Maternal autoimmune thyroid disease and the fetal immune system.

OBJECTIVE: Several studies indicate that in utero exposure to maternal autoimmune diseases and transplacental passage of autoantibodies affect the risk of autoimmunity in the offspring, e. g., maternally derived GAD65 autoantibody correlates with decreased risk of type 1 diabetes, whereas thyroid peroxidase autoantibody (TPOAb) positivity at birth is associated with increased incidence of autoimmune thyroid disease later in life. The aim of this study was to identify immunological changes in children born to mothers with thyroid autoimmunity that may be related to in utero exposure to autoantibodies. DESIGN AND METHOD: Open label prospective analysis of cord blood lymphocytes and serum cytokines by Flow Cytometry in children born to mothers with autoimmune thyroiditis (AIT) (n=31) and to healthy mothers (n=76) and titers of thyroid autoantibodies were determined in cord blood and in maternal peripheral blood at delivery. RESULTS: We found an increase (almost 30%) in the frequency of cord blood natural killer (NK) cells (p=0.0016) and a minor increase in the subset of T cells expressing NK markers (p=0.028), in children born to AIT mothers. There were no detectable differences in the phenotype or frequency of cord blood memory/activated T cells, including CD4 (+)CD25 (+) T cells, between the 2 groups. The levels of pro-inflammatory cytokines TNF-α, IL-10, IL-12p70, IFN-γ and IL-1ß were significantly decreased in offspring of AIT mothers as compared to healthy controls. CONCLUSIONS: Maternal thyroid autoimmunity and transplacental passage of autoantibodies against thyroid antigens may affect the generation or expansion of cells with NK activity and the secretion of inflammatory cytokines.

Glucagon-like peptide-1 receptor signalling selectively regulates murine lymphocyte proliferation and maintenance of peripheral regulatory T cells.

AIMS/HYPOTHESIS: Glucagon-like peptide-1 receptor (GLP-1R) agonists improve glucose control in animals and humans with type 1 diabetes. However, there is little information on the role of the GLP-1R in the immune system. We studied the role of the GLP-1R in immune function in wild-type (WT) and nonobese diabetic (NOD) and Glp1r-/- mice. METHODS: Glp1r mRNA expression was examined in sorted immune subpopulations by RT-PCR. The effects of GLP-1R activation were assessed on cAMP production and proliferation, migration and survival of primary immune cells from WT and NOD mice. The ability of primary cells from Glp1r-/- mice to proliferate, migrate or survive apoptosis was determined. Immunophenotyping studies were performed to assess the frequency of immune subpopulations in Glp1r-/- mice. RESULTS: Ex vivo activation of the GLP-1R resulted in a modest but significant elevation of cAMP in primary thymocytes and splenocytes from both WT and NOD mice. GLP-1R activation did not increase proliferation of primary thymocytes, splenocytes or peripheral lymph node cells. In contrast, Glp1r-/- thymocytes exhibited a hypoproliferative response, whilst peripheral Glp1r-/- lymphocytes were hyperproliferative in response to mitogenic stimulation. Activation or loss of GLP-1R signalling did not modify apoptosis or chemotaxis in primary lymphocytes. Male Glp1r-/- mice exhibited a significantly lower percentage of peripheral regulatory T cells, although no differences were observed in the numbers of CD4+ and CD8+ T cells and B cells in the spleen and lymph nodes of Glp1r-/- mice. CONCLUSIONS/INTERPRETATION: These studies establish that GLP-1R signalling may regulate lymphocyte proliferation and maintenance of peripheral regulatory T cells.

Genetic control of T and B lymphocyte activation in nonobese diabetic mice.

Type 1 diabetes in nonobese diabetic (NOD) mice is characterized by the infiltration of T and B cells into pancreatic islets. T cells bearing the TCR Vbeta3 chain are disproportionately represented in the earliest stages of islet infiltration (insulitis) despite clonal deletion of most Vbeta3(+) immature thymocytes by the mammary tumor virus-3 (Mtv-3) superantigen (SAg). In this report we showed that a high frequency of NOD Vbeta3(+) T cells that escape deletion are activated in vivo and that this phenotype is linked to the Mtv-3 locus. One potential mechanism of SAg presentation to peripheral T cells is by activated B cells. Consistent with this idea, we found that NOD mice harbor a significantly higher frequency of activated B cells than nondiabetes-prone strains. These activated NOD B cells expressed cell surface molecules consistent with APC function. At the molecular level, the IgH repertoire of activated B cells in NOD mice was equivalent to resting B cells, suggesting a polyclonal response in vivo. Genetic analysis of the activated B cell phenotype showed linkage to Idd1, the NOD MHC haplotype (H-2(g7)). Finally, Vbeta3(+) thymocyte deletion and peripheral T cell activation did not require B cells, suggesting that other APC populations are sufficient to generate both Mtv-3-linked phenotypes. These data provide insight into the genetic regulation of NOD autoreactive lymphocyte activation that may contribute to failure of peripheral tolerance and the pathogenesis of type I diabetes.

