Analysis of Ir gene control of cytotoxic response to hapten-modified self: helper T cells specific for a sulfhydryl hapten can substitute for an anti-TNP-H-2b self helper cell defect

Helper T cells specific for N-iodoacetyl-N'-(5-sulfonic 1-naphthyl) ethylene diamine (I-AED) were generated in (C56BL/6 X C3H/He)F1 mice by immunization with I-AED-modified syngeneic cells (AED-self). The requirements for activation of hapten-induced helper cells were investigated. The results demonstrated that activation of AED and trinitrophenyl- (TNP) helper cells was strictly hapten specific. In addition, F1 AEd-helpers could be activated efficiently by either I-AED-modified H-2b or H-2k self components to enhance the anti-AED self-CTL responses. This contrasts with the previous findings demonstrating the failure of TNP-H-2b self to activate F1 TNP-helper cells. After AED-helpers were activated, they were capable of augmenting sensitization of cytotoxic T cells (CTL) against TNP-self. These results indicate that although the activation of hapten-reactive helper cells is antigen (hapten)-specific, the subsequent helper activity, as determined by augmentation of CTL responses against another hapten, is antigen nonspecific. Since helper function was antigen nonspecific, F1 AED-helper cells activated by AED-H-2b or AED-H-2k self were tested for their ability to augment the F1 and anti-TNP-H-2b CTL response. The results indicate that the Ir gene defect in the ability of F1 spleen cells to respond to TNP-H-2b self could not be corrected by these helper cells. These results are discussed in the light of Ir gene controlled differences in the activation of AED and TNP-helper cells and possible models for augmenting CTL responses against various antigens in strains that generate marginal helper activity to TNP-self.     

Insulin-specific tolerance induction. I. Abrogation of helper T cell activity is controlled by H-2-linked Ir genes

Murine antibody responses to heterologous insulins are under H-2-linked immune response (Ir) gene control. We have found that the immune response to insulin in adjuvant can be inhibited by prior i.v. injection of soluble insulin. The effect of i.v. injection of insulin is antigen-specific and dose-dependent and requires the same doses of insulin that are immunogenic if administered with adjuvant. In addition, the inhibitory effect of soluble insulin is dependent upon the route of injection; if soluble insulin is injected i.p., the subsequent response to insulin in adjuvant is augmented rather than inhibited. Unresponsiveness requires at least 4 days after i.v. injection to develop and once induced, it is maintained for 4 wk or more. Unresponsiveness is caused by T cell, but not B cell, tolerance, and we have been unable to demonstrate any role for suppressor T cells in this unresponsiveness. More importantly, analysis of the ability of numerous insulin variants to induce unresponsiveness in several H-2k and H-2b strains of mice has demonstrated that only the variants that were immunogenic in a given strain when administered with adjuvant were able to cause tolerance. This report is, to our knowledge, the first describing that induction of helper T cell tolerance, like the induction of immunity, is controlled by H-2-linked Ir genes.     

人骨髓基质细胞向成骨细胞分化过程中差异表达基因的筛选

为了探讨人骨髓基质细胞（bone marrow stromal cells,BMSCs）向成骨细胞分化过程中差异表达的基因,本实验采用体外培养人BMSCs,诱导向成骨细胞分化。分别选取培养12和21d的细胞作为驱动方（driver）和实验方（tester）,进行抑制消减杂交,构建cDNA消减文库,将挑选出的阳性克隆与GenBank人基因库中己公布的核酸序列进行同源性比较分析。结果表明,从培养21d的BMSCs中,筛查出5个差异基因,与人基因库中己知基因的同源性分别达到90％以上。有兴趣的是,核心蛋白聚糖和Bax inhibitorl在培养2ld的BMSCs中差异表达。RT-PCR检测显示,核心蛋白聚糖基因在培养21d的细胞中高表达,而在12d的细胞中未检测到表达;Bax inhibitorl基因在培养21d细胞中的表达明显高于12d的细胞。     

Genetic control of immune response to sperm whale myoglobin in mice. I. T lymphocyte proliferative response under H-2-linked Ir gene control.

