Type 1 Diabetes (T1D) is an organ-specific autoimmune disease in which T cells are major drivers, leading to the destruction of beta-cells and insulin deficiency. Although the B-chain of insulin is a critical target for islet-reactive CD4 T cells, the native sequence is poorly antigenic and in vivo, insulin elicits only a weak Th1 response. We previously reported that post-translational modification of proinsulin through hybrid insulin peptide (HIP) formation leads to the synthesis of potent epitopes for islet-reactive CD4 T cells, and that cathepsin D (catD), a protease present in beta-cells, can mediate the formation of HIPs through a transpeptidation process. In the present study, we defined the specificity of catD for the insulin B-chain and show a high specificity for proteolytic cleavage following the disease-critical tyrosine 16 residue. We therefore hypothesized that HIP formation occurs at this amino acid. We designed a panel of HIPs containing insB9–16 fragments linked to different secretory granule proteins and challenged splenocytes isolated from diabetic NOD mice with insB9–16 HIPs; ELISPOT analysis showed elevated inflammatory responses to several peptides. Among this panel of HIPs, we identified the 2.40HIP, a fusion peptide between insB9–16 and the C-peptide, as a potent epitope recognized by PD12-2.40, a diabetogenic CD4 T cell clone isolated from the islets of a 12-week-old NOD mouse. These results define a new antigenic region of the insulin B:9–23 peptide and demonstrate that this sequence of the B-chain becomes dramatically more antigenic when post-translationally modified through HIP formation.

Background: Type 1 diabetes (T1D) is an autoimmune disease where autoreactive T cells infiltrate pancreatic islets, resulting in beta-cell destruction. Antigen-specific immunotherapy with tolerogenic peptides to induce peripheral tolerance has shown promise in preclinical studies but has not shown clinical efficacy. Methods: Here, we develop peptide-nanobubbles (NBs) as an image-guided platform for induction of peripheral immune tolerance in mouse models of T1D. Sub-micron sized NB ultrasound contrast agents can passively accumulate in pancreatic islets of non-obese diabetic (NOD) mice during the development of diabetes as a result of increased microvascular permeability. We incorporated an insulin B:9-23 peptide mimotope into NBs to target peptides to pancreatic islets for expansion of islet-resident regulatory T cells. Results: NBs retained normal size distribution and acoustic properties following peptide incorporation. Peptide-NBs accumulated in islets of NOD mice and this accumulation could be visualized in real time using contrast enhanced ultrasound. This resulted in an increased proportion of islet insulin-reactive regulatory T cells. Further, peptide-NBs prepared with a hybrid insulin peptide (HIP) expanded islet HIP-reactive regulatory T cells and substantially delayed diabetes onset in an adoptive transfer mouse model of autoimmune diabetes. Conclusions: Peptide-NBs offer a promising 'theranostic' approach for induction of peripheral tolerance in T1D while monitoring delivery and action via ultrasound contrast.

Pancreatic islet transplantation offers a potential cure for autoimmune type 1 diabetes (T1D) but requires immunosuppression to prevent recurrent, T cell-mediated autoimmunity. Antigen-specific immunotherapy is a targeted alternative that induces peripheral tolerance in autoreactive T cells without broad immunosuppression. Tolerance induction with antigen-specific therapies may induce dominant tolerance, extending to T cell clones other than those targeted by the therapy. However, the mechanism of this suppression remains unclear. Using pancreatic islet transplantation in NOD mice, we show that induction of tolerance to a CD4(+) T cell hybrid insulin peptide neoepitope reprograms the fate of antigen-specific CD8(+) T cells within the grafts. Induction of tolerance to a single hybrid insulin peptide transiently protected islet grafts from autoimmune destruction, and graft survival could be extended with continued therapeutic dosing. Treatment limited a cytolytic/interferon-stimulated differentiation program in antigen-specific CD8(+) T cells and reduced TCR avidity within grafts. This was mediated in part by IL-10-producing regulatory CD4(+) T cells that suppressed dendritic cell activation. IL-10 blockade reversed these effects, restoring CD8(+) T cell differentiation, as well as the licensing of dendritic cells. These findings reveal an IL-10-dependent mechanism by which CD4(+) T cell-targeted immunotherapy restrains pathogenic CD8(+) T cell fates through the suppression of dendritic cells in islet grafts.

