Stimulation of a shorter duration in the state of anergy by an invariant natural killer T cell agonist enhances its efficiency of protection from type 1 diabetes.

We have reported previously that treatment of non-obese diabetic (NOD) mice with the invariant natural killer T (iNK T) cell agonist α-galactosylceramide C26:0 (α-GalCer) or its T helper type 2 (Th2)-biasing derivative α-GalCer C20:2 (C20:2) protects against type 1 diabetes (T1D), with C20:2 yielding greater protection. After an initial response to α-GalCer, iNK T cells become anergic upon restimulation. While such anergic iNK T cells can induce tolerogenic dendritic cells (DCs) that mediate protection from T1D, chronic administration of α-GalCer also results in long-lasting anergy accompanied by significantly reduced iNK T cell frequencies, which raises concerns about its long-term therapeutic use. In this study, our objective was to understand more clearly the roles of anergy and induction of tolerogenic DCs in iNK T cell-mediated protection from T1D and to circumvent potential complications associated with α-GalCer. We demonstrate that NOD iNK T cells activated during multi-dose (MD) treatment in vivo with C20:2 enter into and exit from anergy more rapidly than after activation by α-GalCer. Importantly, this shorter duration of iNK T cells in the anergic state promotes the more rapid induction of tolerogenic DCs and reduced iNK T cell death, and enables C20:2 stimulated iNK T cells to elicit enhanced protection from T1D. Our findings further that suggest C20:2 is a more effective therapeutic drug than α-GalCer for protection from T1D. Moreover, the characteristics of C20:2 provide a basis of selection of next-generation iNK T cell agonists for the prevention of T1D.

Structure-guided design of an invariant natural killer T cell agonist for optimum protection from type 1 diabetes in non-obese diabetic mice.

Because invariant natural killer T (iNK T) cells link innate and adaptive immunity, the structure-dependent design of iNK T cell agonists may have therapeutic value as vaccines for many indications, including autoimmune disease. Previously, we showed that treatment of non-obese diabetic (NOD) mice with the iNK T cell activating prototypic glycolipid α-galactosylceramide (α-GalCer) protects them from type 1 diabetes (T1D). However, α-GalCer is a strong agonist that can hyperactivate iNK T cells, elicit several side effects and has shown only limited success in clinical trials. Here, we used a structure-guided design approach to identify an iNK T cell agonist that optimally protects from T1D with minimal side effects. Analyses of the kinetics and function of a panel of synthetic α-GalCer fatty acyl chain derivatives (C8:0-C16:0) were performed in NOD mice. C16:0 elicited the highest protection from insulitis and T1D, which was associated with a higher frequency and survival of iNK T cells and enhanced activity of tolerogenic dendritic cells (DCs) in draining pancreatic lymph nodes (PLN), inability to transactivate NK cells and a more rapid kinetics of induction and recovery of iNK T cells from anergy. We conclude that the length and structure of the acyl chain of α-GalCer regulates the level of protection against T1D in mice, and propose that the extent of this protection depends on the relative capacity of the acyl chain to accommodate an endogenous spacer lipid of appropriate length and structure. Thus, our findings with the α-GalCer C16:0 derivative suggest strongly that it be considered as a lead glycolipid candidate in clinical trials of T1D.

Activation of natural killer T cells by alpha-galactosylceramide treatment prevents the onset and recurrence of autoimmune Type 1 diabetes.

Type 1 diabetes (T1D) in non-obese diabetic (NOD) mice may be favored by immune dysregulation leading to the hyporesponsiveness of regulatory T cells and activation of effector T-helper type 1 (Th1) cells. The immunoregulatory activity of natural killer T (NKT) cells is well documented, and both interleukin (IL)-4 and IL-10 secreted by NKT cells have important roles in mediating this activity. NKT cells are less frequent and display deficient IL-4 responses in both NOD mice and individuals at risk for T1D (ref. 8), and this deficiency may lead to T1D (refs. 1,6-9). Thus, given that NKT cells respond to the alpha-galactosylceramide (alpha-GalCer) glycolipid in a CD1d-restricted manner by secretion of Th2 cytokines, we reasoned that activation of NKT cells by alpha-GalCer might prevent the onset and/or recurrence of T1D. Here we show that alpha-GalCer treatment, even when initiated after the onset of insulitis, protects female NOD mice from T1D and prolongs the survival of pancreatic islets transplanted into newly diabetic NOD mice. In addition, when administered after the onset of insulitis, alpha-GalCer and IL-7 displayed synergistic effects, possibly via the ability of IL-7 to render NKT cells fully responsive to alpha-GalCer. Protection from T1D by alpha-GalCer was associated with the suppression of both T- and B-cell autoimmunity to islet beta cells and with a polarized Th2-like response in spleen and pancreas of these mice. These findings raise the possibility that alpha-GalCer treatment might be used therapeutically to prevent the onset and recurrence of human T1D.