Studies on the genetic control of immune response to sperm whale myoglobin were initiated. As demonstrated in this paper, the T lymphocyte proliferative response to whale myoglobin is under H-2-linked Ir gene control. Mice of H-2d, H-2f, and H-2s haplotypes were high responders to the myoglobin, whereas haplotypes H-2b, H-2k, H-2p, H-2q, and H-2r were low responders. The Ir gene(s) was localized between H-2K and H2D regions, since the recombinant strain A.TL (KsIkSkDd) was a low responder and A.TH (KsIsSsDd) was a high responder. Further studies with recombinant strains revealed that the expression of the high-responder I-Ad or Ias alleles was sufficient to give a good response, since strains D2.GD (d d b b b b b b) and B10.HTT (s s s s k k k d) were high responders. The expression of the I-Cd allele in strains B10.A (k k k k k d d d) and B10.A(5R) (b b b k k d d d) also gave high response, and thus suggested a second Ir gene, derived from the H-2d haplotype. The finding that expression of the I-Cs allele in B10.S(8R) (k k ? ? s s s s) did not result in high response suggests the lack of the second Ir gene in the high-responder H-2s haplotype.     

Genetic control of experimental autoimmune myasthenia gravis in mice. I. Lymphocyte proliferative response to acetylcholine receptors is under H-2-linked Ir gene control.

As a first step in determining the genetic control of experimental autoimmune myasthenia gravis in mice, we tested the proliferative responses of lymph node cells to torpedo acetylcholine receptors (TAR). Studies with congenic and recombinant inbred strains of mice revealed that T-cell responses to TAR are controlled by an H-2 linked Ir gene, mapping in the I-A subregion of mouse major histocompatibility complex.     

T lymphocyte responses to non-H-2 histocompatibility antigens. I. Role of Ly-1+2+ T cells as cytotoxic effectors and requirement for Ly-1+2+ T cells for optimal generation of cytotoxic effectors

Cytotoxic effector T cells specific for non-H-2 histocompatibility (H) antigens were examined for phenotypic expression of lymphocyte differentiation (Ly) antigens. Virtually all H-Y-specific cytotoxic effectors generated in mixed lymphocyte culture were Ly-1+2+ T cells. H-3-specific effectors comprised both Ly-1+2+ and Ly-1-2+ T cells. However, cytotoxic effectors specific for multiple non-H-2 H antigens were predominantly Ly-1-2+ T cells. The optimal generation of H-Y- and H-3-specific effectors required Ly-1+2+ T cells; optimal generation of multiple non-H-2 H antigen-specific effectors required an interaction between Ly-1+2- and Ly-1-2+ T cells. These observations suggest that the identity of the target H antigen in part determines the Ly type of responsive T cells. Our observations suggest that 2 alternative pathways of T cell response exist for non-H-2 H antigens. The first pathway involves an interaction between Ly-1+2- helper T cells and Ly-1-2+ cytotoxic effector precursors. The 2nd pathway simply involves the response of Ly-1+2+ T cells proliferating and generating H antigen-specific cytotoxic effectors.     

Protein kinase C is required for responses to T cell receptor ligands but not to interleukin-2 in T cells

We have tested the role of protein kinase C in mRNA expression and T cell proliferation mediated through the T cell receptor and through the interleukin-2 (IL-2) receptor. Chronic treatment of a mouse T cell clone with phorbol esters caused a complete loss of protein kinase C activity and a concomitant loss of proliferation to T cell receptor ligands (antigen, lectins, antireceptor antibodies). In contrast, kinase C-depleted T cells retained the ability to proliferate to IL-2. Loss of the T cell receptor response was not due to decreased cell surface expression of receptor or impairment of early receptor function (phosphatidylinositol turnover, calcium mobilization). Kinase C-depleted T cells showed no induction of mRNAs for activation-associated genes on exposure to the T cell receptor ligand Concanavalin A; expression of a subset of the same mRNAs in response to IL-2 was unaffected. We conclude that kinase C is required for mRNA expression and subsequent proliferation mediated through the T cell receptor pathway but is not involved in mRNA expression and proliferation in response to IL-2.     

