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.

Elevated apoptosis of peripheral T lymphocytes in diabetic BB rats.

Thymocytes and peripheral lymphocytes of BioBreeding (BB) diabetes-prone (BBDP) and diabetes-resistant (BBDR) rat were analysed by fluorescence-activated cell sorter (FACS). The number of CD4- CD8-, CD4+ CD8-, CD4- CD8+ and CD4+ CD8+ subsets was not different between BBDP and BBDR rat thymocytes, whereas spleen and lymph nodes in BBDP rats undergo severe T-cell lymphopenia. Notably, mature CD4- CD8+ [T-cell receptor (TCR)-alphabeta+ and CD5+] cells are certainly present in the BBDP rat thymus, unlike some previous reports, suggesting that the differentiation of CD4- CD8+ from CD4+ CD8+ cells occurs normally in the BBDP rat thymus. As a cause of peripheral T-cell lymphopenia we suspected apoptosis of recent thymic emigrants. By FACS analysis with fluorescein isothiocyanate-labelled annexin V, elevated apoptosis was evident in BBDP rat peripheral lymphocytes. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) staining in BBDP rat splenic sections revealed that a number of TUNEL-positive cells were observed in the T-lymphocyte-rich area. From these results, we postulate that an abnormally elevated apoptosis of peripheral T lymphocytes, but not impaired thymocyte differentiation, is a cause of the peripheral T-cell lymphopenia in BBDP rats.

Antigen activation rescues recent thymic emigrants from programmed cell death in the BB rat.

One of the diabetes susceptibility genes of the BB rat is a mutation at the lyp locus that decreases the thymic output of T cells and the life span of most recent thymic emigrants (RTE). Consequently, there is a 10-fold reduction in the number of CD4+ and CD8+ T cells in secondary lymphoid organs. Results presented in this work demonstrate that the BB rat lyp mutation is associated with an accelerated apoptotic death in vitro of mature CD4+ 8- and CD4- 8+ thymocytes and peripheral T cells. The stability of the pool of recirculating T cells (PRL) of BB rats over time results from a > 10-fold increase in the mitotic activity of T cells as assessed in vivo by bromodeoxyuridine incorporation. This increased mitotic activity is not observed when BB T cells develop in the context of a normal sized PRL. MHC haploidentical WF and BB rats differ at minor histocompatibility loci. Intravenous injection of (WF x BB)F1 T cells into euthymic BB rats led to the rejection of donor T cells within 3 wk by unprimed recipients and within 1 wk by primed recipients. This secondary immune response was unaffected by postpriming thymectomy. F1 T cells were not rejected, but rather expanded after their injection into thymectomized BB rats that had been primed as early as 48 h after thymectomy. These results strongly suggest that the BB rat PRL is devoid of long-lived naive T cells and that rescue of recent thymic emigrants from programmed cell death is initiated by Ags, exclusively.

Evidence that Th1 lymphocytes predominate in islet inflammation and thyroiditis in the BioBreeding (BB) rat.

Th1 cytokines are thought to play a key role in islet inflammation and destruction in insulin-dependent diabetes mellitus (IDDM). We studied this hypothesis in the diabetes-prone (DP)-BB and the diabetes-resistant (DR)-BB rats that are used as a model of human IDDM. The DP-BB rat develops spontaneous autoimmune diabetes at the age of 11-14 weeks. In the DR-BB rat, diabetes is inducible by depletion of RT6+ lymphocytes and coadministration of polyinosinic:polycytidylic acid (Poly I:C). We used reverse transcriptase-polymerase chain reaction (RT-PCR) and semi-quantitative PCR techniques to examine mRNA expression of Th1 and Th2 cytokines in inflamed islets and thyroids from DP-BB and DR-BB rats. We observed that in DP-BB and in treated DR-BB rats, the levels of TCR beta, IFN-gamma and IL-12p40 mRNA increase with disease progression. In contrast, expression of message for IL-2 and IL-4 is minimal to undetectable in DP-BB and RT6-depleted DR-BB animals at any age. Message for IL-10 is detectable in DP and DR islets; however, its level of expression does not change with disease progression. A similar cytokine mRNA profile is observed in inflamed thyroids from acutely diabetic RT6-depleted DR-BB rats. Incubation of 10 wk old DP islets for 48 h in the presence of anti-CD3 antibody, followed by an incubation with rIL-2 for an additional 5-7 days, results in an expansion of T lymphocytes, and these cells express high levels of IFN-gamma and IL-10 mRNA. Our results suggest that autoimmunity in DP-BB and DR-BB rats is mediated by Th1 lymphocytes and that IFN-gamma and IL-12 are likely to play a key role in islet and thyroid inflammation and destruction in IDDM.