Evidence for two homologous, but nonidentical, Ia molecules determined by the I-EC subregion.

Sequential immunoprecipitation, two-dimensional gel electrophoresis and peptide mapping analyses of B10A(3R), 35S-methionine-labeled, I-EC subregion products were performed. Evidence is presented here for the presence of two structurally homologous, but nonidentical, gene products of the I-EC subregion. These two Ia molecules are independently immunoprecipitable, identical in molecular size and charge, but differ by approximately equal to 20% in their peptides obtained by partial digestion with Staphylococcus aureus protease V8.

In vitro analysis of allogeneic lymphocyte interaction. II. I-region control of the activity of a B-cell-derived H-2-restricted allogeneic effect factor and its receptor during B-cell activation.

A genetically restricted allogeneic effect factor (AEF) derived from a mixed lymphocyte culture reaction between Ia-negative activated responder cells and irradiated T-cell-depleted stimulator cells was characterized. Restricted AEF is a B-cell-derived soluble helper factor which consists in part of Ia antigens controlled by the I-A subregion of the stimulator haplotype; additional control by the I-B, I-E, and I-C subregions, although unlikely, could not be excluded. This factor helps B cells of only its own haplotype or of haplotypes which carry an I-A and/or I-B subregion identity. Unprimed as well as hapten-primed Ia-positive B cells express a receptor for restricted AEF. The results indicate that the B-cell receptor for AEF is determined by the I-A subregion. Both restricted AEF and its receptor may therefore be products of the same I-region gene(s). The data are compatible with the hypothesis that the AEF Ia antigens serve as a second signal required for B-cell activation to IgG antibody production.

In vitro analysis of allogeneic lymphocyte interaction. VII. I-A-restricted self-reactive and alloreactive helper components of allogeneic effect factor are distinct donor T cell-derived Ia-molecules that recognize Ia determinants on antigen-presenting cells.

An allogeneic effect factor (AEF) derived from mixed lymphocyte reaction (MLR) cultures of alloactivated A.SW (H-2s) responder T cells and irradiated T cell-depleted A/WySn (H-2a) stimulator spleen cells was fractionated on the basis of molecular size and charge into two I-A-restricted helper components. The cellular origin of these components is believed to be an Lyt-1+2- -activated responder T helper (TH) cell. One alloreactive component, TIAH-1, recognizes allo-I-A determinants on an A/WySn antigen-presenting cell (APC). The other self-reactive component, TIAH-2, recognizes self-I-A determinants on an A.SW APC. The interaction of each of these components with the appropriate APC subsequently activates an in vitro primary anti-SRBC PFC response of either stimulator haplotype- or responder haplotype-derived B cells. These data demonstrate that the activity of TIAH-1 and TIAH-2 is dependent on the genotype of the APC and not the B cell, and that the target cell of action of these AEF TH components is an APC. TIAH-1 and TIAH-2 are 68,000 mol wt single polypeptide chains that have an isoelectric point (pI) of 5.8 and 5.5, respectively. Their charge difference is not attributable to altered amounts of sialylation or phosphorylation, but probably is due to other forms of altered glycosylation and/or to changes in their amino acid sequence. They share approximately 80% of their tryptic peptides and likely constitute homologous but nonidentical molecules. Papain cleaves TIAH-1 and TIAH-2 into a 40,000 mol wt fragment. TIAH-1 and TIAH-2 may represent structurally very related but nonidentical secreted forms of activated responder TH cell-derived receptors for allo-I-A and self-I-A determinants, respectively.

In vitro analysis of allogeneic lymphocyte interaction. I. Characterization and cellular origin of an Ia-positive helper factor-allogeneic effect factor.

