Disease diagnostics using machine learning of immune receptors.

Clinical diagnosis typically incorporates physical examination, patient history, and various laboratory tests and imaging studies, but makes limited use of the human system's own record of antigen exposures encoded by receptors on B cells and T cells. We analyzed immune receptor datasets from 593 individuals to develop MAchine Learning for Immunological Diagnosis (Mal-ID) , an interpretive framework to screen for multiple illnesses simultaneously or precisely test for one condition. This approach detects specific infections, autoimmune disorders, vaccine responses, and disease severity differences. Human-interpretable features of the model recapitulate known immune responses to SARS-CoV-2, Influenza, and HIV, highlight antigen-specific receptors, and reveal distinct characteristics of Systemic Lupus Erythematosus and Type-1 Diabetes autoreactivity. This analysis framework has broad potential for scientific and clinical interpretation of human immune responses.

Helical TomoTherapy Total Lymphoid Irradiation and Hematopoietic Cell Transplantation for Kidney Transplant Tolerance in Rhesus Macaques.

Development of a post-transplant kidney transplant tolerance induction protocol involving a novel total lymphoid irradiation (TLI) conditioning method in a rhesus macaque model is described. We examined the feasibility of acheiving tolerance to MHC 1-haplotype matched kidney transplants by establishing a mixed chimeric state with infusion of donor hematopoietic cells (HC) using TomoTherapy TLI. The chimeric state was hypothesized to permit the elimination of all immunosuppressive (IS) medications while preserving allograft function long-term without development of graft-versus-host-disease (GVHD) or rejection. An experimental group of 11 renal transplant recipients received the tolerance induction protocol and outcomes were compared to a control group (n = 7) that received the same conditioning but without donor HC infusion. Development of mixed chimerism and operational tolerance was accomplished in two recipients in the experimental group. Both recipients were withdrawn from all IS and continued to maintain normal renal allograft function for 4 years without rejection or GVHD. None of the animals in the control group achieved tolerance when IS was eliminated. This novel experimental model demonstrated the feasibility for inducing of long-term operational tolerance when mixed chimerism is achieved using a TLI post-transplant conditioning protocol in 1-haplotype matched non-human primate recipients of combined kidney and HC transplantation.

Development of immunosuppressive myeloid cells to induce tolerance in solid organ and hematopoietic cell transplant recipients.

Replacement of failed organs followed by safe withdrawal of immunosuppressive drugs has long been the goal of organ transplantation. We studied changes in the balance of T cells and myeloid cells in the blood of HLA-matched and -mismatched patients given living donor kidney transplants followed by total lymphoid irradiation, anti-thymocyte globulin conditioning, and donor hematopoietic cell transplant to induce mixed chimerism and immune tolerance. The clinical trials were based on a conditioning regimen used to establish mixed chimerism and tolerance in mice. In preclinical murine studies, there was a profound depletion of T cells and an increase in immunosuppressive polymorphonuclear (pmn) myeloid-derived suppressor cells (MDSCs) in the spleen and blood following transplant. Selective depletion of pmn MDSCs in mice abrogated mixed chimerism and tolerance. In our clinical trials, patients given an analogous tolerance conditioning regimen developed similar changes, including profound depletion of T cells and a marked increase in MDSCs in blood posttransplant. Posttransplant pmn MDSCs transiently increased expression of lectin-type oxidized LDL receptor-1, a marker of immunosuppression, and production of the T-cell inhibitor arginase-1. These posttransplant pmn MDSCs suppressed the activation, proliferation, and inflammatory cytokine secretion of autologous T-cell receptor microbead-stimulated pretransplant T cells when cocultured in vitro. In conclusion, we elucidated changes in receptors and function of immunosuppressive myeloid cells in patients enrolled in the tolerance protocol that were nearly identical to those of MDSCs required for tolerance in mice. These trials were registered at www.clinicaltrials.gov as #NCT00319657 and #NCT01165762.

Mixed chimerism and acceptance of kidney transplants after immunosuppressive drug withdrawal.