Temporary anti-CD25/CsA therapy induces a CD4+ T-cell-mediated tolerance in BB/OK rats.

Pancreatic islets obtained from the congenic LEW.1BB/OK rat strain (MHC identical, different in genetic background from BB/OK rats) were grated into diabetic BB/OK rats. The recipients were treated for 10 d with 1.0 mg/kg bw anti-IL2 receptor monoclonal antibody (ART-18) in combination with 1.5 mg/kg bw cyclosporin A, which resulted in indefinite graft survival in the majority of animals. The successfully treated recipients (normoglycaemia for > 120 days) relapsed immediately into hyperglycaemia after graft removal. A second donor-identical graft was accepted without any further immunotherapy, whereas MHC-different islet grafts (obtained from LEW.1A or DA rats) were rejected, demonstrating the induction of donor-specific tolerance. Splenocytes or thoracic duct lymphocytes (TDL) obtained from successfully treated recipients were transfused into naive diabetic BB/OK rats grafted with LEW.1BB/OK islets. Independent of the origin of the transfused cells, 64% of recipients maintained normoglycaemia for more than 120 days. To characterize the cell population(s) responsible for transfer of tolerance, B-lymphocytes were removed from the TDLs using the monoclonal antibody OX33 and magnetic beads. When OX33-depleted TDLs were transfused into naive diabetic BB/OK with a LEW.1BB/OK islet graft, all recipients maintained normoglycaemia. The OX33-depleted TDLs consisted only of CD4+ T-lymphocytes, which were either negative for CD45RC or coexpressed the CD45RC at low levels. We conclude that the cell-dependent tolerance transfer is mediated by a TH2-like suppressor cell.

Adoptive transfer of autoimmune diabetes mellitus to athymic rats: synergy of CD4+ and CD8+ T cells and prevention by RT6+ T cells.

We describe the induction and prevention of autoimmune insulin dependent diabetes mellitus (IDDM), and its pathological substrate, insulitis, in congenitally athymic nude rats following injections of major histocompatibility complex (MHC) compatible lymph node T cells. The cells capable of adoptive transfer of autoimmunity were obtained from diabetes resistant (DR) BB rats that had been rendered hyperglycemic by in vivo depletion of the RT6+ regulatory T cell subset. We first established that our adoptive transfer assay system is cell dose- and time dependent and therefore amenable to quantitative analysis. It was also observed that both CD4+ and CD8+ T cells are required for efficient transfer of autoimmunity. The data indicate that, as in the NOD mouse, a synergistic interaction between CD4+ and CD8+ T cells is important for beta cell destruction. Finally, we demonstrated that the admixture of equal numbers of lymph node T cells, 60% of which were RT6+, from intact, non-diabetic DR rats prevented the adoptive transfer of IDDM mediated by diabetogenic T cells from RT6-depleted DR-BB rats. We conclude that an equilibrium between autoreactive and regulatory cells determines the expression of autoimmunity in the DR-BB rat and in the adoptive transfer of diabetes in quantitative analytical systems.

Spontaneous reestablishment of self-tolerance in BB/Pfd rats.

