic T cells in spleens of scid recipients; in contrast, i.v. injection of 10(5) nonfractionated B6 SC or 3 x 10(5) cell sorter-purified, naive or anti-H-2d-primed splenic CD4+ or CD8+ B6 T cells led to rejection by young scid recipient mice. B6 T cells engrafted into spleens of scid mice after i.v. injection showed proliferative anti-host alloreactivity in vitro. No cytotoxic reactivity against host-type alloantigens was found in standard 4-h 51Cr-release assays. These data demonstrate that allogeneic T cells injected i.v. into immunodeficient scid mice are partially tolerized against host-type alloantigens.     

Fate of potentially self-reactive T cells in neonatal mice: analysis of V beta 6+ T cells in Mlsa mice

FACS analysis of either CD4 or CD8 depleted thymocytes revealed, that in adult Mlsa mice among CD4+ or CD8+ subpopulation of mature T cells V beta 6+ cells were absent. In contrast neonatal Mlsa mice had CD4+ V beta 6+ cells that rapidly decreased after day 4. On thymus cryosections of neonatal DBA/2 (Mlsa) mice V beta 6+ cells were found both in the cortex and the medulla of the thymus, in addition V beta 6+ cells were also detected in lymph nodes. After day 7-8 V beta 6+ cells disappeared from the medulla and the periphery. In the adult thymus only dull staining V beta 6+ cells were present in the cortex. Comparison of the fluorescence intensities of V beta 6 staining thymocytes from neonatally with Mlsa spleen cells tolerized mice with that of untreated BALB/c controls showed a significant decrease in T cell receptor density. Neonatal V beta 6+ T cells could be shown to be autoreactive against Mlsa, this reactivity declined at about the same time when the stimulatory capacity of Mlsa was established and could be first shown on day 4.     

Establishment of stable CD8+ suppressor T cell clones and the analysis of their suppressive function.

Stable CD8+ suppressor T cell (Ts) clones were established by a relatively simple method. Keyhole limpet hemocyanin (KLH)-primed spleen cells from C3H mice were depleted of B cells and CD4+ T cells by panning and cytotoxic treatment, and the resulting CD8+ T cells were periodically stimulated with antigen and irradiated syngeneic spleen cells followed by manifestation in interleukin-2 (IL-2) containing medium. T cell clones with a definite suppressor function were established by limiting dilution. They were defined as classical effector type Ts of CD8+ phenotype as they had constant and definite suppressor functions in antigen-induced T cell proliferation and specific antibody response against T cell-dependent antigens without detectable cytotoxic activity against both antigen presenting cells (APC) and helper T cells (Th). They showed no helper activity for B cells and produced no detectable helper type lymphokines such as IL-2 and IL-4. CD8+ Ts clones were able to inhibit the antigen-induced IL-2 production of normal and cloned T cells. Their suppressive activity was antigen-nonspecific and major histocompatibility complex-unrestricted. CD8+ Ts clones were also able to suppress the proliferative response of Th clones induced by immobilized anti-T cell receptor (TcR) and anti-CD3 mAbs but not the response induced by concanavalin A (ConA) and IL-2. All the CD8+ T cell clones established independently utilized the TcR V beta 8 gene. Syngeneic antigen presenting cells could induce proliferation of these CD8+ clones, which was blocked by anti-CD8 and anti-I-Ak monoclonal antibody (mAb) but not by anti-class I mAbs. The stimulation of CD8+ Ts clones with immobilized anti-CD3 resulted in the release of a suppressor factor(s) that potently inhibited the antigen-induced proliferation of CD4+ Th clones and the in vitro secondary antibody formation.