In this review, we cover the discovery of hybrid insulin peptides (HIPs) as antigens for CD4 T cells involved in pathogenesis and regulation of autoimmune diabetes. HIPs represent a unique posttranslational modification in autoimmunity and consist of peptide sequences from two beta-cell proteins, one being proinsulin, covalently joined to form new nongenomic peptides. Using the nonobese diabetic (NOD) mouse model, we showed that HIPs are target antigens for a panel of diabetogenic CD4 T-cell clones. The prototype clone of this panel is BDC-2.5, and the first HIP identified was the peptide ligand for BDC-2.5, the 2.5HIP, consisting of an insulin C-peptide fragment combined with a natural cleavage product of chromogranin A. T cells with different TCRs, all specific for the 2.5HIP, were shown to be a dominant population among the T cells infiltrating the islets of NOD mice. T cells reactive to HIPs are significantly elevated in the PBMC of newly diagnosed patients with type 1 diabetes (T1D) and in at-risk subjects, an important finding from a clinical standpoint. When coupled to biodegradable nanoparticles (NPs), HIPs can serve as epitopes to induce antigen-specific tolerance. 2.5HIP NPs not only prevent transfer of disease by BDC-2.5 T cells but also prolong islet graft survival in diabetic NOD mice. Investigation of the mechanisms underlying 2.5HIP NP-induced tolerance revealed that protection occurs through an IL-10-dependent process in which regulatory T cells in the graft tissue are increased, limiting dendritic cell licensing and the subsequent terminal differentiation of both CD4 and CD8 islet-specific T cells.

Persistent antigen exposure during chronic viral infection and tumor development drives CD8 T cells into an exhausted, hypofunctional state. Understanding the molecular pathways that enforce T-cell exhaustion is critical for improving current immunotherapies. Previously, we have shown the bioactive lipid lysophosphatidic acid (LPA) regulates CD8 T-cell function through LPA receptor 5 (LPAR5) signaling, including demonstrating that Lpar5-/- CD8 T cells exhibit enhanced tumor clearance in murine models of melanoma. Importantly, significantly elevated levels of LPA have been identified in individuals with different cancers and persistent viral infections such as HIV, hepatitis C virus, and hepatitis B virus. To investigate the role of Lpar5 in the differentiation and maintenance of exhausted CD8 T cells, we utilized the lymphocytic choriomeningitis virus (LCMV) infection model. In response to infection with LCMV Clone 13, but not Armstrong, one-quarter of Lpar5-/- animals succumbed to infection, and this was accompanied by an increased frequency of LCMV-specific Lpar5-/- CD8 T cells maintained in a less terminally exhausted state. Using P14 transgenic mice, we demonstrate that Lpar5 acts in a cell-intrinsic and temporal manner to regulate CD8 T-cell accumulation and exhaustion programming during Clone 13 infection. The enhanced accumulation of Lpar5-/- P14 cells during the acute phase of Clone 13 infection appears to be regulated by Lpar5-mediated changes in T-cell survival and not through trafficking or proliferation. RNA sequencing analyses and surface phenotyping show that Lpar5 likely regulates CD8 T-cell exhaustion through modulation of NK receptor expression, including the CD94/NKG2A inhibitory axis.

Type 1 diabetes (T1D) is an autoimmune disease wherein insulin-producing beta cells are destroyed by self- reactive T cells, but how loss of tolerance to beta cells occurs is poorly understood. Increasing evidence in mice and humans has shown that post-translational fusion of insulin with beta cell secretory granule proteins produces a class of neo-self-antigens termed Hybrid Insulin Peptides (HIPs). We hypothesize that islet- infiltrating CD4+ T cells react to a new class of HIPs containing sequences from the B-chain of insulin (B-chain HIPs). We carried out antigen discovery using signaling and antigen-presenting bifunctional receptors (SABRs) developed by the Joglekar lab on islet-infiltrating CD4+ T cell isolated from non-obese diabetic (NOD) mice. Here, we show that multiple highly expanded CD4+ T clones specific for unmodified insulin B-chain are also cross-reactive with B-chain HIPs and more specifically, we found that several intra-islet, clonally-expanded CD4+ T cells were reactive to a novel B-chain/Calreticulin HIP. Using an I-Ag7 tetramer loaded with the B- chain/Calreticulin HIP, we detected tetramer-positive CD4+ T cells in islets and pancreatic lymph nodes of NOD mice displaying an antigen-experienced, effector phenotype. Currently, we are using tetramers paired with scRNAseq to determine the TCR repertoire of B-chain HIP-reactive T cells, explore their transcriptional phenotypes within the islets, and investigate their diabetogenic potential. JDRF (Breakthrough T1D) 3-SRA-2023-1354-S-B; Training Grant (Autoimmunity and Immunopathology) 5T32AI089443-15 Basic Autoimmunity (BA)

