Absence of the RT-6 T cell subset in diabetes-prone BB/W rats

Diabetes-prone Bio-breeding/Worcester (DP) rats exhibit a severe T cell lymphopenia and autoimmune pancreatic insulitis. The present results indicate that the T cell lymphopenia is due in large part, if not entirely, to the absence of the RT-6+ peripheral T cell subset, which includes members of both the helper/inducer (W3/25) and suppressor/cytotoxic (OX 8) antigenic phenotypes. Delineation of the causal mechanism(s) for the selective absence of RT-6+ T cells in DP rats may provide important insights into the cellular basis of the insulin-dependent diabetes mellitus syndrome in these animals.     

Adoptive transfer of autoimmune diabetes mellitus in biobreeding/Worcester (BB/W) inbred and hybrid rats

Adoptive transfer of diabetes was accomplished by the injection of Con A-activated acutely diabetic BB/W rat spleen cells into immunosuppressed diabetes-resistant BB/W control rats and F1 hybrid offspring produced by BB/W X Lewis, BN, Yashida, and NEDH matings. Immune suppression methods that facilitated adoptive transfer of diabetes included neonatal thymectomy, cyclophosphamide, and splenectomy plus rabbit anti-rat lymphocyte serum injections. The successful transfer of BB/W diabetes to otherwise normal (BB/W X inbred)F1 rats and to diabetes-resistant BB/W animals suggests that antigenically normal pancreatic beta cells were destroyed by the injected effector cells. Diabetes-resistant BB/W control rats also evidenced diabetes after the injection of cyclophosphamide alone. The requirement for immunosuppression suggests that an intact immune system protects against adoptive transfer and diabetes in diabetes-resistant BB/W rats.     

Impaired antigen-induced CD8+ T cell clonal expansion in aging is due to defects in antigen presenting cell function

CD8+ T cell activation depends on interaction with antigen-presenting cells (APCs) and this interaction leads to the expansion of T cells with the capacity to control infection. Using professional APCs, we demonstrate that with age, the duration of APC-T cell contact time required to achieve clonal expansion increases. Na?ve CD8+ T cells from aged mice showed no defect in antigen-induced proliferation when stimulated with APC from young mice. In contrast, CD8+ T cells from young mice exhibited reduced clonal expansion and secreted significantly lower amounts of IFN-gamma when stimulated by APCs from aged mice. The aged APCs were defective in costimulatory molecule expression and cytokine and chemokine secretion. These data indicate that defects in APC function lead to poor T cell clonal expansion and function in aging.     

T cell immunity to type II collagen in the biobreeding rat: the identification and characterization of RT1u-restricted T cell epitopes on alpha 1(II)

Susceptibility to experimental collagen-induced arthritis in rodents is dependent on MHC class II elements to bind peptides from the type II collagen (CII) molecule. Although a substantial body of data has been reported in mice defining these peptide Ags, little has been reported in rats. In this study, we investigate the locations and sequences of CII peptides, which are bound by RT1(u) molecules, expressed by diabetic-resistant, arthritis-susceptible Biobreeding rats, and, in turn, stimulate CII-specific T cells. By using overlapping and substituted peptide homologues of CII, we have identified and characterized an immunodominant and five subdominant epitopes on CII, which stimulate RT1(u)-restricted T cell proliferation. The immunodominant epitope, CII (186-192), contains a QGPRG core sequence, which was found in a subdominant epitope CII (906-916). Similar sequences containing single conservative substitutions were identified in three other epitopes. One, CII (263-272), contained a conservatively substituted R-->K substitution, whereas CII (880-889) and CII (906-916) contained nonconservative substitutions, i.e., P-->D and R-->M, respectively. Homologue peptides containing these sequences stimulated T cell proliferative responses, although less intensely than peptides containing CII (186-192). Substituting QGR residues in the QGPRG core with alanine, isoleucine, or proline reduced proliferation, as did substituting flanking E and G residues at the N terminus and E at the C terminus. Collectively, these data indicate that RT1(u)-restricted immunodominant and several subdominant epitopes on CII often share a QGPRG-like motif, with conservative substitutions present at either P or R positions. This motif is similar to one recognized by collagen-induced arthritis-susceptible HLA-DR1- and HLA-DR4-transgenic mice.     

Absence of T cell tolerance to pancreatic islet cells.