A soluble allogeneic effect factor (AEF) was produced by using H-2 congenic mouse strains and a serum.free cell culture medium. An AEF derived from untreated activated responder cells and irradiated stimulator cells provided helper cell function in a primary and secondary antibody response for both T-cell-depleted responder B cells and stimulator B cells. This interaction may be determined by genes situated in the I-A and I-B regions: additional K-region control was not excluded. Ia antigens, but neither H-2 nor Ig determinants are molecular constituents of AEF. The active components of this AEF consist, in part, of Ia antigens derived from both the activated responder cell population and irradiated stimulator cell population. An AEF derived from Ia negative responder cells and irradiated T-cell- depleted stimulator cells helps a secondary antibody response of T-cell- depleted stimulator B cells but not responder B cells. This genetically restricted AEF contains Ia antigens determined by the stimulator haplotype but not the responder haplotype. The priming antigen, DNP- keyhole limpet hemocyanin, is not a component of restricted AEF. The data suggest that restricted AEF may be a product of a stimulator B cell and/or macrophage. They support the hypothesis that the recognition by allogeneic T cells of Ia antigens on B cells activates the B cell to IgG antibody production.

The role of H-2 and Ia antigens in graft-versus-host reactions (GVHR). Presence of host alloantigens on donor cells after GVHR and suppression of GVHR with an anti-Ia antiserum against hose Ia antigens.

By using an indirect immunofluorescence technique, the presence of host cell derived H-2K, H-2D, and Ia alloantigens on donor cells recovered from recipient spleens after a graft-versus-host response (GVHR) was demonstrated. Mapping studies indicated that only host K, D, and I-A region gene products could be identified on the donor cells. Host I-E/C- and I-J-subregion products were not absorbed by donor cells. Treatment of activated donor cells with anti-Ly sera plus C' revealed that donor cells carrying host Ia antigens have a Ly-1+,2-,3- phenotype, whereas donor cells carrying H-2K and H-2D host antigens have a Ly-1-,2+,3+ phenotype. A GVHR that resulted from only an I-region incompatibility was suppressed by the injection of recipient mice with an anti-Ia antiserum directed against self Ia antigens. The degree of suppression was proportional to the amount of anti-Ia antiserum administered.

Role of Ia antigens in graft vs. host reactions. II. Molecular and functional analysis of T cell alloreactivity by the characterization of host Ia antigens on alloactivated donor T cells.

Graft vs. host response (GVHR)-activated donor T cells bind to stimulatory host cell-derived Ia antigens. Radioimmune cell-binding assays demonstrate that activated donor T cells acquire both host I-A and I-E alloantigens on their surface. Approximately threefold to fivefold less I-E products than I-A products are transferred. Immunoprecipitation and one-dimensional and two-dimensional gel electrophoresis analyses show that radioiodinated alpha and beta polypeptide chains of both I-A and I-E-encoded host Ia molecules may be transferred in an apparently structurally unaltered form from host cells to donor cells. Biosynthetic studies indicate that [35S]methionine-labeled activated donor T cells do not synthesize Ia antigens of the donor haplotype. Functional analyses with fluorescence-activated cell sorter sorted donor T cell subpopulations show that donor T cells that bind host I-A antigens preferentially cooperate with nonimmune host B cells. Donor T cells that do not bind detectable amounts of host I-A antigens preferentially help nonimmune donor B cells. By contrast, donor T cells that either bind or do not bind host I-A antigens display no H-2-restricted interaction and help both donor and host immune B cells. These data reveal that the Ia antigen-binding specificity of distinct functional subpopulations of alloactivated donor T cells regulates their I-region-restricted (self or allo) helper activity for nonimmune B cells but not immune B cells. Furthermore, they suggest that T cell-macrophage and T cell-B cell collaboration is mediated by a complementary anti-Ia:Ia receptor:ligand type of interaction in which the receptor of a T cell binds to the ligand of an antigen-presenting macrophage and/or B cell.

Antigen-induced B lymphocyte activation involves the p21ras and ras.GAP signaling pathway.