CD1 is an antigen presenting glycoprotein homologous to MHC I; however, CD1 proteins present lipid rather than peptide antigen. CD1 proteins are well established to present lipid antigens of Mycobacterium tuberculosis (Mtb) to T cells, but understanding the role of CD1-restricted immunity in vivo in response to Mtb infection has been limited by availability of animal models naturally expressing the CD1 proteins implicated in human response: CD1a, CD1b and CD1c. Guinea pigs, in contrast to other rodent models, express four CD1b orthologs, and here we utilize the guinea pig to establish the kinetics of gene and protein expression of CD1b orthologs, as well as the Mtb lipid-antigen and CD1b-restricted immune response at the tissue level over the course of Mtb infection. Our results indicate transient upregulation of CD1b expression during the effector phase of adaptive immunity that wanes with disease chronicity. Gene expression indicates that upregulation of CD1b is the result of transcriptional induction across all CD1b orthologs. We show high CD1b3 expression on B cells, and identify CD1b3 as the predominant CD1b ortholog in pulmonary granuloma lesions. We identify ex vivo cytotoxic activity directed against CD1b that closely paralleled the kinetic changes in CD1b expression in Mtb infected lung and spleen. This study confirms that CD1b expression is modulated by Mtb infection in lung and spleen, leading to pulmonary and extrapulmonary CD1b-restricted immunity as a component of the antigen-specific response to Mtb infection.

There is accumulating evidence that pathogenic T cells in T1D recognize epitopes formed by post-translational modifications of β-cell antigens, including hybrid insulin peptides (HIPs). The ligands for several CD4 T-cell clones derived from the NOD mouse are HIPs composed of a fragment of proinsulin joined to peptides from endogenous β-cell granule proteins. The diabetogenic T-cell clone BDC-6.9 reacts to a fragment of C-peptide fused to a cleavage product of pro-islet amyloid polypeptide (6.9HIP). In this study, we used a monoclonal antibody (MAb) to the 6.9HIP to determine when and where HIP antigens are present in NOD islets during disease progression and with which immune cells they associate. Immunogold labeling of the 6.9HIP MAb and organelle-specific markers for electron microscopy were employed to map the subcellular compartment(s) in which the HIP is localized within β-cells. While the insulin B9-23 peptide was present in nearly all islets, the 6.9HIP MAb stained infiltrated islets only in NOD mice at advanced stages of T1D development. Islets co-stained with the 6.9HIP MAb and antibodies to mark insulin, macrophages, and dendritic cells indicate that 6.9HIP co-localizes within insulin-positive β-cells as well as intra-islet antigen-presenting cells (APCs). In electron micrographs, the 6.9HIP co-localized with granule structures containing insulin alone or both insulin and LAMP1 within β-cells. Exposing NOD islets to the endoplasmic reticulum (ER) stress inducer tunicamycin significantly increased levels of 6.9HIP in subcellular fractions containing crinosomes and dense-core granules (DCGs). This work demonstrates that the 6.9HIP can be visualized in the infiltrated islets and suggests that intra-islet APCs may acquire and present HIP antigens within islets.