Development and function of diabetogenic T-cells in B-cell-deficient nonobese diabetic mice.

Insulin-dependent diabetes (type 1 diabetes) in the NOD mouse is a T-cell-mediated autoimmune disease. However, B-cells may also play a critical role in disease pathogenesis, as genetically B-cell-deficient NOD mice (NOD.microMT) have been shown to be protected from type 1 diabetes and to display reduced responses to certain islet autoantigens. To examine the requirements for B-cells in the development of type 1 diabetes, we generated a B-cell-naive T-cell repertoire by transplantation of NOD fetal thymuses (FTs) into NOD.scid recipients. Surprisingly, these FT-derived NOD T-cells were diabetogenic in 36% of NOD.scid recipients, despite the absence of B-cells. In addition, T-cells isolated from NOD.microMT mice were diabetogenic in 22% of NOD.scid recipients. Together, these results indicate that B-cells are not an absolute requirement for the generation or effector function of an islet-reactive T-cell repertoire in NOD mice. We suggest that conditions favoring rapid lymphocyte expansion can reveal autoreactive T-cell activity and precipitate disease in genetically susceptible individuals.

Irradiation promotes V(D)J joining and RAG-dependent neoplastic transformation in SCID T-cell precursors.

Defects in the nonhomologous end-joining (NHEJ) pathway of double-stranded DNA break repair severely impair V(D)J joining and selectively predispose mice to the development of lymphoid neoplasia. This connection was first noted in mice with the severe combined immune deficient (SCID) mutation in the DNA-dependent protein kinase (DNA-PK). SCID mice spontaneously develop thymic lymphoma with low incidence and long latency. However, we and others showed that low-dose irradiation of SCID mice dramatically increases the frequency and decreases the latency of thymic lymphomagenesis, but irradiation does not promote the development of other tumors. We have used this model to explore the mechanistic basis by which defects in NHEJ confer selective and profound susceptibility to lymphoid oncogenesis. Here, we show that radiation quantitatively and qualitatively improves V(D)J joining in SCID cells, in the absence of T-cell receptor-mediated cellular selection. Furthermore, we show that the lymphocyte-specific endonuclease encoded by the recombinase-activating genes (RAG-1 and RAG-2) is required for radiation-induced thymic lymphomagenesis in SCID mice. Collectively, these data suggest that irradiation induces a DNA-PK-independent NHEJ pathway that facilitates V(D)J joining, but also promotes oncogenic misjoining of RAG-1/2-induced breaks in SCID T-cell precursors.

Two genetic loci regulate T cell-dependent islet inflammation and drive autoimmune diabetes pathogenesis.

Insulin-dependent diabetes mellitus (IDDM) is a polygenic disease caused by progressive autoimmune infiltration (insulitis) of the pancreatic islets of Langerhan, culminating in the destruction of insulin-producing beta cells. Genome scans of families with diabetes suggest that multiple loci make incremental contributions to disease susceptibility. However, only the IDDM1 locus is well characterized, at a molecular and functional level, as alleleic variants of the major histocompatibility complex (MHC) class II HLA-DQB1, DRB1, and DPB1 genes that mediate antigen presentation to T cells. In the nonobese diabetic (NOD) mouse model, the Idd1 locus was shown to be the orthologous MHC gene I-Ab. Inheritance of susceptibility alleles at IDDM1/Idd1 is insufficient for disease development in humans and NOD mice. However, the identities and functions of the remaining diabetes loci (Idd2-Idd19 in NOD mice) are largely undefined. A crucial limitation in previous genetic linkage studies of this disease has been reliance on a single complex phenotype-diabetes that displays low penetrance and is of limited utility for high-resolution genetic mapping. Using the NOD model, we have identified an early step in diabetes pathogenesis that behaves as a highly penetrant trait. We report that NOD-derived alleles at both the Idd5 and Idd13 loci regulate a T lymphocyte-dependent progression from a benign to a destructive stage of insulitis. Human chromosomal regions orthologous to the Idd5 and -13 intervals are also linked to diabetes risk, suggesting that conserved genes encoded at these loci are central regulators of disease pathogenesis. These data are the first to reveal a role for individual non-MHC Idd loci in a specific, critical step in diabetes pathogenesis-T cell recruitment to islet lesions driving destructive inflammation. Importantly, identification of intermediate phenotypes in complex disease pathogenesis provides the tools required to progress toward gene identification at these loci.