Ligation of a B lymphocyte surface immunoglobulin (sIg) antigen receptor (AgR) by its specific Ag ligand initiates a signaling pathway that culminates in B cell activation. However, many events of this pathway have not been elucidated. Here we present three novel findings that demonstrate directly that AgR-mediated signaling in B cells functions by the p21ras/ras.GAP-dependent pathway. First, stimulation of TA3 7.9 Ag-specific murine B lymphoma cells for 2 min with either Ag or F(ab')2 anti-IgM induces p21ras activation as measured by an increase in the GTP/GDP ratio of its bound nucleotides. This activation of p21ras does not occur via a change in its guanine nucleotide exchange rate. Second, Ag stimulation results in the inhibition of activity of p120 ras.GAP, a protein that regulates p21ras activation. Tyrosine phosphorylation of ras.GAP occurs within 1 min after Ag stimulation but is no longer detectable at 20 min after stimulation, at which time ras.GAP activity remains inhibited. Thus, tyrosine phosphorylation of ras.GAP is not required for the inhibition of its activity. Third, despite the role proposed for a ras.GAP-associated p190 protein in the control of ras.GAP activity in B cells, p190 was not detectable either in anti-ras.GAP immunoprecipitates of [35S]methionine labeled lysates of Ag-stimulated or -unstimulated 7.9 cells or as a tyrosine phosphoprotein in Western blots of anti-ras.GAP immunoprecipitates of Ag-stimulated 7.9 cell lysates. Inasmuch as the TA3 7.9 B lymphoma is representative of a mature, sIgM-bearing B cell, our observations raise the intriguing possibility that the capacity of p190 to associate with ras.GAP and regulate the activities of ras.GAP and p21ras in a B cell is dependent on the stage of differentiation of the B cell.

Immunochemical evidence for three Ia loci in the I-region of the H-2 complex.

Sequential immunoprecipitation analyses of Ia antigens were performed with 125I-labeled B10, B10.A(3R), and B10.A(5R) spleen cell lysates. The results suggest the presence of one I-region associated (Ia) locus (Ia-1) in the I-A subregion. In addition, they indicate that two Ia loci map between the I-J and S regions. One locus, Ia-5, maps in the I-E subregion. The second locus, most likely Ia-3, maps in either the I-E or I-C subregion. Taken together, the data demonstrate that at least three Ia loci in the I-region of the H-2 complex code for molecules detectable by immunoprecipitation.

In vitro analysis of allogeneic lymphocyte interaction. V. Identification and characterization of two components of allogeneic effect factor, one of which displays H-2-restricted helper activity and the other, T cell-growth factor activity.

An allogeneic effect factor (AEF) derived from mixed lymphocyte reaction (MLR) cultures of alloactivated A.SW (H-2s) responder T cells and irradiated A/WySn (H-2a) stimulator spleen cells helps an in vitro primary anti-erythrocyte plaque-forming cell PFC response of BALB/c nude spleen cels and also A/WySn but not A.SW T cell-depleted spleen cells. AEF activity is adsorbed by anti-Ik and anti-I-Ak but not by anti-I-Jk, anti-I-ECk, and anti-Is. Gel filtration of ACA 54 resolves AEF into two main components that which appear in the 50,000- to 70,000-mol wt (component I) and 30,000- to 35,000-mol wt (component II) regions, respectively. Component I has a mol wt of 68,000, elutes from DEAE-Sephacel at 0.05-0.1 M NaCl, and has an isoelectric point (pI) of 5.8. It helps A/WySn but not A.SW B cells and, therefore, is H-2 restricted. Component II is not H-2 restricted, because it helps both A.SW and A/WySn B cells. It also stimulates (a) the growth of a long-term cytotoxic cell line in vitro, (b) Con A-induced thymocyte mitogenesis, and (c) the generation of cytotoxic T cells. The latter three properties of component II are not shared by component I. In addition, component II elutes from DEAE-Sephacel at 0.15-0.2 M NaCl and has a pI of 4.3 and 4.9. Ia determinants and Ig VH, CH, L-chain, and idiotypic determinants are not present on either component I or component II. The properties of component II are identical to that of a T cell growth factor produced by Con A-stimulated spleen cells. It is suggested that the H-2-restricted component I of AEF might be an MLR-activated responder T cell-derived Ia alloantigen receptor.

Distinct antibody specificities to a 64-kD islet cell antigen in type 1 diabetes as revealed by trypsin treatment.