An autoimmune-mediated death of insulin-producing beta cells, orchestrated by effector CD4 and CD8 T cells that recognize islet antigens, results in Type 1 Diabetes. A dominant CD4 islet autoantigen in the NOD mouse model of autoimmune diabetes is a neoepitope termed the 2.5 Hybrid Insulin Peptide (2.5HIP). By delivering the 2.5HIP as an antigen-specific immunotherapy on tolerogenic PLG-nanoparticles, islet grafts in diabetic NOD mice survived longer and cytokine production in autoreactive CD4 and CD8 T cells was suppressed, including the well-studied IGRP tetramer+ (tet+) CD8 T cells. Using both diabetic transplant recipients and prediabetic NOD mice treated with 2.5HIP nanoparticles, we examined mechanisms of peripheral tolerance induction to a dominant CD4 neoepitope and the impact on the function of IGRP CD8 T cells. Following induction of tolerance to the 2.5HIP, there was an increase in dysfunctional surface marker expression (PD1+ TIM3+) on effector 2.5HIP tet+ CD4 T cells in the spleen of treated mice. Along with a decrease in effector function, we observed an increase in the fraction of both Treg and Tr1 2.5HIP tet+ T cells expressing IL10 in the islets, spleen, and draining lymph nodes of tolerized mice. Concurrently, IGRP tet+ CD8 T cells accumulated in the draining lymph nodes and were less able to traffic and infiltrate islets. Of the IGRP tet+ cells that entered islets, there were fewer cytolytic CX 3CR1 +effector cells in tolerized mice. The robust functional increase in both Treg and Tr1 2.5HIP tet+ T cells may provide a potential mechanism for the accumulation of IGRP tet+ CD8 T cells in the draining lymph node and explain their inefficient trafficking into, and function within, islets following tolerance induction. NIH - T32 5T32DK120520-03, R01 2R01DK081166-11) JDRF 2-SRA-2020-907-S-B

Type 1 diabetes is the culmination of T cell mediated autoimmunity resulting in the destruction of insulin-producing beta cells. Islet-derived hybrid insulin peptides (HIPs), recognized by autoreactive CD4 T cells, are likely dominant T cell epitopes in the NOD mouse model of autoimmune diabetes. The 2.5HIP is formed by the fusion of a fragment of insulin C-peptide with a natural cleavage product of chromogranin A, generating a neoepitope. When the 2.5HIP was delivered as an immunotherapy on tolerance inducing PLG-nanoparticles, islet grafts functioned longer in transplanted, diabetic NOD mice. Tolerance induction to this single neoepitope induced dysfunctional cytokine production in autoreactive CD4 and CD8 T cells specific for other known islet antigens. Here we used both prediabetic NOD mice and diabetic transplant recipients to examine mechanisms of peripheral tolerance induction to a HIP. Using the Tiger IL10-GFP reporter mouse, we observed a substantial increase in the proportion of (CD25+) Treg and (CD25-) Tr1 2.5HIP tetramer+ CD4 T cells that expressed IL10 in the islets, spleen, and draining lymph node of 2.5HIP nanoparticle treated mice. Autoreactive, IGRP tetramer+ CD8 T cells displayed a trafficking defect and accumulated in secondary lymphoid tissue with a concomitant reduction within islets. The proportion of cytolytic, CX3CR1+ IGRP tetramer+ CD8 T cells that infiltrated islets was also reduced. IL10 generated by both Treg and Tr1 2.5HIP tet+ T cells may directly suppress IGRP tetramer+ CD8 T cells or suppress an intermediate cell type such as a dendritic cell, causing CD8 T cell dysfunction. Examining how antigen-specific immunotherapies suppress multiple autoreactive T cells specificities may help inform future therapies without the need for immunosuppression. Disclosure J.E.Dilisio: None. R.L.Baker: None. K.M.Haskins: None. Funding National Institutes of Health (5T32DK120520-03, 2R01DK081166-11); JDRF (2-SRA-2020-907-S-B)

Type 1 diabetes is the culmination of T cell mediated autoimmunity resulting in the destruction of insulin-producing beta cells. Islet-derived hybrid insulin peptides (HIPs), recognized by autoreactive CD4 T cells, are likely dominant T cell epitopes in the NOD mouse model of autoimmune diabetes. The 2.5HIP is formed by the fusion of a fragment of insulin C-peptide with a natural cleavage product of chromogranin A, generating a neoepitope. When the 2.5HIP was delivered as an immunotherapy on tolerance inducing PLG-nanoparticles, islet grafts functioned longer in transplanted, diabetic NOD mice. Tolerance induction to this single neoepitope induced dysfunctional cytokine production in autoreactive CD4 and CD8 T cells specific for other known islet antigens. Here we used both prediabetic NOD mice and diabetic transplant recipients to examine mechanisms of peripheral tolerance induction to a HIP. Using the Tiger IL10-GFP reporter mouse, we observed a substantial increase in the proportion of (CD25+) Treg and (CD25-) Tr1 2.5HIP tetramer+ CD4 T cells that expressed IL10 in the islets, spleen, and draining lymph node of 2.5HIP nanoparticle treated mice. Autoreactive, IGRP tetramer+ CD8 T cells displayed a trafficking defect and accumulated in secondary lymphoid tissue with a concomitant reduction within islets. The proportion of cytolytic, CX3CR1+ IGRP tetramer+ CD8 T cells that infiltrated islets was also reduced. IL10 generated by both Treg and Tr1 2.5HIP tet+ T cells may directly suppress IGRP tetramer+ CD8 T cells or suppress an intermediate cell type such as a dendritic cell, causing CD8 T cell dysfunction. Examining how antigen-specific immunotherapies suppress multiple autoreactive T cells specificities may help inform future therapies without the need for immunosuppression. Disclosure J.E.Dilisio: None. R.L.Baker: None. K.M.Haskins: None. Funding National Institutes of Health (5T32DK120520-03, 2R01DK081166-11); JDRF (2-SRA-2020-907-S-B)