Preclinical studies have shown that persistent mixed chimerism is linked to acceptance of organ allografts without immunosuppressive (IS) drugs. Mixed chimerism refers to continued mixing of donor and recipient hematopoietic cells in recipient tissues after transplantation of donor cells. To determine whether persistent mixed chimerism and tolerance can be established in patients undergoing living donor kidney transplantation, we infused allograft recipients with donor T cells and hematopoietic progenitors after posttransplant lymphoid irradiation. In 24 of 29 fully human leukocyte antigen (HLA)-matched patients who had persistent mixed chimerism for at least 6 months, complete IS drug withdrawal was achieved without subsequent evidence of rejection for at least 2 years. In 10 of 22 HLA haplotype-matched patients with persistent mixed chimerism for at least 12 months, reduction of IS drugs to tacrolimus monotherapy was achieved. Withdrawal of tacrolimus during the second year resulted in loss of detectable chimerism and subsequent rejection episodes, unless tacrolimus therapy was reinstituted. Posttransplant immune reconstitution of naïve B cells and B cell precursors was more rapid than the reconstitution of naïve T cells and thymic T cell precursors. Robust chimerism was observed only among naïve T and B cells but not among memory T cells. No evidence of rejection was observed in all surveillance graft biopsies obtained from mixed chimeric patients withdrawn from IS drugs, and none developed graft-versus-host disease. In conclusion, persistent mixed chimerism established in fully HLA- or haplotype-matched patients allowed for complete or partial IS drug withdrawal without rejection.

High-parametric evaluation of human invariant natural killer T cells to delineate heterogeneity in allo- and autoimmunity.

Human invariant natural killer T (iNKT) cells are a rare innate-like lymphocyte population that recognizes glycolipids presented on CD1d. Studies in mice have shown that these cells are heterogeneous and are capable of enacting diverse functions, and the composition of iNKT cell subsets can alter disease outcomes. In contrast, far less is known about how heterogeneity in human iNKT cells relates to disease. To address this, we used a high-dimensional, data-driven approach to devise a framework for parsing human iNKT heterogeneity. Our data revealed novel and previously described iNKT cell phenotypes with distinct functions. In particular, we found 2 phenotypes of interest: (1) a population with T helper 1 function that was increased with iNKT activation characterized by HLA-II+CD161- expression, and (2) a population with enhanced cytotoxic function characterized by CD4-CD94+ expression. These populations correlate with acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation and with new onset type 1 diabetes, respectively. Our study identifies human iNKT cell phenotypes associated with human disease that could aid in the development of biomarkers or therapeutics targeting iNKT cells.

Accelerated, but not conventional, radiotherapy of murine B-cell lymphoma induces potent T cell-mediated remissions.

Conventional local tumor irradiation (LTI), delivered in small daily doses over several weeks, is used clinically as a palliative, rather than curative, treatment for chemotherapy-resistant diffuse large B-cell lymphoma (DLBCL) for patients who are ineligible for hematopoietic cell transplantation. Our goal was to test the hypothesis that accelerated, but not conventional, LTI would be more curative by inducing T cell-mediated durable remissions. We irradiated subcutaneous A20 and BL3750 lymphoma tumors in mice with a clinically relevant total radiation dose of 30 Gy LTI, delivered in 10 doses of 3 Gy over 4 days (accelerated irradiation) or as 10 doses of 3 Gy over 12 days (conventional irradiation). Compared with conventional LTI, accelerated LTI resulted in more complete and durable tumor remissions. The majority of these mice were resistant to rechallenge with lymphoma cells, demonstrating the induction of memory antitumor immunity. The increased efficacy of accelerated LTI correlated with higher levels of tumor cell necrosis vs apoptosis and expression of "immunogenic cell death" markers, including calreticulin, heat shock protein 70 (Hsp70), and Hsp90. Accelerated LTI-induced remissions were not seen in immunodeficient Rag-2 -/- mice, CD8+ T-cell-depleted mice, or Batf-3 -/- mice lacking CD8α+ and CD103+ dendritic cells. Accelerated, but not conventional, LTI in immunocompetent hosts induced marked increases in tumor-infiltrating CD4+ and CD8+ T cells and MHCII+CD103+CD11c+ dendritic cells and corresponding reductions in exhausted PD-1+Eomes+CD8+ T cells and CD4+CD25+FOXP3+ regulatory T cells. These findings raise the possibility that accelerated LTI can provide effective immune control of human DLBCL.

Infusion of donor-derived CD8+ memory T cells for relapse following allogeneic hematopoietic cell transplantation.