Type 1 diabetes is associated with antibodies that immunoprecipitate a 64-kD islet cell membrane protein from detergent extracts of pancreatic islets. In this study we have determined whether mild trypsin treatment of islet membranes can release fragments of the antigen that bind antibodies in the serum of Type 1 diabetic patients. Partial tryptic proteolysis of [35S]methionine-labeled 64-kD antigen immunoprecipitated from detergent extracts of rat islets resulted in the formation of 50-, 40-, and 37-kD fragments. Similar sized fragments were recovered when sera from diabetic patients were employed to immunoprecipitate polypeptides solubilized by mild trypsin treatment of a particulate fraction of radiolabeled rat islets. Of 27 diabetic patients, 22 possessed antibodies to the 50-kD polypeptide and 21 to the 40- and 37-kD polypeptides. A positive association was found between 64k antibodies and antibodies to the 50-kD fragment but not between 64k antibodies and antibodies to the 40- or 37-kD fragments. Some 64k antibody negative patients possessed antibodies that efficiently immunoprecipitated the latter fragments. Serum from 25 of 27 (93%) diabetic patients immunoprecipitated at least one of the three tryptic polypeptides. One of 20 nondiabetic controls immunoprecipitated a 50-kD polypeptide and all controls were negative for antibodies to 40- and 37-kD fragments. Thus, Type 1 diabetes is associated with the presence of at least two antibody reactivities to distinct determinants of the 64-kD antigen, and some patients may possess antibodies to a cryptic epitope on the detergent-solubilized molecule. These data suggest that the detection of antibodies (present in 93% of patients) to epitopes on tryptic polypeptides of the 64-kD antigen may be of even greater diagnostic value for the onset of Type 1 diabetes than analyses of antibodies reactive with the intact 64-kD antigen.

Impaired plasma membrane targeting of Grb2-murine son of sevenless (mSOS) complex and differential activation of the Fyn-T cell receptor (TCR)-zeta-Cbl pathway mediate T cell hyporesponsiveness in autoimmune nonobese diabetic mice.

Nonobese diabetic (NOD) mouse thymocytes are hyporesponsive to T cell antigen receptor (TCR)-mediated stimulation of proliferation, and this T cell hyporesponsiveness may be causal to the onset of autoimmune diabetes in NOD mice. We previously showed that TCR-induced NOD T cell hyporesponsiveness is associated with a block in Ras activation and defective signaling along the PKC/Ras/MAPK pathway. Here, we report that several sequential changes in TCR-proximal signaling events may mediate this block in Ras activation. We demonstrate that NOD T cell hyporesponsiveness is associated with the (a) enhanced TCR-beta-associated Fyn kinase activity and the differential activation of the Fyn-TCR-zeta-Cbl pathway, which may account for the impaired recruitment of ZAP70 to membrane-bound TCR-zeta; (b) relative inability of the murine son of sevenless (mSOS) Ras GDP releasing factor activity to translocate from the cytoplasm to the plasma membrane; and (c) exclusion of mSOS and PLC-gamma1 from the TCR-zeta-associated Grb2/pp36-38/ZAP70 signaling complex. Our data suggest that altered tyrosine phosphorylation and targeting of the Grb2/pp36-38/ZAP70 complex to the plasma membrane and cytoskeleton and the deficient association of mSOS with this Grb2-containing complex may block the downstream activation of Ras and Ras-mediated amplification of TCR/CD3-mediated signals in hyporesponsive NOD T cells. These findings implicate mSOS as an important mediator of downregulation of Ras signaling in hyporesponsive NOD T cells.

Thymic T cell anergy in autoimmune nonobese diabetic mice is mediated by deficient T cell receptor regulation of the pathway of p21ras activation.