AbstractCD1 is an antigen presenting glycoprotein homologous to MHC I; however, CD1 proteins present lipid rather than peptide antigen. CD1 proteins are well established to present lipid antigens ofMycobacterium tuberculosis(Mtb) to T cells, but understanding the role of CD1-restricted immunityin vivoin response to Mtb infection has been limited by availability of animal models naturally expressing the CD1 proteins implicated in human response: CD1a, CD1b and CD1c. Guinea pigs, in contrast to other rodent models, express four CD1b orthologs, and here we utilize the guinea pig to establish the kinetics of gene and protein expression of CD1b orthologs, as well as the Mtb lipid-antigen and CD1b-restricted immune response at the tissue level over the course of Mtb infection. Our results indicate transient upregulation of CD1b expression during the effector phase of adaptive immunity that wanes with disease chronicity. Gene expression indicates that upregulation of CD1b is the result of transcriptional induction across all CD1b orthologs. We show high CD1b3 expression on B cells, and identify CD1b3 as the predominant CD1b ortholog in pulmonary granuloma lesions. We identifyex vivocytotoxic activity directed against CD1b that closely paralleled the kinetic changes in CD1b expression in Mtb infected lung and spleen. This study confirms that CD1b expression is modulated by Mtb infection in lung and spleen, leading to pulmonary and extrapulmonary CD1b-restricted immunity as a component of the antigen-specific response to Mtb infection.

Abstract Autoreactive T cells are thought to drive autoimmune diabetes by recognizing beta cell-derived peptide antigens. We previously discovered that hybrid insulin peptides (HIPs) are potent neoantigen peptide ligands for a subset of autoreactive CD4 T cells in both the NOD mouse model and human type 1 diabetes patients. Inducing tolerance to prominent T cell autoantigens through antigen-specific immunotherapy could prevent disease onset or recurrence after islet transplantation without the need for broad immunosuppression. Here we show that tolerogenic nanoparticle (NP) delivery of the 2.5HIP, a dominant CD4 T cell HIP epitope in the NOD mouse, can prolong islet graft survival in transplanted diabetic NOD mice. Tolerance induction to the 2.5HIP not only suppressed 2.5HIP tetramer+ CD4 T cells but also autoreactive CD4 and CD8 T cells specific for different islet antigens. 2.5HIP NP treatment induced a dysfunctional state in graft-infiltrating T cells characterized by increased expression of anergic markers on CD4 T cells and reduced inflammatory cytokine production in 2.5HIP tetramer+ CD4 T cells as well as IGRP (islet specific glucose-6-phosphatase catalytic subunit-related protein) tetramer+ CD8 T cells. In conclusion, we demonstrate that antigen-specific immunotherapy aimed at inducing tolerance to a single CD4 T cell HIP epitope can prolong islet graft survival and suppress autoreactive CD8 T cells in the NOD mouse model of autoimmune diabetes. Supported by grants from NIH (R01 DK122566, R01 DK081166, T32 DK120520) and JDRF (2-SRA-2018-566-S-B)

Bacillary dysentery, caused by Shigella bacteria, is a major enteric disease responsible for over 200 million infections annually with 650,000 fatal cases. Due to its high communicability, improvement of hygienic standards alone should reduce the spread of dysentery. However, such measures are expensive, and in the communities (e.g. penitentiaries and asylums) or in the areas of the world where bacillary dysentery is most frequently encountered (e.g. in the developing countries) they are not likely to take effect in the reasonably near future. Therefore the possibility of other preventive means such as anti-dysentery vaccines have been explored over the past 40 years. Recently, increased understanding of the molecular biology of bacillary dysentery and the possibility of designing well characterized vaccine strains have increased interest in the field. Several promising vaccine candidates are at various levels of investigations, but to date no Shigella vaccines are available for public health purposes. In this review, beyond the relevant basic information about the pathology, pathomechanism and molecular biology of bacillary dysentery, the various approaches and strategies to construct a safe and immunogenic anti-dysentery vaccine are critically discussed.