To examine whether the lack of self-tolerance to beta cells is responsible for the development of type I diabetes in nonobese diabetic (NOD) mice, we attempted to induce T cell responses to cells from the islets of Langerhans. The data show that all NOD mice, irrespective of age, sex, and disease progression, possess islet cell-specific CD4+, MHC class II-restricted T cells. Both primary and secondary proliferative responses to islet cells were readily induced. The activation of T cells required presentation of islet cell Ag by APC in the responding lymph node cell population. Cells from other tissues, e.g., salivary gland, adrenal gland, and spleen, failed to activate autologous T lymphocytes. T cells specific for other Ag did not respond to islet cells, indicating that the proliferation is not the result of nonspecific stimulation by islet cell products. The presence of islet cell-reactive T cells is, however, not unique to NOD mice, because similar T cell reactivity was also demonstrated in non-diabetes-prone mouse strains. Hence, self-tolerance to islet cells appears to be absent. The results indicate a normal occurrence of islet cell-reactive T cells in both diabetes-prone as well as non-diabetes-prone mice. Thus, the lack of tolerance cannot be the initial cause of diabetes, but the activation of such autoreactive T cells may be important for the development of the disease.     

BioBreeding/Worcester (BB/Wor) rats are deficient in the generation of functional cytotoxic T cells

The BioBreeding/Worcester (BB/Wor) rat provides a good model of spontaneous autoimmune diabetes. There are several sublines of the BB/Wor rat. The diabetes prone (DP) sublines develop diabetes at a frequency of 50 to 80% from 60 to 120 days of age. The DP rats are lymphopenic, have a severe deficit in phenotypic OX 19+ OX 8+ cytotoxic T cells (Tc), and lack RT 6.1 T cells. These rats have a relative increase in OX 19- OX 8+ natural killer (NK) cells and in NK activity as compared with the diabetes resistant (DR) sublines. The DR sublines have a normal complement of phenotypic Tc and RT 6.1 T cells, fewer NK cells, and lower NK activity than the DP rat. The ability to elicit functional Tc in the BB/Wor rat has not been well studied. In these experiments, by using a model of lymphocytic choriomeningitis virus (LCMV) infection in DP and DR rats, we have studied the functional activity of Tc in these lines. Seven days after infection with LCMV, DR rats develop lymphocytes which are cytotoxic for LCMV-infected syngeneic fibroblasts. These cytotoxic lymphocytes are phenotypic Tc (OX 19+ OX 8+), and do not kill Pichinde virus-infected syngeneic fibroblasts or LCMV-infected allogeneic fibroblasts. This cytotoxic activity is accompanied by an increase in phenotypic Tc from 17 to 33%. DP rats produced neither functional nor phenotypic Tc. These studies confirm that NK cells are the predominant cytotoxic lymphocyte in the BB/Wor rat and suggest that these rats may not utilize a Tc mechanism in islet destruction or another immunologic process such as graft rejection.     

Rapamycin-mediated enrichment of T cells with regulatory activity in stimulated CD4+ T cell cultures is not due to the selective expansion of naturally occurring regulatory T cells but to the induction of regulatory functions in conventional CD4+ T cells

Rapamycin is an immunosuppressive drug currently used in different clinical settings. Although the capacity of rapamycin to inhibit the mammalian target of rapamycin serine/threonine protein kinase and therefore T cell cycle progression is well known, its effects are complex and not completely understood. It has been reported recently that TCR-mediated stimulation of murine CD4+ T cells in the presence of rapamycin results in increased proportions of CD4+ T cells with suppressive functions, suggesting that the drug may also exert its immunosuppressive activity by promoting the selective expansion of naturally occurring CD4+ regulatory T cells (Treg). In this study, we show that stimulation of human circulating CD4+ T cells in the presence of rapamycin results indeed in highly increased suppressor activity. By assessing the effect of rapamycin on the growth of nonregulatory and Treg populations of defined differentiation stages purified ex vivo from circulating CD4+ T cells, we could demonstrate that this phenomenon is not due to a selective expansion of naturally occurring Tregs, but to the capacity of rapamycin to induce, upon TCR-mediated stimulation, suppressor functions in conventional CD4+ T cells. This condition, however, is temporary and reversible as it is dependent upon the continuous presence of rapamycin.     