Independent genetic regulation of T-cell and antigen-presenting cell participation in autoimmune islet inflammation.

Genetic susceptibility to type 1 diabetes in the nonobese diabetic (NOD) mouse involves at least 17 Idd loci. Idd1 has been mapped to a class II gene in the major histocompatibility complex (MHC), whereas the products and functions of the remaining Idd loci are unresolved. To investigate how non-MHC Idd genes regulate islet inflammation and IDDM progression, NOD mice were compared with the nonobese diabetes-resistant (NOR) mouse, a related MHC-identical strain that possesses a subset of the NOD-derived alleles at the Idd loci. Using quantitative reverse transcriptase-polymerase chain reaction amplification and immunohistochemistry, we observed that disease resistance in NOR mice is reflected by a protracted block at the earliest stage of insulitis. In NOD islets, early antigen-presenting cell (APC) recruitment to islet lesions was temporally coincident with progressive T-cell infiltration. In striking contrast, islet infiltrates in NOR mice were composed of APCs with minimal contribution from T-cells and T-cell-derived inflammatory cytokines, conferring apparent resistance to invasive insulitis and beta-cell destruction. This is the first evidence that a subset of Idd susceptibility loci independently regulate T-cell and APC participation in insulitis progression. As progress is made toward identification of the Idd gene products, it will be crucial to determine how they regulate diabetogenesis. Our data define distinct cellular stages of IDDM pathogenesis in which the impact of Idd genes can be readily analyzed.

The absolute number of trans-rearrangements between the TCRG and TCRB loci is predictive of lymphoma risk: a severe combined immune deficiency (SCID) murine model.

Pilot studies in human populations have demonstrated a correlation between the level of antigen receptor trans-rearrangements and risk (at the population level) of lymphoid malignancy. Irradiation of newborn severe combined immune deficiency mice results in an increased risk of subsequent development of thymic lymphoma (100% of mice so irradiated are dead of thymic lymphoma by 20 weeks of age). We, therefore, assayed the occurrence of trans-rearrangements in this well-controlled mouse mutant system and found a 50-100-fold increase in the absolute number of TCRGV-TCRBJ trans-rearrangements compared to unirradiated littermates (and a comparable fold increase over age-matched BALB/c mice) at 2 weeks following irradiation. We also found a marked disproportion in generating trans-rearrangements versus intralocus rearrangements in the severe combined immune deficiency system compared to BALB/c, independent of irradiation. The trans-rearrangements noted were polyclonal in nature. These data, again, suggest that the absolute level of antigen receptor trans-rearrangements may serve as a biomarker of lymphoma risk.

Induction of a heterogeneous TCR repertoire in (PL/JXSJL/J)F1 mice by myelin basic protein peptide Ac1-11 and its analog Ac1-11[4A].

Experimental autoimmune encephalomyelitis (EAE) serves as a rodent model of the autoimmune disease multiple sclerosis. In mice, EAE is induced by immunizing with spinal cord homogenate, components of the myelin sheath, such as myelin basic protein (MBP) or proteolipid protein (PLP), or peptides derived from these components. EAE can be induced in H-2u or (H-2u x H-2s)F1 mice with the N-terminal peptide of MBP, Ac1-11. Coimmunization with Ac1-11 and Ac1-11[4A], an analog in which lysine at position four is substituted with alanine, prevents EAE. The mechanism of inhibition has not been elucidated, but probably does not work through MHC blockade, T cell anergy or clonal elimination of encephalitogenic T cells. We have isolated T cell clones and hybridomas from (PL/J x SJL/J)F1 mice immunized with either Ac1-11 alone or Ac1-11 and Ac1-11[4A] and analysed these cells for differences in their T cell receptor repertoire and in vitro response. Although T cells elicited by coinjection of Ac1-11 and Ac1-11[4A] expressed TCR that used V alpha and Vbeta gene elements similar to those elicited by Ac1-11 alone, they differed in the sequences of the junctional region of the alpha chain. Most of these T cells also responded less well to Ac1-11 in vitro, suggesting that coinjection of Ac1-11 and Ac1-11[4A] preferentially activates T cells bearing TCR of different affinity for Ac1-11 bound to I-A(u), and which may therefore be less encephalitogenic. Furthermore, our results show that a more diverse repertoire of V alpha and Vbeta genes are elicited by Ac1-11 in (PL/J x SJL/J)F1 mice compared to PL/J and B10.PL mice, providing further evidence that a restricted TCR repertoire is not required for the development of autoimmune disease.