We describe a substrain of BB rats, BB/Pfd, characterized by a loss of autoimmune potential soon after onset of diabetes. When fresh syngeneic (BB/Pfd) islets (6 islets/g body weight) were transplanted under the kidney capsule of diabetic BB/Pfd rats 1-2 weeks after diabetes onset (n = 14), no recurrence of diabetes occurred. When, however, islets were transplanted on the day of diabetes diagnosis (n = 16), 10 animals were able to destroy the transplant (P < 0.005 vs. previous group). Pancreatic biopsies taken at the moment of transplantation in both groups showed an almost complete disappearance of beta cells and also insulitis in the pancreata of the 1- to 2-week diabetic rats, while the acutely diabetic rats still conserved a certain amount of beta cells and a florid insulitis. The development of a general immune defect was not the cause of this nonrecurrence, since allogeneic islet grafts were easily rejected (7 of 8), nor was there a general defect in mounting immune memory, since second set skin grafts could be rejected in an accelerated manner (9.7 vs. 15.8 days). The development of suppressor mechanisms as cause for nonrecurrence could not be demonstrated, since transfer of lymphocytes taken from 1- to 2-week diabetic rats into acutely diabetic rats at the moment of syngeneic islet transplantation was unable to prevent recurrence of disease (6 recurrences in 10 animals). We conclude that in the BB/Pfd substrain, the autoimmune capacity wanes rapidly after the onset of diabetes. This loss of autoimmune potential is parallelled by a disappearance of insulitis in the native pancreas, but the exact mechanisms of the spontaneous reestablishment of self-tolerance remain unclear.

Predictive value of lymphocyte antibodies for the appearance of diabetes in BB rats.

Lymphocyte antibodies have been described in autoimmune disorders, including insulin-dependent diabetes mellitus (IDDM). We have developed a quantitative method to measure autoantibodies directed against T-lymphocytes, based on two-color fluorescence labeling of Wistar mononuclear cells and analysis of fluorescence by flow cytometry. The lymphocyte antibody levels were determined retrospectively in the serum of 73 BB and 18 Wistar rats. We demonstrated the binding of the lymphocyte autoantibodies of both CD4+ and CD8+ T-cells. Lymphocyte antibodies were present in 90% of the BB rats at diabetes onset, compared with 11% of the Wistar rats. At 75 days of age, 83% of the BB rats, which later became diabetic, were positive for the lymphocyte antibodies, compared with 15% of their littermates who maintained a normal glucose tolerance. In all cases, the antibodies were of the immunoglobulin M isotype. We conclude that lymphocyte antibodies are present before diabetes onset and, using this method, that their presence can predict the development of diabetes with a sensitivity of 83% and a specificity of 85% in BB rats.

Peculiar T-cell signaling does not preclude positive selection in the diabetes-prone BB rat.

Various studies have provided evidence that peripheral T-cells from the diabetes-prone BB-DP rat are abnormal in function and cell surface phenotype. These characteristics have often been interpreted as indicators of immaturity and/or short life span. In this study, we describe a CD4-dependent signaling abnormality in BB-DP peripheral T-cells. In spite of the fact that CD4 plays a critical role in thymocyte development, the abnormal signaling does not appear to influence thymocyte development at the stage when the T-cell receptor is rearranged and the recombinase enzymes RAG-1 and RAG-2 transcripts are downregulated. Therefore, if a maturation defect leading to the seeding of the periphery with immature T-cells occurs in the BB-DP rat, it does not preclude the initial selection of the self major histocompatibility complex-restricted T-cell repertoire.

Quantitative phenotypic and functional analyses of islet immune cells before and after diabetes onset in the BB rat.

Inflammatory cells invading islets are thought to be mediators of islet destruction in spontaneous autoimmune diabetes mellitus. Thus methods were developed to isolate and characterize in situ islet inflammatory cells from 75-95-day-old prediabetic and diabetic BB rats. Islet inflammatory cells were structurally examined using single- and double-colour flow cytometry. Functional studies consisted of cytolytic assays using normal rat islet target cells and in situ islet or spleen effector cells. Structural data reveal natural killer cells to be the major cell population (70%) of total immune cells present in inflamed islets during prediabetes. At diabetes onset, the natural killer cell population remained at a high level (47%), but an increasing population of T cells (40%) was noted also. Analyses of T-cell subsets before and after diabetes onset revealed CD4+ T cells as predominant (50-55% of total T cells) with double-negative (CD4-CD8-) T cells (25-30%) and CD8+ T cells (15-20%) also present in significant quantities. Activated T cells accounted only for a minority of T cells (< 3%). Functional studies indicate that in situ islet-derived cytolytic effector cells are more potent killers (ten-fold) of normal islet target cells than are splenic effector cells. These data suggest that in situ islet inflammatory cells (a) can be quantitatively studied both structurally and functionally; (b) express structural phenotypes differing substantially from splenic mononuclear cell populations; (c) are considerably more cytolytic than splenic effectors; and (d) should prove informative in determining the most significant autoimmune functional events prior to and during islet beta-cell destruction.