Thymic T cell anergy, as manifested by thymocyte proliferative unresponsiveness to antigens expressed in the thymic environment, is commonly believed to mediate the acquisition of immunological self-tolerance. However, we previously found that thymic T cell anergy may lead to the breakdown of tolerance and predispose to autoimmunity in nonobese diabetic (NOD) mice. Here, we show that NOD thymic T cell anergy, as revealed by proliferative unresponsiveness in vitro after stimulation through the T cell receptor (TCR), is associated with defective TCR-mediated signal transduction along the PKC/p21ras/p42mapk pathway of T cell activation. PKC activity is reduced in NOD thymocytes. Activation of p21ras is deficient in quiescent and stimulated NOD T cells, and this is correlated with a significant reduction in the tyrosine phosphorylation of p42mapk, a serine/threonine kinase active downstream of p21ras. Treatment of NOD T cells with a phorbol ester not only enhances their p21ras activity and p42mapk tyrosine phosphorylation but also restores their proliferative responsiveness. Since p42mapk activity is required for progression through to S phase of the cell cycle, our data suggest that reduced tyrosine phosphorylation of p42mapk in stimulated NOD T cells may abrogate its activity and elicit the proliferative unresponsiveness of these cells.

Regulation of fas ligand expression during activation-induced cell death in T cells by p38 mitogen-activated protein kinase and c-Jun NH2-terminal kinase.

Activation-induced cell death (AICD) is a mechanism of peripheral T cell tolerance that depends upon an interaction between Fas and Fas ligand (FasL). Although c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) may be involved in apoptosis in various cell types, the mode of regulation of FasL expression during AICD in T cells by these two MAPKs is incompletely understood. To investigate the regulatory roles of these two MAPKs, we analyzed the kinetics of TCR-induced p38 MAPK and JNK activity and their regulation of FasL expression and AICD. We report that both JNK and p38 MAPK regulate AICD in T cells. Our data suggest a novel model of T cell AICD in which p38 MAPK acts early to initiate FasL expression and the Fas-mediated activation of caspases. Subsequently, caspases stimulate JNK to further upregulate FasL expression. Thus, p38 MAPK and downstream JNK converge to regulate FasL expression at different times after T cell receptor stimulation to elicit maximum AICD.

Interleukin 4 reverses T cell proliferative unresponsiveness and prevents the onset of diabetes in nonobese diabetic mice.

Beginning at the time of insulitis (7 wk of age), CD4+ and CD8+ mature thymocytes from nonobese diabetic (NOD) mice exhibit a proliferative unresponsiveness in vitro after T cell receptor (TCR) crosslinking. This unresponsiveness does not result from either insulitis or thymic involution and is long lasting, i.e., persists until diabetes onset (24 wk of age). We previously proposed that it represents a form of thymic T cell anergy that predisposes to diabetes onset. This hypothesis was tested in the present study by further investigating the mechanism responsible for NOD thymic T cell proliferative unresponsiveness and determining whether reversal of this unresponsiveness protects NOD mice from diabetes. Interleukin 4 (IL-4) secretion by thymocytes from > 7-wk-old NOD mice was virtually undetectable after treatment with either anti-TCR alpha/beta, anti-CD3, or Concanavalin A (Con A) compared with those by thymocytes from age- and sex-matched control BALB/c mice stimulated under identical conditions. NOD thymocytes stimulated by anti-TCR alpha/beta or anti-CD3 secreted less IL-2 than did similarly activated BALB/c thymocytes. However, since equivalent levels of IL-3 were secreted by Con A-activated NOD and BALB/c thymocytes, the unresponsiveness of NOD thymic T cells does not appear to be dependent on reduced IL-2 secretion. The surface density and dissociation constant of the high affinity IL-2 receptor of Con A-activated thymocytes from both strains are also similar. The patterns of unresponsiveness and lymphokine secretion seen in anti-TCR/CD3-activated NOD thymic T cells were also observed in activated NOD peripheral spleen T cells. Exogenous recombinant (r)IL-2 only partially reverses NOD thymocyte proliferative unresponsiveness to anti-CD3, and this is mediated by the inability of IL-2 to stimulate a complete IL-4 secretion response. In contrast, exogenous IL-4 reverses the unresponsiveness of both NOD thymic and peripheral T cells completely, and this is associated with the complete restoration of an IL-2 secretion response. Furthermore, the in vivo administration of rIL-4 to prediabetic NOD mice protects them from diabetes. Thus, the ability of rIL-4 to reverse completely the NOD thymic and peripheral T cell proliferative defect in vitro and protect against diabetes in vivo provides further support for a causal relationship between this T cell proliferative unresponsiveness and susceptibility to diabetes in NOD mice.