Essential and perilous: V(D)J recombination and DNA damage checkpoints in lymphocyte precursors.

V(D)J recombination generates a diverse array of antigen-binding specificities, but breakage and re-joining of DNA segments have grave implications for the maintenance of genomic stability and oncogenic risk. Exposure of eukaryotic cells to genotoxic agents activates a DNA damage checkpoint that induces cell-cycle arrest and DNA repair, or apoptosis. We discuss several lines of evidence implicating DNA-dependent protein kinase (DNA-PK), and the gene mutated in ataxia telangiectasia (ATM), two mammalian homologues of yeast DNA damage-checkpoint genes, in regulating the response to intrinsic DNA damage that occurs during V(D)J recombination.

IL-4 expression at the onset of islet inflammation predicts nondestructive insulitis in nonobese diabetic mice.

In nonobese diabetic mice, autoimmune diabetes progresses in an age-linked and gender-dependent manner. Insulitis begins in male and female mice at approximately 1 mo of age; however, 70 to 90% of females, but only 10 to 20% of males, become diabetic by 6 mo. Multiple studies propose that proinflammatory Th1 and immunomodulatory Th2 cytokines impact diabetes pathogenesis, but the role of these cytokines in spontaneous diabetes progression is not yet clear. We used quantitative reverse-transcriptase-coupled PCR to analyze expression of cytokines and APC costimulatory molecules in the islets of 20- to 180-day-old male and female nonobese diabetic littermates, and identified three stages in diabetes progression. At 1 to 2 mo of age, islet-infiltrating T cells displayed a Th1 cytokine bias in females, and a Th2 cytokine bias in males. In females, stage II (2-3 mo of age) was characterized by an increase in islet-infiltrating T cells, APC, and Th1 cytokines, whereas male infiltrates did not increase in size, and Th1 cytokine expression continued to decline during this interval. Islet infiltration reached a plateau (stage III) in 3- to 4-mo-old females, months before overt diabetes onset. Our data imply that Th cytokine expression in early insulitis exerts substantial impact on beta cell destruction and overt diabetes. A clinical implication of our results is that young individuals in the early stages of insulitis are ideal candidates for therapeutic intervention to minimize beta cell destruction and morbidity.

Biochemical and genetic defects in the DNA-dependent protein kinase in murine scid lymphocytes.

The scid gene product has been identified as the 460-kDa catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs p460), a member of the phosphatidylinositol 3-kinase family. DNA-PK activity is undetectable in scid cells, but the molecular basis for this defect has not been identified. Here we report that expression of p460 in scid lymphocyte precursors is detectable but is reduced at least 10-fold relative to that in wild-type lymphocytes. In addition, we show that the scid mutation disturbs p460 nuclear association, presumably affecting its role in DNA repair pathways. To examine the molecular basis for our observations, we used a degenerate PCR strategy to clone the C-terminal p460 kinase domain from wild-type and scid thymocytes. Northern (RNA) analysis with these probes revealed normal steady-state p460 mRNA levels in scid cells, suggesting that the reduced abundance of p460 protein is due to a posttranscriptional defect. Sequence comparisons identified a single-base-pair alteration in the scid C-terminal p460 kinase domain, resulting in a premature stop codon. This mutation is predicted to truncate p460 by approximately 8 kDa, but it preserves the conserved motifs required for kinase activity in members of the phosphoinositidyl 3-kinase family. Despite a computed molecular weight alteration of less than 2%, we were able to visualize this difference by Western blot (immunoblot) analysis of wild-type and scid p460. These data demonstrate that the scid DNA-PKes mutation is not a null allele and suggest a molecular rationale for the well-described leakiness of the scid phenotype.

V(D)J recombination activates a p53-dependent DNA damage checkpoint in scid lymphocyte precursors.