Development of memory-like autoregulatory CD8+ T cells is CD4+ T cell dependent

Progression of spontaneous autoimmune diabetes is associated with development of a disease-countering negative-feedback regulatory loop that involves differentiation of low-avidity autoreactive CD8(+) cells into memory-like autoregulatory T cells. Such T cells blunt diabetes progression by suppressing the presentation of both cognate and noncognate Ags to pathogenic high-avidity autoreactive CD8(+) T cells in the pancreas-draining lymph nodes. In this study, we show that development of autoregulatory CD8(+) T cell memory is CD4(+) T cell dependent. Transgenic (TG) NOD mice expressing a low-affinity autoreactive TCR were completely resistant to autoimmune diabetes, even after systemic treatment of the mice with agonistic anti-CD40 or anti-4-1BB mAbs or autoantigen-pulsed dendritic cells, strategies that dramatically accelerate diabetes development in TG NOD mice expressing a higher affinity TCR for the same autoantigenic specificity. Furthermore, whereas abrogation of RAG-2 expression, hence endogenous CD4(+) T cell and B cell development, decelerated disease progression in high-affinity TCR-TG NOD mice, it converted the low-affinity TCR into a pathogenic one. In agreement with these data, polyclonal CD4(+) T cells from prediabetic NOD mice promoted disease in high-affinity TCR-TG NOD.Rag2(-/-) mice, but inhibited it in low-affinity TCR-TG NOD.Rag2(-/-) mice. Thus, in chronic autoimmune responses, CD4(+) Th cells contribute to both promoting and suppressing pathogenic autoimmunity.     

CD8+ T cell immunity against a tumor/self-antigen is augmented by CD4+ T helper cells and hindered by naturally occurring T regulatory cells

CD4(+) T cells control the effector function, memory, and maintenance of CD8(+) T cells. Paradoxically, we found that absence of CD4(+) T cells enhanced adoptive immunotherapy of cancer when using CD8(+) T cells directed against a persisting tumor/self-Ag. However, adoptive transfer of CD4(+)CD25(-) Th cells (Th cells) with tumor/self-reactive CD8(+) T cells and vaccination into CD4(+) T cell-deficient hosts induced autoimmunity and regression of established melanoma. Transfer of CD4(+) T cells that contained a mixture of Th and CD4(+)CD25(+) T regulatory cells (T(reg) cells) or T(reg) cells alone prevented effective adoptive immunotherapy. Maintenance of CD8(+) T cell numbers and function was dependent on Th cells that were capable of IL-2 production because therapy failed when Th cells were derived from IL-2(-/-) mice. These findings reveal that Th cells can help break tolerance to a persisting self-Ag and treat established tumors through an IL-2-dependent mechanism, but requires simultaneous absence of naturally occurring T(reg) cells to be effective.     

CD40-CD40 ligand interaction between dendritic cells and CD8+ T cells is needed to stimulate maximal T cell responses in the absence of CD4+ T cell help

Stimulation of CD40 on APCs through CD40L expressed on helper CD4+ T cells activates and "licenses" the APCs to prime CD8+ T cell responses. Although other stimuli, such as TLR agonists, can also activate APCs, it is unclear to what extent they can replace the signals provided by CD40-CD40L interactions. In this study, we used an adoptive transfer system to re-examine the role of CD40 in the priming of naive CD8+ T cells. We find an approximately 50% reduction in expansion and cytokine production in TCR-transgenic T cells in the absence of CD40 on all APCs, and on dendritic cells in particular. Moreover, CD40-deficient and CD40L-deficient mice fail to develop endogenous CTL responses after immunization. Surprisingly, the role for CD40 and CD40L are observed even in the absence of CD4+ T cells; in this situation, the CD8+ T cell itself provides CD40L. Furthermore, we show that although TLR stimulation improves T cell responses, it cannot fully substitute for CD40. Altogether, these results reveal a direct and unique role for CD40L on CD8+ T cells interacting with CD40 on APCs that affects the magnitude and quality of CD8+ T cell responses.     

Congenital developmental defects in the genetic monitoring system. I. The selection of developmental defects subject to dynamic observation

The requirements for selection of congenital malformations in the system of genetic monitoring as models have been studied. Model malformations have been shown to be easily and reliably diagnosed even in infancy. Their rate should be not less than 1:5000 births. The precise knowledge of the genetics of model forms is needed as well. Down's syndrome registration is the most convenient method for evaluation of mutations in a genome. We recommend registration in total all multiple congenital malformations (without Down's syndrome) for estimation of dominant mutability dynamics, as it was shown that more than 11% of all multiple malformations are caused by sporadic dominant mutations.     