Heterogeneity of CD4+ T cells involved in anti-allo-class I H-2 immune responses. Functional discrimination between the major proliferating cells and helper cells assisting cytotoxic T cell responses.

MLR in various combinations with class I H-2 disparity revealed that there are three patterns of MLR in the aspect of responding T subset (CD4 vs CD8) dominance. Irrespective of the CD8 vs CD4 dominance, a single i.v. administration of class I-disparate allogeneic spleen cells resulted in almost complete abrogation of anti-class I proliferative capacity of both CD4+ and CD8+ T cells in six combinations. The suppression of proliferative responses was correlated with the striking reduction in the ability to produce IL-2 upon stimulation with the relevant class I alloantigens. In contrast, i.v. presensitized recipient mice exhibiting only marginal MLR/Il-2 production could generate comparable magnitudes of anti-allo class I CTL as well as graft rejection responses to those induced by normal unpresensitized mice. The administration in vivo of anti-CD4 antibody along with the i.v. presensitization not only suppressed the generation of CTL responses by spleen cells but also induced appreciable prolongation of allo-class I-disparate skin grafts under conditions in which neither alone did it. These results demonstrate that 1) the suppression of graft rejection responses is not necessarily reflected on the reduction of MLR; 2) CD8+ CTL precursors responsible for graft rejection can be activated by either allo-class I-reactive CD8+ or CD4+ Th cells; 3) i.v. presensitization induces functional elimination of CD8+ and CD4+ proliferative/IL-2-producing T cells but not of CD8+ CTL precursors and CD4+ Th whose capacity is expressed by assistance of CTL induction but not by their own proliferation. Thus, this study illustrates the heterogeneity of class I alloantigen-reactive CD4+ T cells in the aspect of their capacity to proliferate themselves vs contribute to CTL induction as well as graft rejection.     

The immunoregulatory role of bone marrow. I. Suppression of the induction of antibody responses to T-dependent and T-independent antigens by cells in the bone marrow.

Bone marrow cells (BMC) from normal mice suppressed the in vitro IgM, but not the IgG, antibody (Ab) response of spleen cells. BMC were inhibitory only when added during the first 24 hr of culture, and inhibition was not due to an induced shift in the kinetics of the response. Addition of specifically activated T cells or nonspecific T-cell-replacing factors to normal or T-depleted spleen cell cultures did not abrogate suppression while the response to the T-independent antigen DNP-polymerized flagellin or lipopolysaccharide was also suppressed. BMC did not inhibit background Ab synthesis by normal or primed cells in the absence of antigen and did not inhibit, but stimulated, DNA synthesis in normal spleen cell cultures. In addition, high-avidity Ab synthesis was preferentially suppressed. A possible role for the bone marrow suppressor cell in the induction of B cell tolerance is discussed.     

Class I MHC-restricted cytotoxic T lymphocyte recognition of cells infected with human cytomegalovirus does not require endogenous viral gene expression.

The human pathogen CMV, is a major cause of morbidity and mortality in immunocompromised hosts. The CD8+ class I-restricted CTL response to CMV assists in preventing progression of CMV infection to life-threatening disease; however, the viral Ag recognized by CD8+ CTL are not well characterized. In general, virus-specific CTL recognize endogenously synthesized viral proteins processed and presented associated with class I MHC molecules. Although proteins or inactivated virions have been experimentally delivered to the cytoplasm to result in class I MHC presentation, this mode of Ag delivery to the class I processing pathway after natural viral entry has not been documented in humans. Our data demonstrate that the CMV-specific class I-restricted CTL response in individuals latently infected with CMV is predominantly specific for selected structural virion proteins introduced into the cell after viral penetration and efficient recognition occurs in the absence of de novo viral gene expression. This CTL response may provide a biological advantage for limiting the spread of infection after CMV reactivation because infected cells are lysed before viral assembly.     