The induction of antigen (Ag)-specific tolerance and replacement of islet β-cells are major ongoing goals for the treatment of Type 1 Diabetes (T1D). Our group previously showed that a hybrid insulin peptide (2.5HIP) is a critical autoantigen for diabetogenic CD4+ T cells in the non-obese diabetic (NOD) mouse model. In this study, we investigated whether induction of Ag-specific tolerance using 2.5HIP-coupled tolerogenic nanoparticles (NPs) could protect diabetic NOD mice from disease recurrence upon syngeneic islet transplantation. Islet graft survival was significantly prolonged in mice treated with 2.5HIP NPs, but not NPs containing the insulin B chain peptide 9-23. Protection in 2.5HIP NP-treated mice was attributed both to the simultaneous induction of anergy in 2.5HIP-specific effector T cells and to the expansion of Foxp3+ regulatory T cells specific for the same antigen. Notably, our results indicate that effector function of graft-infiltrating CD4+ and CD8+ T cells specific for other β-cell epitopes was significantly impaired, suggesting a novel mechanism of therapeutically induced linked suppression. This work establishes that tolerance induction with a hybrid insulin peptide can delay recurrent autoimmunity in NOD mice, which could inform the development of an Ag-specific therapy for T1D.

Insulin is considered to be a key antigenic target of T cells in Type 1 Diabetes (T1D) and autoimmune diabetes in the NOD mouse with particular focus on the B-chain amino acid sequence B:9-23 as the primary epitope. Our lab previously discovered that hybrid insulin peptides (HIPs), comprised of insulin C-peptide fragments fused to other β-cell granule peptides, are ligands for several pathogenic CD4 T cell clones derived from NOD mice and for autoreactive CD4 T cells from T1D patients. A subset of CD4 T cell clones from our panel react to insulin and B:9-23 but only at high concentrations of antigen. We hypothesized that HIPs might also be formed from insulin B-chain sequences covalently bound to other endogenously cleaved ß-cell proteins. We report here on the identification of a B-chain HIP, termed the 6.3HIP, containing a fragment of B:9-23 joined to an endogenously processed peptide of ProSAAS, as a strong neo-epitope for the insulin-reactive CD4 T cell clone BDC-6.3. Using an I-A g7 tetramer loaded with the 6.3HIP, we demonstrate that T cells reactive to this B-chain HIP can be readily detected in NOD mouse islet infiltrates. This work suggests that some portion of autoreactive T cells stimulated by insulin B:9-23 may be responding to B-chain HIPs as peptide ligands.

Prions are infectious proteins causing fatal, transmissible neurodegenerative diseases of animals and humans. Replication involves template-directed refolding of host encoded prion protein, PrPC, by its infectious conformation, PrPSc. Following its discovery in captive Colorado deer in 1967, uncontrollable contagious transmission of chronic wasting disease (CWD) led to an expanded geographic range in increasing numbers of free-ranging and captive North American (NA) cervids. Some five decades later, detection of PrPSc in free-ranging Norwegian (NO) reindeer and moose marked the first indication of CWD in Europe. To assess the properties of these emergent NO prions and compare them with NA CWD we used transgenic (Tg) and gene targeted (Gt) mice expressing PrP with glutamine (Q) or glutamate (E) at residue 226, a variation in wild type cervid PrP which influences prion strain selection in NA deer and elk. Transmissions of NO moose and reindeer prions to Tg and Gt mice recapitulated the characteristic features of CWD in natural hosts, revealing novel prion strains with disease kinetics, neuropathological profiles, and capacities to infect lymphoid tissues and cultured cells that were distinct from those causing NA CWD. In support of strain variation, PrPSc conformers comprising emergent NO moose and reindeer CWD were subject to selective effects imposed by variation at residue 226 that were different from those controlling established NA CWD. Transmission of particular NO moose CWD prions in mice expressing E at 226 resulted in selection of a kinetically optimized conformer, subsequent transmission of which revealed properties consistent with NA CWD. These findings illustrate the potential for adaptive selection of strain conformers with improved fitness during propagation of unstable NO prions. Their potential for contagious transmission has implications for risk analyses and management of emergent European CWD. Finally, we found that Gt mice expressing physiologically controlled PrP levels recapitulated the lymphotropic properties of naturally occurring CWD strains resulting in improved susceptibilities to emergent NO reindeer prions compared with over-expressing Tg counterparts. These findings underscore the refined advantages of Gt models for exploring the mechanisms and impacts of strain selection in peripheral compartments during natural prion transmission.