Naive CD4+ T cells from lupus-prone Fas-intact MRL mice display TCR-mediated hyperproliferation due to intrinsic threshold defects in activation

Autoreactive T cell activation is a consistent feature of murine lupus; however, the mechanism of such activation remains unclear. We hypothesized that naive CD4+ T cells in lupus have a lower threshold of activation through their TCR-CD3 complex that renders them more susceptible to stimulation with self-Ags. To test this hypothesis, we compared proliferation, IL-2 production, and single cell calcium signaling of naive CD4+ T cells isolated from Fas-intact MRL/+(Fas-lpr) mice with H-2k-matched B10.BR and CBA/CaJ controls, following anti-CD3 stimulation in the presence or absence of anti-CD28. We also assessed the responsiveness of naive CD4+ T cells isolated from Fas-intact MRL and control mice bearing a rearranged TCR specific for amino acids 88-104 of pigeon cytochrome c to cognate and low affinity peptide Ags presented by bone marrow-matured dendritic cells. TCR transgenic and wild-type CD4+ T cells from MRL mice displayed a lower threshold of activation than control cells, a response that was class II MHC dependent. The rise in intracellular calcium in MRL vs controls was enhanced and prolonged following anti-CD3 triggering, suggestive of proximal defects in TCR-engendered signaling as the mechanism for the observed hyperactivity. These findings were observed as early as 1-2 mo postweaning and, based on analysis of F1 T cells, appeared to be dominantly expressed. This genetically altered threshold for activation of MRL T cells, a consequence of a proximal defect in CD3-mediated signal transduction, may contribute to the abrogation of T cell tolerance to self-Ags in lupus.     

CD31 is required on CD4+ T cells to promote T cell survival during Salmonella infection

Hematopoietic cells constitutively express CD31/PECAM1, a signaling adhesion receptor associated with controlling responses to inflammatory stimuli. Although expressed on CD4(+) T cells, its function on these cells is unclear. To address this, we have used a model of systemic Salmonella infection that induces high levels of T cell activation and depends on CD4(+) T cells for resolution. Infection of CD31-deficient (CD31KO) mice demonstrates that these mice fail to control infection effectively. During infection, CD31KO mice have diminished numbers of total CD4(+) T cells and IFN-γ-secreting Th1 cells. This is despite a higher proportion of CD31KO CD4(+) T cells exhibiting an activated phenotype and an undiminished capacity to prime normally and polarize to Th1. Reduced numbers of T cells reflected the increased propensity of naive and activated CD31KO T cells to undergo apoptosis postinfection compared with wild-type T cells. Using adoptive transfer experiments, we show that loss of CD31 on CD4(+) T cells alone is sufficient to account for the defective CD31KO T cell accumulation. These data are consistent with CD31 helping to control T cell activation, because in its absence, T cells have a greater propensity to become activated, resulting in increased susceptibility to become apoptotic. The impact of CD31 loss on T cell homeostasis becomes most pronounced during severe, inflammatory, and immunological stresses such as those caused by systemic Salmonella infection. This identifies a novel role for CD31 in regulating CD4 T cell homeostasis.     

GRAIL is up-regulated in CD4+ CD25+ T regulatory cells and is sufficient for conversion of T cells to a regulatory phenotype

GRAIL (gene related to anergy in lymphocytes) is an ubiquitin-protein isopeptide ligase (E3) ubiquitin ligase necessary for the induction of CD4(+) T cell anergy in vivo. We have extended our previous studies to characterize the expression pattern of GRAIL in other murine CD4(+) T cell types with a described anergic phenotype. These studies revealed that GRAIL expression is increased in naturally occurring (thymically derived) CD4(+) CD25(+) T regulatory cells (mRNA levels 10-fold higher than naive CD25(-) T cells). Further investigation demonstrated that CD25(+) Foxp3(+) antigen-specific T cells were induced after a "tolerizing-administration" of antigen and that GRAIL expression correlated with the CD25(+) Foxp3(+) antigen-specific subset. Lastly, using retroviral transduction, we demonstrated that forced expression of GRAIL in a T cell line was sufficient for conversion of these cells to a regulatory phenotype in the absence of detectable Foxp3. These data demonstrate that GRAIL is differentially expressed in naturally occurring and peripherally induced CD25(+) T regulatory cells and that the expression of GRAIL is linked to their functional regulatory activity.     

Rapid G0/1 transition and cell cycle progression in CD8+ T cells compared to CD4+ T cells following in vitro stimulation

T‐cell population consists of two major subsets, CD4⁺ T cells and CD8⁺ T cells, which can be distinguished by the expression of CD4 or CD8 molecules, respectively. Although they play quite different roles in the immune system, many of their basic cellular processes such as proliferation following stimulation are presumably common. In this study, we have carefully analyzed time–course of G0/1 transition as well as cell cycle progression in the two subsets of quiescent T‐cell population following in vitro growth stimulation. We found that CD8⁺ T cells promote G0/1 transition more rapidly and drive their cell cycle progression faster compared to CD4⁺ T cells. In addition, expression of CD25 and effects of its blockade revealed that IL‐2 is implicated in the rapid progression, but not the earlier G0/1 transition, of CD8⁺ T cells.     