Murine models showed that CD8+CD44hi memory T (TM) cells could eradicate malignant cells without inducing graft-versus-host disease (GVHD). We evaluated the feasibility and safety of infusing freshly isolated and purified donor-derived phenotypic CD8+ TM cells into adults with disease relapse after allogeneic hematopoietic cell transplantation (HCT). Phenotypic CD8 TM cells were isolated after unmobilized donor apheresis using a tandem immunomagnetic selection strategy of CD45RA depletion followed by CD8+ enrichment. Fifteen patients received CD8+ TM cells at escalating doses (1 × 106, 5 × 106, or 10 × 106 cells per kg). Thirteen received cytoreduction before CD8+ TM cell infusion, and 9 had active disease at the time of infusion. Mean yield and purity of the CD8+ TM infusion were 38.1% and 92.8%, respectively; >90% had CD8+ T effector memory phenotype, cytokine expression, and secretion profile. No adverse infusional events or dose-limiting toxicities occurred; GVHD developed in 1 patient (grade 2 liver). Ten patients (67%) maintained or achieved response (7 complete response, 1 partial response, 2 stable disease) for at least 3 months after infusion; 4 of the responders had active disease at the time of infusion. With a median follow-up from infusion of 328 days (range, 118-1328 days), median event-free survival and overall survival were 4.9 months (95% confidence interval [CI], 1-19.3 months) and 19.6 months (95% CI, 5.6 months to not reached), respectively. Collection and enrichment of phenotypic CD8+ TM cells is feasible, well tolerated, and associated with a low incidence of GVHD when administered as a manipulated infusion of donor lymphocytes in patients who have relapsed after HCT. This trial was registered at www.clinicaltrials.gov as #NCT01523223.

Macrochimerism and clinical transplant tolerance.

Current theory holds that macrochimerism is essential to the development of transplant tolerance. Hematopoietic cell transplantation from the solid organ donor is necessary to achieve macrochimerism. Over the last 10-20 years, trials of tolerance induction with combined kidney and hematopoietic cell transplantation have moved from the preclinical to the clinical arena. The achievement of macrochimerism in the clinical setting is challenging, and potentially toxic due to the conditioning regimen necessary to hematopoietic cell transplantation and due to the risk of graft-versus-host disease. There are differences in chimerism goals and methods of the three major clinical stage tolerance induction strategies in both HLA-matched and HLA-mismatched living donor kidney transplantation, with consequent differences in efficacy and safety. The Stanford protocol has proven efficacious in the induction of tolerance in HLA-matched kidney transplantation, allowing cessation of immunosuppressive drug therapy in 80% of study participants, with the safety profile of conventional transplantation. In HLA-mismatched transplantation, multi-lineage macrochimerism of over a year's duration can now be consistently achieved with the Stanford protocol, with complete withdrawal of immunosuppressive drug therapy during the second post-transplant year as the next experimental step and test of tolerance.

Deep Immune Profiling of an Arginine-Enriched Nutritional Intervention in Patients Undergoing Surgery.

Application of high-content immune profiling technologies has enormous potential to advance medicine. Whether these technologies reveal pertinent biology when implemented in interventional clinical trials is an important question. The beneficial effects of preoperative arginine-enriched dietary supplements (AES) are highly context specific, as they reduce infection rates in elective surgery, but possibly increase morbidity in critically ill patients. This study combined single-cell mass cytometry with the multiplex analysis of relevant plasma cytokines to comprehensively profile the immune-modifying effects of this much-debated intervention in patients undergoing surgery. An elastic net algorithm applied to the high-dimensional mass cytometry dataset identified a cross-validated model consisting of 20 interrelated immune features that separated patients assigned to AES from controls. The model revealed wide-ranging effects of AES on innate and adaptive immune compartments. Notably, AES increased STAT1 and STAT3 signaling responses in lymphoid cell subsets after surgery, consistent with enhanced adaptive mechanisms that may protect against postsurgical infection. Unexpectedly, AES also increased ERK and P38 MAPK signaling responses in monocytic myeloid-derived suppressor cells, which was paired with their pronounced expansion. These results provide novel mechanistic arguments as to why AES may exert context-specific beneficial or adverse effects in patients with critical illness. This study lays out an analytical framework to distill high-dimensional datasets gathered in an interventional clinical trial into a fairly simple model that converges with known biology and provides insight into novel and clinically relevant cellular mechanisms.

Lack of IL7Rα expression in T cells is a hallmark of T-cell immunodeficiency in Schimke immuno-osseous dysplasia (SIOD).