An alpha-galactosylceramide C20:2 N-acyl variant enhances anti-inflammatory and regulatory T cell-independent responses that prevent type 1 diabetes.

Protection from type 1 diabetes (T1D), a T helper type 1 (Th1)-mediated disease, is achievable in non-obese diabetic (NOD) mice by treatment with alpha-galactosylceramide (alpha-GalCer) glycolipids that stimulate CD1d-restricted invariant natural killer T (iNK T) cells. While we have reported previously that the C20:2 N-acyl variant of alpha-GalCer elicits a Th2-biased cytokine response and protects NOD mice from T1D more effectively than a form of alpha-GalCer that induces mixed Th1 and Th2 responses, it remained to determine whether this protection is accompanied by heightened anti-inflammatory responses. We show that treatment of NOD mice with C20:2 diminished the activation of 'inflammatory' interleukin (IL)-12 producing CD11c(high)CD8+ myeloid dendritic cells (mDCs) and augmented the function of 'tolerogenic' DCs more effectively than treatment with the prototypical iNKT cell activator KRN7000 (alpha-GalCer C26:0) that induces Th1- and Th2-type responses. These findings correlate with a reduced capacity of C20:2 to sustain the early transactivation of T, B and NK cells. They may also explain our observation that C20:2 activated iNK T cells depend less than KRN7000 activated iNK T cells upon regulation by regulatory T cells for cytokine secretion and protection from T1D. The enhanced anti-inflammatory properties of C20:2 relative to KRN7000 suggest that C20:2 should be evaluated further as a drug to induce iNK T cell-mediated protection from T1D in humans.

Sulfated beef insulin treatment elicits CD8+ T cells that may abrogate immunologic insulin resistance in type I diabetes.

The in vitro responses of T cells from 13 insulin-nonresistant and 1 immunologically insulin-resistant (IIR) type I diabetes patients to sulfated beef insulin (SBI) were analyzed. Insulin A-loop specific CD4+ T cells from these patients did not respond to SBI. After 1 yr of treatment with SBI the IIR patient's T cell and antibody responses to beef, pork, and human insulin progressed from very high to nondetectable levels. This occurred in parallel to the appearance of her insulin-specific CD8+ T cells, which inhibited the response of her A-loop-specific CD4+ T cells to insulin. A transient increase in her CD8+ anti-insulin antibody activity coincided with a relative lack of her CD8+ T cell activity. CD8+ T cells that regulate T cell responsiveness to insulin are probably present but difficult to detect in most type I diabetes patients. These T cells were identified in only 2 of 13 insulin-nonresistant patients who presented with lipoatrophy and insulin allergy, respectively, and who possessed high-titered, anti-insulin antibodies. Our data demonstrate that CD8+ T cells play an important role in controlling peripheral tolerance to insulin and may abrogate IIR in a diabetic patient treated with SBI.

Detection of pancreatic islet 64,000 M(r) autoantigens in insulin-dependent diabetes distinct from glutamate decarboxylase.

Patients with insulin-dependent diabetes (IDDM) possess antibodies to islet proteins of M(r)-64,000. Potential autoantigens of this M(r) include glutamate decarboxylase (GAD) and 65 kD heat shock protein. We have detected two distinct antibody specificities in IDDM that bind 50,000 M(r) or 37,000/40,000 M(r) proteolytic fragments of 64,000 M(r) proteins. In this study, we investigated relationships of these proteolytic fragments to GAD and heat shock proteins. Polyclonal antibodies to GAD bound 50,000 M(r) fragments of islet antigen. Recombinant GAD65, but not GAD67, blocked binding to this antigen, suggesting that 50,000 M(r) fragments are derived from islet GAD65. In contrast, GAD antibodies did not recognize 37,000/40,000 M(r) fragments, and neither GAD isoforms blocked autoantibody binding to precursors of these fragments. The 37,000/40,000 M(r) fragments, but not the 50,000 M(r) fragments, were detected after trypsin treatment of immunoprecipitates from insulinoma cells that lacked expression of major GAD isoforms. Antibodies in IDDM did not bind native or trypsinized islet heat shock proteins. Thus, IDDM patients possess antibodies to GAD, but also distinct antibodies to a 64,000 M(r) protein that is not related to known GAD isoforms or heat shock proteins.