Double-stranded DNA breaks (DSBs) trigger p53-mediated cell cycle arrest or apoptosis pathways that limit the oncogenic consequences of exposure to genotoxic agents, but p53-mediated responses to DSB generated by normal physiologic events have not been documented. "Broken" V(D)J coding ends accumulate in scid lymphocyte precursors as a consequence of a mutation in DNA-dependent protein kinase (DNA-PK). The ensuing failure to rearrange efficiently antigen receptors arrests lymphoid development. Here we show that scid thymocytes express high levels of p53 protein, attributable to recombinase activating gene (RAG)-dependent generation of DSB adjacent to V, D, and J gene segments. To examine the functional importance of p53 expression in vivo, we bred p53-/- scid mice. The absence of p53 facilitated production of in-frame V(D)Jbeta coding joints and developmental progression of scid thymocytes, in addition to a dramatic accumulation of pro-B cells. All mice developed disseminated pro-B or immature T cell lymphoma/leukemia by 7-12 weeks of age. We present evidence that p53 deficiency prolongs the survival of scid lymphocyte precursors harboring broken V(D)J coding ends, allowing the accumulation of aneuploid cells. These results demonstrate that a p53-mediated DNA damage checkpoint contributes to the immune deficiency characteristic of the scid mutation and limits the oncogenic potential of DSBs generated during V(D)J recombination.

Transient restoration of gene rearrangement at multiple T cell receptor loci in gamma-irradiated scid mice.

The developmental arrest of thymocytes from scid mice, deficient in variable, (diversity), and joining, or V(D)J recombination, can be overcome by sublethal gamma-irradiation. Since previous studies focused on restoration of rearrangement of the T cell receptor (TCR) beta locus, productive rearrangement of which is selected for, we sought to examine to what extent locus specificity and cellular selection contributed to the observed effects. We report here that irradiation of newborn scid mice induces normal V-D-J rearrangements of the TCR delta locus, which like TCR beta, is also actively rearranged in CD(4-)CD(8-) (double negative) thymocytes. In contrast, no complete V-J alpha rearrangements were detected. Instead, we detected substantial levels of hairpin-terminated coding ends at the 5' end of the J alpha locus, demonstrating that TCR alpha rearrangements manifest the effects of the scid mutation. Irradiation, therefore, transiently compensates for the effects of the scid mutation in a locus-nonspecific manner in thymocytes, resulting in a burst of normal TCR beta and delta rearrangements. Irradiation also allows the development of cells that can initiate but fail to complete V(D)J recombination events at the TCR alpha locus, which is normally inaccessible in scid thymocytes.

Lck dependence of signaling pathways activated by gamma-irradiation and CD3 epsilon engagement in RAG-1(-/-)-immature thymocytes.

The src family tyrosine kinase, Lck, transduces signals from the pre-TCR complex which regulate the development and expansion of CD4/CD8 double-positive (DP) thymocytes from CD25(+) CD4/CD8 double-negative progenitors. We and others have recently shown that sublethal gamma-irradiation bypasses the need for TCRbeta expression to promote the development and expansion of DP thymocytes in scid or recombinase-activating gene (RAG)-deficient mice. Here we demonstrate that gamma-irradiation activates an Lck-dependent signaling process in immature thymocytes similar to that initiated physiologically by the pre-TCR complex.

Microvascular transplantation of physeal allografts.

We compared growth in vascularised allograft transplants, autografts and in non-operated physes in rabbits immunosuppressed with cyclosporin A and in non-immunosuppressed animals. Molecular haplotyping was undertaken before operation to ensure allogenicity. Postoperative bone scans and fluorochrome labelling were used to confirm physeal vascularity. The animals were killed at three or five weeks. Proximal tibial physeal autografts, with or without cyclosporin A, or allografts with cyclosporin A, grew at similar rates to the physes of non-operated rabbits. All the operated physes grew at rates significantly greater than their contralateral controls. 99mTc-MDP bone scans accurately predicted the viability of the epiphyseal plate. Quantitative histomorphological analysis of the heights of the physeal proliferative and hypertrophic zones showed that successful physeal transplants have a normal appearance, but when unsuccessful have thickened hypertrophic zones compatible with physeal ischaemia. We discuss the significance of these results in relation to the transplantation of physes in children.

An alternative view of T-cell receptor-MHC interaction: T-cell receptor binds transversally to the alpha-helices of the MHC molecule.