Molecular mechanisms underlying diabetes and other autoimmune diseases.

Autoimmunity is a fascinating and complex phenomenon. The various mechanisms such as self tolerance, genetic and environmental regulation of the autoimmune phenotype, which govern the susceptibility to autoimmune disease were discussed at a recent workshop. Here, Ron Gill and Kathryn Haskins report on the new advances in this area.

Prevention of diabetes and induction of non-specific suppressor cell activity in the BB rat by an immunomodulatory azaspirane, SK&F 106610.

Immunomodulatory azaspirane compounds have immunosuppressive activity in animal models of autoimmune disease such as adjuvant-induced arthritis and experimental autoimmune encephalomyelitis. The mechanism of action of azaspiranes appears to be the induction of antigen non-specific (natural) suppressor cell activity. In this study, we tested the azaspirane, SK&F 106610 in an animal model of autoimmune (type 1) diabetes, the BB rat. Oral administration of SK&F 106610 (15 mg/kg/day) to diabetes-prone BB rats, from age 30 days, significantly decreased diabetes incidence at 100 days from 80% (24 of 30 control rats) to 32% (10 of 31 drug-treated rats, P < 0.001). Protection from diabetes by SK&F 106610 was accompanied by decreased lymphocytic infiltration of the pancreatic islets (insulitis). No changes occurred in splenic T cell, B cell or macrophage subsets, or in proliferative responses to the mitogens lipopolysaccharide and concanavalin A (Con-A). Cell mixing experiments in vitro, however, revealed increased antigen non-specific suppressor activity (suppression of splenic lymphoproliferative response to Con-A) in spleens of SK&F 106610-treated rats. The suppressor cell activity was enriched in a low density fraction of splenic cells relatively depleted of T cells, B cells, macrophages and natural killer cells. These results indicate that the azaspirane compound, SK&F 106610 can prevent insulitis and autoimmune diabetes in BB rats and that these actions may be related to the activation of non-specific (natural) suppressor cells.

Characterization of monoclonal islet cell reactive autoantibodies from the diabetic biobreeding (BB/OK) rat.

Two fusion experiments using the heteromyeloma cell line CB-F7 and splenocytes from two diabetes-prone BB (BioBreeding) rats at the onset of diabetes resulted in 128 islet cell reactive autoantibodies primarily detected with permeabilized insulin-producing rat insulinoma cells (RIN) by a cellular enzyme-linked immunosorbent assay. Seventy-nine (62%) of 128 RIN cell reactive supernatants exhibited a cross-reactivity with rat splenic lymphocytes. Six stable hybridomas secreting monoclonal ICSA (islet cell surface antibodies) were established, but only one monoclonal antibody, R4B10, showed preferential beta-cell binding. Six monoclonal antibodies showed a dual reactivity as ICA (islet cell cytoplasmic antibodies) detected by immunostaining of pancreatic islet cryosections and as ICSA on the surface of viable islet cells, whereas two reacted only with an ICA-like pattern. One monoclonal ICSA was specifically displaced from the RIN cell surface by sera of type 1 diabetic patients.

Polygenic nature of spontaneous diabetes in the rat. Permissive MHC haplotype and presence of the lymphopenic trait of the BB rat are not sufficient to produce susceptibility.

We describe the phenotypic characteristics of animals in the fifth backcross-intercross generation of a breeding program in which the RT1 u haplotype and the phenotypic trait responsible for the T-lymphopenia of BB rats have been transferred to the ACI background. In this generation of animals, 24% were lymphopenic with decreased numbers of PBL expressing CD5, TCR alpha, and RT6. The PBL of the lymphopenic animals had a decreased mitogenic response to ConA. All of the nonlymphopenic animals were homozygous for RT6.2. Phenotypic analysis of intestinal IEL revealed that this was also the case for the lymphopenic animals. Moreover, IEL of the lymphopenic animals exhibited a pattern of staining (increased numbers of TCR alpha beta+CD4+CD8+ and decreased numbers of TCR alpha beta+CD4-CD8+) similar to that of BB DP animals. The ACI.1U(BB)-lymphopenic animals, although having two of the genetic traits associated with the expression of spontaneous diabetes mellitus, uniformly fail to develop diabetes. Breeding studies in which these animals were crossed with BB and hBB rats suggest that other genes are necessary for development of overt diabetes.