Autoreactive T cells are thought to orchestrate the onset and progression of autoimmune diabetes. Key cognate antigens of these diabetogenic T cells include hybrid insulin peptides, formed by the fusion of insulin fragments to cleavage products of other β-cell granule proteins. Here we review initial work exploring tolerance induction to a hybrid insulin peptide using a biodegradable, nanoparticle delivery system in non-obese diabetic (NOD) mice. The immune phenotype(s) and possible mechanism(s) behind antigen-specific tolerance induction were dissected with a disease transfer model using transgenic autoreactive mouse T cells. Treatment of NOD mice with peptide-coupled nanoparticles appeared to have a dual function in preventing diabetes onset, inducing anergy in effector T cells and enhancing the activity of regulatory T cells. Importantly, the ratio of these two cell types in the pancreas was pushed toward tolerance. Antigen-specific tolerance induction to hybrid insulin peptides has the translational potential to preserve islet β-cells in new-onset or at-risk patients and prevent recurrent autoimmunity in transplant patients.

ABSTRACT IMPACT: Examining lipid immunity for Mycobacterium tuberculosis in a translatable Guinea pig model may serve as a critical foundation for the creation of an efficacious human lipid based vaccine against tuberculosis. OBJECTIVES/GOALS: CD1 is a group of glycoproteins on antigen-presenting cells (APCs) that present lipid antigens to T cells. Mycobacterium tuberculosis (Mtb) has a lipid-rich cell wall which is essential for the pathogenesis of tuberculosis. Our goal is to determine the frequency, phenotypes, and functionality of CD1 T cells against Mtb using the guinea pig model. METHODS/STUDY POPULATION: Guinea pigs serve as the best translational model for CD1 immunology as they have both group 1 and group 2 CD1 complexes, comparable to human CD1. We performed ex-vivo and in-vivo experiments to analyze lipid antigen-specific CD1 T cell responses with Mtb infection. Assays to detect lipid-specific CD1 T cell activation include cellular proliferation, cytotoxicity assays, and interferon-gamma (IFNγ) release assay (Elispot) using both synthetic and Mtb-derived lipids. We isolated and characterized CD1 T cells using tetramerized CD1 complexes loaded with specific Mtb lipids. Spatial interaction between lipid loaded CD1 APCs with CD1 T cells were demonstrated by immunohistochemistry (IHC). Lastly, we will investigate the impact of lipid-based immunology via knockdown and overexpression of CD1 complexes. RESULTS/ANTICIPATED RESULTS: The cytotoxicity assay demonstrated that the CD1b1 and CD1b3 complexes play roles in the presentation of Mtb lipids, specifically glucose monomycolate, and mycolic acid, as noted by T cell killing of fibroblasts that express specific CD1 complexes that can present Mtb lipids. Similarly, cellular proliferation exhibited lipid specific T cell proliferation. IFNγ production by the stimulated CD1-restricted T cells (Elispot) was weak indicating CD1 T cells may not produce IFNγ. IHC successfully showed CD1 APCs in lungs and spleens of infected guinea pigs. It is anticipated that knocking out CD1 expression will lead to impaired immunity, and increase severity of disease as noted by pathologic lesions/bacterial burden, and systemic spread; in contrast, CD1 enhancement will limit the severity of tuberculosis. DISCUSSION/SIGNIFICANCE OF FINDINGS: We characterized CD1 T cells in infected guinea pigs at the tissue level, demonstrating Mtb lipid immunology. As a result, we laid the groundwork for investigating whether augmenting lipid immunity in the guinea pig model will enhance immunity against tuberculosis. Fruition of such work may lead to the development of effective tuberculosis vaccines.