A VH12 transgenic mouse exhibits defects in pre-B cell development and is unable to make IgM+ B cells

V(H)12 B cells undergo stringent selection at multiple checkpoints to favor development of B-1 cells that bind phosphatidylcholine. Selection begins with the V(H) third complementarity-determining region (CDR3) at the pre-B cell stage, in which most V(H)12 pre-B cells are selectively eliminated, enriching for those with V(H)CDR3s of 10 aa and a fourth position Gly (designated 10/G4). To understand this selection, we compared B cell differentiation in mice of two V(H)12 transgenic lines, one with the favored 10/G4 V(H)CDR3 and one with a non-10/G4 V(H)CDR3 of 8 aa and no Gly (8/G0). Both H chains drive B cell differentiation to the small pre-BII cell stage, and induce allelic exclusion and L chain gene rearrangement. However, unlike 10/G4 pre-B cells, 8/G0 pre-B cells are deficient in cell division and unable to differentiate to B cells. We suggest that this is due to poor 8/G0 pre-B cell receptor expression and to an inability to form an 8/G0 B cell receptor. Our findings also suggest that V(H)12 H chains have evolved such that association with surrogate and conventional L chains is most efficient with a 10/G4 CDR3. Thus, selection for phosphatidylcholine-binding B-1 cells is most likely the underlying evolutionary basis for the loss of non-10/G4 pre-B cells.     

B cells are crucial for determinant spreading of T cell autoimmunity among beta cell antigens in diabetes-prone nonobese diabetic mice

The determinant spreading of T cell autoimmunity plays an important role in the pathogenesis of type 1 diabetes and in the protective mechanism of Ag-based immunotherapy in NOD mice. However, little is known about the role of APCs, particularly B cells, in the spreading of T cell autoimmunity. We studied determinant spreading in NOD/scid or Igmu(-/-) NOD mice reconstituted with NOD T and/or B cells and found that mice with mature B cells (TB NOD/scid and BMB Igmu(-/-) NOD), but not mice that lacked mature B cells (T NOD/scid and BM Igmu(-/-) NOD), spontaneously developed Th1 autoimmunity, which spread sequentially among different beta cell Ags. Immunization of T NOD/scid and BM Igmu(-/-) NOD mice with a beta cell Ag could prime Ag-specific Th1 or Th2 responses, but those T cell responses did not spread to other beta cell Ags. In contrast, immunization of TB NOD/scid and BMB Igmu(-/-) NOD mice with a beta cell Ag in IFA induced Th2 responses, which spread to other beta cell Ags. Furthermore, we found that while macrophages and dendritic cells could evoke memory and effector T cell responses in vitro, B cells significantly enhanced the detection of spontaneously primed and induced Th1 responses to beta cell Ags. Our data suggest that B cells, but not other APCs, mediate the spreading of T cell responses during the type 1 diabetes process and following Ag-based immunotherapy. Conceivably, the modulation of the capacity of B cells to present Ag may provide new interventions for enhancing Ag-based immunotherapy and controlling autoimmune diseases.     

Enzyme histochemistry of malignant T cell lymphoma due to chronic magnesium deficiency in rats

Summary The lymphocytes of the rat thymus can be grossly differentiated by their cell membrane-bound proteinases. Subcapsular thymocytes lack aminopeptidase A (APA) and AMP and -glutamyltranspeptidase (GGT). Cortical thymocytes show a high activity of APA but no APM and no GGT. Medullar thymocytes posses a high GGT and APM activity but are free of APA. Under Mg deficiency, the APA-negative subcapsular thymocytes are reduced. In lymphoma and beginning lymphoma, APA, APM and GGT are absent. In lymphoma, the alkaline phosphatase activity is increased. Differences are found for dipeptidylpeptidase IV (DPP IV). In some lymphoma, its activity is reduced, in others the DPP IV activity is increased.     

Enzyme histochemistry of malignant T cell lymphoma due to chronic magnesium deficiency in rats

The lymphocytes of the rat thymus can be grossly differentiated by their cell membrane-bound proteinases. Subcapsular thymocytes lack aminopeptidase A (APA) and AMP and gamma-glutamyltranspeptidase (GGT). Cortical thymocytes show a high activity of APA but no APM and no GGT. Medullar thymocytes possess a high GGT and APM activity but are free of APA. Under Mg deficiency, the APA-negative subcapsular thymocytes are reduced. In lymphoma and beginning lymphoma, APA, APM and GGT are absent. In lymphoma, the alkaline phosphatase activity is increased. Differences are found for dipeptidylpeptidase IV (DPP IV). In some lymphoma, its activity is reduced, in others the DPP IV activity is increased.