Schimke immuno-osseous dysplasia (SIOD) is an autosomal recessive, fatal childhood disorder associated with skeletal dysplasia, renal dysfunction, and T-cell immunodeficiency. This disease is linked to biallelic loss-of-function mutations of the SMARCAL1 gene. Although recurrent infection, due to T-cell deficiency, is a leading cause of morbidity and mortality, the etiology of the T-cell immunodeficiency is unclear. Here, we demonstrate that the T cells of SIOD patients have undetectable levels of protein and mRNA for the IL-7 receptor alpha chain (IL7Rα) and are unresponsive to stimulation with IL-7, indicating a loss of functional receptor. No pathogenic mutations were detected in the exons of IL7R in these patients; however, CpG sites in the IL7R promoter were hypermethylated in SIOD T cells. We propose therefore that the lack of IL7Rα expression, associated with hypermethylation of the IL7R promoter, in T cells and possibly their earlier progenitors, restricts T-cell development in SIOD patients.

Buteo Nesting Ecology: Evaluating Nesting of Swainson's Hawks in the Northern Great Plains.

Swainson's hawks (Buteo swainsoni) are long-distance migratory raptors that nest primarily in isolated trees located in areas of high grassland density. In recent years, anthropogenic conversion of grassland habitat has raised concerns about the status of the breeding population in the northern Great Plains. In 2013, we initiated a study to investigate the influence of extrinsic factors influencing Swainson's hawk nesting ecology in north-central South Dakota and south-central North Dakota. Using ground and aerial surveys, we located and monitored nesting Swainson's hawk pairs: 73 in 2013 and 120 in 2014. We documented 98 successful breeding attempts that fledged 163 chicks; 1.52 and 1.72 fledglings per successful nest in 2013 and 2014, respectively. We used Program MARK to evaluate the influence of land cover on nest survival. The top model, SDist2Farm+%Hay, indicated that nest survival (fledging at least one chick) decreased as nests were located farther from farm sites and as the percent of hay cover increased within 1200-m of the nest site (34.4%; 95% CI = 27.6%-42.3%). We used logistic regression analysis to evaluate the influence of landscape variables on nest-site selection; Swainson's hawks selected for nest sites located closer to roads. We suggest that tree belts associated with farm sites, whether occupied or not, provide critical breeding sites for Swainson's hawks. Additionally, poor breeding success may be related to the late migratory behavior of this species which requires them to occupy marginal habitat due to other raptors occupying the most suitable habitat prior to Swainson's hawks arriving to the breeding grounds.

Ablative Tumor Radiation Can Change the Tumor Immune Cell Microenvironment to Induce Durable Complete Remissions.

PURPOSE: The goals of the study were to elucidate the immune mechanisms that contribute to desirable complete remissions of murine colon tumors treated with single radiation dose of 30 Gy. This dose is at the upper end of the ablative range used clinically to treat advanced or metastatic colorectal, liver, and non-small cell lung tumors. EXPERIMENTAL DESIGN: Changes in the tumor immune microenvironment of single tumor nodules exposed to radiation were studied using 21-day (>1 cm in diameter) CT26 and MC38 colon tumors. These are well-characterized weakly immunogenic tumors. RESULTS: We found that the high-dose radiation transformed the immunosuppressive tumor microenvironment resulting in an intense CD8(+) T-cell tumor infiltrate, and a loss of myeloid-derived suppressor cells (MDSC). The change was dependent on antigen cross-presenting CD8(+) dendritic cells, secretion of IFNγ, and CD4(+)T cells expressing CD40L. Antitumor CD8(+) T cells entered tumors shortly after radiotherapy, reversed MDSC infiltration, and mediated durable remissions in an IFNγ-dependent manner. Interestingly, extended fractionated radiation regimen did not result in robust CD8(+) T-cell infiltration. CONCLUSIONS: For immunologically sensitive tumors, these results indicate that remissions induced by a short course of high-dose radiotherapy depend on the development of antitumor immunity that is reflected by the nature and kinetics of changes induced in the tumor cell microenvironment. These results suggest that systematic examination of the tumor immune microenvironment may help in optimizing the radiation regimen used to treat tumors by adding a robust immune response.

Treatment of 4T1 metastatic breast cancer with combined hypofractionated irradiation and autologous T-cell infusion.