We have attempted to elucidate the relative orientation of the T-cell receptor (TCR) to the major histocompatibility complex (MHC)-antigen complex during antigen recognition, using the T-cell response to B10.A (I-Ek) and B10.A(5R) (I-Eb) mice to the 1-23(H) peptide derived from glycoprotein D of the herpes simplex virus. The 1-23(H)-specific T-cells derived from both B10.A and B10.A(5R) mice use the same set of V alpha genes and a different array of V beta genes. The CDR1s of these TCR beta chains share residues at particular positions. The CDR2s of the TCR beta chains have a negative charge, which correlates with I-Eb reactivity and with the positively charged polymorphic residues residing at the C-terminal end of the alpha-helix of the I-Eb beta chain of the class II molecule. Taken together, the data suggest that the TCR beta chain interacts with both the alpha and beta chains of the MHC class II molecule, as does the TCR alpha chain.

Restriction fragment-length polymorphism analysis of the major histocompatibility complex in New Zealand white rabbits.

As a model system, rabbits are particularly useful in transplantation studies because of their size and accessibility. However, inbred rabbits of known MHC haplotype are not commercially available, and there are few monoclonal antibody reagents available to permit serological typing of experimental animals prior to transplant. Here we present a rapid and reliable method to distinguish rabbits that differ at their MHC class I and class II loci, and present 34 class I and 16 class II haplotypes determined by restriction fragment-length polymorphism analysis of outbred heterozygous rabbits. The applicability of this molecular typing system to transplantation experiments in the New Zealand White rabbit is discussed.

Development of CD4+CD8+ thymocytes in RAG-deficient mice through a T cell receptor beta chain-independent pathway.

Antigen-binding diversity is generated by site-specific V(D)J recombination of the T cell receptor (TCR) and immunoglobulin loci in lymphocyte precursors. Coordinate expression of two structurally distinct recombinase activating genes, RAG-1 and RAG-2, is necessary for activation of site-specific V(D)J recombination. In mice bearing targeted disruptions of either the RAG-1 or RAG-2 genes, T and B lymphocyte development is arrested at the CD4-8- double negative (DN) thymocyte or B220+/CD43+ pro-B cell stage. Development of CD4+CD8+ double positive (DP) thymocytes is restored by expression of a functionally rearranged TCR beta transgene, suggesting that TCR beta expression is critical for this developmental transition. We have found that treatment of adult or newborn RAG-deficient mice with a single sublethal dose of gamma-irradiation rescues the DN to DP transition in early thymocytes, and this is accompanied by a dramatic increase in thymus cellularity. In contrast to the observed induction of thymocyte maturation, there was no phenotypic or functional evidence of coincident B lymphocyte development in irradiated RAG-deficient mice. Interestingly, maturation of DP thymocytes occurred without expression of TCR beta protein in the cytoplasm or on the cell surface. These results suggest an in vivo pathway for DP thymocyte development which is TCR beta chain independent.

Peri-islet infiltrates of young non-obese diabetic mice display restricted TCR beta-chain diversity.

To define the clonal diversity of autoreactive T cells associated with the induction of type 1 diabetes, we characterized TCR expression in the earliest detectable islet infiltrates of non-obese diabetic (NOD) mice. The islets of young NOD females were examined for V beta and J beta germ-line gene usage and V(D)J beta junctional sequence diversity. The results from 7-wk-old mice corroborate prior studies demonstrating that the T cell repertoire of islet infiltrates diversifies early in the inflammatory process. In contrast, examination of 4-wk-old NOD mice showed that TCR-beta expression in the peri-islet infiltrates was restricted both in V beta and J beta gene utilization and, most significantly, in V(D)J junctional sequence diversity. Islet-infiltrating T cells from young mice included V beta 3+ T cells, despite the presence of a mammary tumor virus-3-associated superantigen that deletes the majority of immature V beta 3+ thymocytes in NOD mice. Few other TCR V beta types were repeatedly detectable in early stage infiltrates. V(D)J junctional sequence diversity was evaluated in cDNA libraries made from the islets of young NOD mice. Analysis of these clones revealed limited junctional CDR3 diversity in early-infiltrating T cells, as compared with lymph node T cell libraries. Evaluation of TCR expression in individual islets revealed CDR3 sequence conservation between animals and among islets from a single animal. These results suggest that T cells bearing limited TCR-beta-chain diversity contribute to the inductive phases of autoimmune diabetes.