Surface markers on the T cells that regulate cytotoxic T-cell responses I. The Ly phenotype of suppressor T cells changes as a function of time, and is distinct from that of helper or cytotoxic T cells

Regulatory T cells can be obtained from primary mixed lymphocyte cultures of CBA spleen cells responding to BALB/c stimulators. At day 3 of culture, T cells are generated which can either help or suppress the generation of cytotoxic T cells in a second primary MLC culture. The regulatory activity observed depends on the conditions employed in the assay system allowing independent assay of different functional cell types which coexist in the cultures. Both the helper activity and the suppressor activity are mediated by differentiated antigen-specific T cells whose function is radioresistant. The Ly phenotype of these regulatory cells was tested. At day 3 of the first-step culture, the phenotype of the helper cells is Ly 1.1+ Ly 2.1-, whereas the inhibitory cells are Ly 1.1 Ly 2.1+. At day 5 of M LC culture, suppressor activity and helper activity are also observed. However, at this point, a suppressor cell which is Ly 1.1-Ly 2.1+ represents the major inhibitory activity. It is not clear whether this change in suppressor cell phenotype as a function of time in culture represents one differentiation pathway or cells derived from two different precursor cells. The Ly phenotype of helper or cytotoxic T cells did not change as a function of time in culture. In day 5 first-step cells, the cytotoxic cells were typed as Ly 1.1+ 2.1+, whereas the inhibitory cells present in aliquots of the same treated cell population expressed the Ly 1.1- Ly 2.1 phenotype. Taken together, these observations show that the antigen-specific suppressor cells and helper cells which regulate the generation of cytotoxicity, and the cytotoxic cells themselves represent physically distinct subclasses of T cells.     

Development of diabetogenic T cells from NOD/Lt marrow is blocked when an allo-H-2 haplotype is expressed on cells of hemopoietic origin, but not on thymic epithelium

Diabetes is a T cell-mediated process in NOD/Lt mice, with a major genetically recessive component of susceptibility linked to homozygous expression of the unique H-2g7 MHC haplotype. Heterozygous expression of the H-2nb1 haplotype derived from the NON/Lt strain confers diabetes resistance both in (NOD x NON)F1 hybrids and in NOD mice congenic for the H-2nb1 haplotype. However, diabetes resistance is abrogated in F1 hybrids by NOD/Lt bone marrow reconstitution. To establish whether the generation of beta cell autoreactive T cells from NOD/Lt bone marrow-derived precursors required at least heterozygous expression of the H-2g7 haplotype on thymic epithelium, adolescent thymectomized (NOD x NON)F1 mice were implanted with neonatal NON/Lt thymus grafts before lethal radiation and reconstitution with NOD/Lt bone marrow. Peripheral T cells maturing through this ectopic thymic implant exclusively expressed the NOD H-2g7 haplotype and were tolerant to H-2nb1 skin grafts. Nevertheless, diabetes developed in 32% of the NON/Lt thymus-grafted chimeras vs 38% of the sham-thymectomized NOD bone marrow chimeras. Thus, homozygous expression of the diabetes-resistant H-2nb1 haplotype on thymic epithelium failed to block development of a diabetogenic T cell repertoire. To examine if expression of H-2nb1 on hemopoietically derived APC could alter the diabetogenic potential of NOD/Lt marrow, diabetes-resistant NOD.NON-H-2nb1 congenic mice were mated with NOD/Lt mice to produce NOD-H-2g7/H-2nb1 heterozygous recipients. These were lethally irradiated and reconstituted with either NOD/Lt marrow alone, NOD.H-2nb1 homozygous congenic marrow alone, or a 1:1 mixture of the two marrow populations. By 25 wk of age, all of the MHC heterozygous recipients of NOD.NON-H-2nb1 marrow remained diabetes-free whereas 75% of the MHC heterozygous recipients of NOD/Lt marrow developed diabetes. A striking decrease in diabetes was observed when T cell precursors derived from NOD/Lt marrow interacted with H-2nb1 gene products on hemopoietically derived APC, inasmuch as only 7% of the MHC heterozygous recipients reconstituted with a 1:1 mixture of NOD/Lt and NOD.NON-H-2nb1 marrow developed diabetes. Peripheral leukocytes in all reconstitution classes expressed the MHC phenotype(s) of the marrow donor(s). Skin grafting confirmed that all reconstitution classes of MHC heterozygous recipients were tolerant to the H-2nb1 haplotype.(ABSTRACT TRUNCATED AT 400 WORDS)     