Quantitative analyses comparing all major spleen cell phenotypes in BB and normal rats: autoimmune imbalance and double negative T cells associated with resistant, prone and diabetic animals.

The BB rat is a model of spontaneous autoimmune diabetes. To characterize quantitatively all known immune cell subsets involved in disease pathogenesis, FACS analysis of spleen cells was performed in diabetes-prone (DP) and acutely diabetic (D) BB rats and compared with diabetes-resistant (DR) BB and normal Wistar-Furth (WF) strains. We observed increased percentages of splenic NK cells in DP and D animals compared with DR rats using an NK-specific monoclonal antibody. We found increased proportions of splenic macrophages in the T-lymphopenic DP and D rats and low macrophage contents in DR spleens compared with WF spleens. We observed that percentages of the CD4-CD8- T cell receptor alpha/beta+ (double-negative) T cell subset were strikingly increased in the lymphopenic DP and D animals, compared with DR animals. We observed increased percentages of activated splenic CD5+ T cells expressing the IL-2 receptor and MHC class II antigen in DP and D rats compared with DR animals. Our studies suggest that (a) splenic NK cells and macrophages quantitatively appear to be involved in the pathogenesis of diabetes; (b) double-negative T cells escape from the T cell depletion process; (c) a marked increase of activated splenic T cells suggests diabetes is associated with general T cell activation processes; and (d) an altered balance among the different immune cell subsets may in part explain the pathogenesis of diabetes, since marked relative changes are observed when comparing the DR strain to the DP strain in both the prediabetic and diabetic stages.

Major-histocompatibility-complex restricted and nonrestricted autoimmune effector mechanisms in BB rats.

To investigate whether the immunologic mechanisms of autoimmune pancreatic beta-cell destruction are MHC restricted, we examined the relative vulnerability of islet allografts from a panel of MHC-compatible and -incompatible donors to autoimmune damage after transplantation to spontaneously diabetic BB recipients. To circumvent a potentially confounding allograft response to the foreign islet graft, we utilized two strategies: (1) pretransplant in vitro culture of islets to delete intraislet APCs; and (2) induction of islet donor-specific immunologic tolerance in diabetes-prone BB rats. Experiments employing organ culture to prevent rejection demonstrated that MHC-incompatible grafts were significantly less vulnerable to autoimmunity than MHC-compatible grafts. In contrast, when we used the model of immunologic tolerance to exclude rejection, both MHC-compatible and -incompatible islet grafts were equally susceptible to autoimmune damage. The reason for this discrepancy has not been defined fully but may be related to our observation that tolerant BB animals exhibit increased peripheral blood NK-cell activity. NK cells are known to be cytotoxic to islets in vitro and could play a role in a non-MHC-restricted diabetogenic response in vivo. We conclude that both MHC-restricted and nonrestricted mechanisms are capable of contributing to anti-beta-cell autoimmunity in BB rats.

Diabetes-prone BB rats express the RT6 alloantigen on intestinal intraepithelial lymphocytes.

The RT6 alloantigen of the rat is expressed on most peripheral T cells but not on thymocytes and thus represents a marker for postthymic T lymphocyte maturation in this species. Diabetes-prone (DP) BB rats exhibit a genetically determined T cell lymphopenia associated with a deficiency of RT6+ T cells. In this study we have analyzed the expression of RT6 on lymph node (LN) cells and intestinal intraepithelial lymphocytes (IEL) in two DP BB strains (BB/OK and BB/Mol) and two control strains (non-lymphopenic BB/PhiK and LEW) by flow cytometry. In the DP BB rats the number of LN T cells was substantially reduced (less than 25% TcR2+ cells) and completely lacked RT6 expression. The IEL population was also reduced in number and in marked contrast to normal rats consisted predominantly of CD4+ cells. The majority of IEL, however clearly expressed RT6. Treatment with a phosphatidylinositol (PI)-specific phospholipase C markedly reduced the RT6 density showing that PI-mediated anchoring of RT6 in the cell membrane also applies to IEL of DP BB rats. The results demonstrate that the DP BB strains possess a functional RT6 gene and are also able to generate the PI anchor. The defect in RT6 expression is thus unlikely to be the primary cause of the T cell lymphopenia.