The goal of this study was to determine whether a combination of local tumor irradiation and autologous T-cell transplantation can effectively treat metastatic 4T1 breast cancer in mice. BALB/c mice were injected subcutaneously with luciferase-labeled 4T1 breast tumor cells and allowed to grow for 21 days, at which time metastases appeared in the lungs. Primary tumors were treated at that time with 3 daily fractions of 20 Gy of radiation each. Although this approach could eradicate primary tumors, tumors in the lungs grew progressively. We attempted to improve efficacy of the radiation by adding autologous T-cell infusions. Accordingly, T cells were purified from the spleens of tumor-bearing mice after completion of irradiation and cryopreserved. Cyclophosphamide was administered thereafter to induce lymphodepletion, followed by T-cell infusion. Although the addition of cyclophosphamide to irradiation did not improve survival or reduce tumor progression, the combination of radiation, cyclophosphamide and autologous T-cell infusion induced durable remissions and markedly improved survival. We conclude that the combination of radiation and autologous T-cell infusion is an effective treatment for metastatic 4T1 breast cancer.

Generation and expansion of regulatory human CD4(+) T-cell clones specific for pancreatic islet autoantigens.

Autoantigen-specific regulatory T cells (Treg) are a potential cell therapy for human autoimmune disease, provided they could be generated in adequate numbers and with stable function. To this end, we determined the feasibility of cloning and expanding human CD4(+) Treg specific for the type 1 diabetes autoantigens, GAD65 and proinsulin. Blood CD4(+) cells stimulated to divide in response to GAD65 (in three healthy individuals) or proinsulin (in one type 1 diabetic) were flow sorted into single cells and cultured on feeder cells in the presence of anti-CD3 monoclonal antibody, IL-2 and IL-4. Clones were expanded over 4-6 weeks and screened for autoantigen-dependent suppression of tetanus toxoid-specific T-cell proliferation. Suppression by Treg clones was then confirmed against autoantigen-specific non-Treg clones. Of a total of 447 clones generated, 98 (21.9%) had autoantigen-dependent suppressor function. Treg clones were anergic but proliferated to autoantigen after addition of IL-2 or in co-culture with stimulated bulk T cells, without loss of suppressor function. Treg clones were stored over liquid N(2), thawed and further expanded over 12 days, whereupon they exhibited decreased suppressor function. Expansion of Treg clones overall was in the order 107-108-fold. Treg clones were not distinguished by markers of conventional CD4(+)CD25(+) Treg and suppressed independently of cell-cell contact but not via known soluble suppressor factors. This study demonstrates that autoantigen-specific CD4(+) Treg clones with potential application as a cell therapy for autoimmune disease can be generated and expanded from human blood.

The dendritic cell subtype-restricted C-type lectin Clec9A is a target for vaccine enhancement.

A novel dendritic cell (DC)-restricted molecule, Clec9A, was identified by gene expression profiling of mouse DC subtypes. Based on sequence similarity, a human ortholog was identified. Clec9A encodes a type II membrane protein with a single extracellular C-type lectin domain. Both the mouse Clec9A and human CLEC9A were cloned and expressed, and monoclonal antibodies (mAbs) against each were generated. Surface staining revealed that Clec9A was selective for mouse DCs and was restricted to the CD8(+) conventional DC and plasmacytoid DC subtypes. A subset of human blood DCs also expressed CLEC9A. A single injection of mice with a mAb against Clec9A, which targets antigens (Ags) to the DCs, produced a striking enhancement of antibody responses in the absence of added adjuvants or danger signals, even in mice lacking Toll-like receptor signaling pathways. Such targeting also enhanced CD4 and CD8 T-cell responses. Thus, Clec9A serves as a new marker to distinguish subtypes of both mouse and human DCs. Furthermore, targeting Ags to DCs with antibodies to Clec9A is a promising strategy to enhance the efficiency of vaccines, even in the absence of adjuvants.

N-terminal flanking residues of a diabetes-associated GAD65 determinant are necessary for activation of antigen-specific T cells in diabetes-resistant mice.

A diabetes-associated peptide in the glutamic acid decarboxylase 65 (GAD65) molecule, p524-543, activates two distinct populations of T cells, which apparently play opposite roles in the development of diabetes in NOD mice. By comparing the fine specificity of these two T cell repertoires using a nested set of truncated peptides that cover the p524-543 region, we found, surprisingly, that all clones recognized the same core within this peptide, p530-539. The core itself was non-immunogenic, but the residues flanking this shared sequence played the crucial role in selecting T cells to activate. A peptide missing N-terminal flanking residues at position 528 and 529 was stimulatory in NOD but not in MHC-matched, NOD-resistant (NOR) mice, suggesting that a protective response in the resistant mice may require T cell recognition of one or more of the N-terminal flanking residues. T cell repertoire studies demonstrated selective clonal expansions within the BV4 TCR family that dominates the p524-543 response in NOD but not in NOR mice. These data suggest that processing or trimming events affecting T cell recognition of very few flanking residues of diabetes-associated determinants might be involved in the protective response in NOR mice.