H-2 linked Ir gene control of antibody responses to porcine insulin. I. Development of insulin-specific antibodies in some but not all nonresponder strains injected with proinsulin

Cell-mediated and humoral immune responses to heterologous insulins in mice are controlled by H-2 linked, dominant, immune response (Ir) genes. For example, mice bearing the H-2d haplotype develop T cell proliferative responses and produce antibody after injection with porcine insulin, whereas mice bearing other H-2 haplotypes do not. Data presented in this communication demonstrate that homozygous and heterozygous H-2d mice produce insulin-binding antibodies when immunized with porcine insulin or proinsulin. Some (H-2b,k,s) insulin-nonresponder mice produce insulin-binding antibodies after injection of proinsulin, whereas other insulin-nonresponder strains (H-2q) do not. All strains, except homozygous H-2q mice, produce antibodies specific for proinsulin, suggesting that the response to porcine proinsulin is also controlled by H-2-linked Ir genes. More importantly, F1 hybrids between insulin-nonresponder C57BL/10 (H-2b) and DBA/1 (H-2q) produce no insulin-binding antibodies when injected with proinsulin, despite the fact that proinsulin-binding antibodies are produced by these mice.     

Cell-mediated immune responses to syngeneic tumors. I. Identification of two distinct CTL effector pathways which differ in antigen specificity, genetic regulation, and cell surface phenotype

It has been demonstrated previously that draining lymph nodes (DLN) from tumor-immunized mice contain a population of lymphoid cells that are capable of differentiating into functional antitumor cytotoxic T lymphocytes (CTL) during in vitro culture. In the present studies, it was observed that DLN cells from either C57BL/10 (B10) or C3H mice that had been footpad-immunized with syngeneic tumor cells differentiated into CTL during a 4-day in vitro culture in the absence of added antigen. The specificity patterns of the CTL thus generated, however, were quite different in the two strains. DLN from B10 mice immunized with ultraviolet light-induced fibrosarcoma cells of B10 origin differentiated into CTL which were only capable of lysing target cells from the tumor used for immunization. Thus, the antitumor CTL which differentiate from B10 DLN appeared to be specific for the tumor-specific antigen (TSA) expressed by these tumor cells. In contrast, DLN from C3H mice immunized with a syngeneic ultraviolet light-induced fibrosarcoma differentiated into CTL which effectively lysed not only target cells from the immunizing tumor, but several other fibrosarcomas of both B10 and C3H origin, and which did not lyse normal nontumor targets. These C3H effectors thus appeared to be specific for a tumor-associated antigen (TAA) which is widely shared by a number of tumors. Cold target-blocking studies demonstrated that the CTL generated by C3H DLN cells contained a subpopulation of TSA-specific cells in addition to cross-reactive TAA-specific effectors. (B6 X C3H)F1 (B6C3F1) mice generated cross-reactive TAA-specific CTL in response to in vivo challenge with either B10 or C3H tumors, indicating that the ability to generate a TAA-specific CTL response behaves as a dominant trait of the responding mouse strain and not as a function of the tumor used for immunization. TSA-specific CTL and cross-reactive TAA-specific CTL were distinguishable on the basis of their cell surface phenotypes, because the TSA-specific CTL generated by B10 DLN cells were Thy-1.2+ Lyt-2.2+, whereas TAA-specific B6C3F1 CTL were Thy-1.2+ Lyt-2.2-; alloantigen-specific CTL generated from the same B6C3F1 lymph nodes were Thy-1.2+ Lyt-2.2+.(ABSTRACT TRUNCATED AT 400 WORDS)