Cytotoxic phenylpropanoids and an additional thapsigargin analogue isolated from Thapsia garganica.

Four phenylpropanoids and a thapsigargin analogue have been isolated from the fruits of Thapsia garganica. A spectroscopic method for elucidating the relative stereochemistry at the two pairs of stereogenic centers in the phenylpropanoids has been developed. The phenylpropanoids were found to be potent cytotoxins.

Proinsulin is encoded by an RNA splice variant in human blood myeloid cells.

Genes for peripheral tissue-restricted self-antigens are expressed in thymic and hematopoietic cells. In thymic medullary epithelial cells, self-antigen expression imposes selection on developing autoreactive T cells and regulates susceptibility to autoimmune disease in mouse models. Less is known about the role of self-antigen expression by hematopoietic cells. Here we demonstrate that one of the endocrine self-antigens expressed by human blood myeloid cells, proinsulin, is encoded by an RNA splice variant. The surface expression of immunoreactive proinsulin was significantly decreased after transfection of monocytes with small interfering RNA to proinsulin. Furthermore, analogous to proinsulin transcripts in the thymus, the abundance of the proinsulin RNA splice variant in blood cells corresponded with the length of the variable number of tandem repeats 5' of the proinsulin gene, known to be associated with type 1 diabetes susceptibility. Self-antigen expression by peripheral myeloid cells extends the umbrella of "immunological self" and, by analogy with the thymus, may be implicated in peripheral immune tolerance.

The insulin A-chain epitope recognized by human T cells is posttranslationally modified.

The autoimmune process that destroys the insulin-producing pancreatic beta cells in type 1 diabetes (T1D) is targeted at insulin and its precursor, proinsulin. T cells that recognize the proximal A-chain of human insulin were identified recently in the pancreatic lymph nodes of subjects who had T1D. To investigate the specificity of proinsulin-specific T cells in T1D, we isolated human CD4(+) T cell clones to proinsulin from the blood of a donor who had T1D. The clones recognized a naturally processed, HLA DR4-restricted epitope within the first 13 amino acids of the A-chain (A1-13) of human insulin. T cell recognition was dependent on the formation of a vicinal disulfide bond between adjacent cysteine residues at A6 and A7, which did not alter binding of the peptide to HLA DR4. CD4(+) T cell clones that recognized this epitope were isolated from an HLA DR4(+) child with autoantibodies to insulin, and therefore, at risk for T1D, but not from two healthy HLA DR4(+) donors. We define for the first time a novel posttranslational modification that is required for T cell recognition of the insulin A-chain in T1D.

A peptide of glutamic acid decarboxylase 65 can recruit and expand a diabetogenic T cell clone, BDC2.5, in the pancreas.

Self peptide-MHC ligands create and maintain the mature T cell repertoire by positive selection in the thymus and by homeostatic proliferation in the periphery. A low affinity/avidity interaction among T cells, self peptides, and MHC molecules has been suggested for these events, but it remains unknown whether or how this self-interaction is involved in tolerance and/or autoimmunity. Several lines of evidence implicate the glutamic acid decarboxylase 65 (GAD-65) peptide, p524-543, as a specific, possibly low affinity, stimulus for the spontaneously arising, diabetogenic T cell clone BDC2.5. Interestingly, BDC2.5 T cells, which normally are unresponsive to p524-543 stimulation, react to the peptide when provided with splenic APC obtained from mice immunized with the same peptide, p524-543, but not, for example, with hen egg white lysozyme. Immunization with p524-543 increases the susceptibility of the NOD mice to type 1 diabetes induced by the adoptive transfer of BDC2.5 T cells. In addition, very few CFSE-labeled BDC2.5 T cells divide in the recipient's pancreas after transfer into a transgenic mouse that overexpresses GAD-65 in B cells, whereas they divide vigorously in the pancreas of normal NOD recipients. A special relationship between the BDC2.5 clone and the GAD-65 molecule is further demonstrated by generation of a double-transgenic mouse line carrying both the BDC2.5 TCR and GAD-65 transgenes, in which a significant reduction of BDC2.5 cells in the pancreas has been observed, presumably due to tolerance induction. These data suggest that unique and/or altered processing of self Ags may play an essential role in the development and expansion of autoreactive T cells.