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Thymic presentation of self-antigens is critical for establishing a functional yet self-tolerant T-cell population. Hybrid peptides formed through transpeptidation within pancreatic β-cell lysosomes have been proposed as a new class of autoantigens in type 1 diabetes (T1D). While the production of hybrid peptides in the thymus has not been explored, due to the nature of their generation, it is thought to be highly unlikely. Therefore, hybrid peptide-reactive thymocytes may preferentially escape thymic selection and contribute significantly to T1D progression. Using an antibody-peptide conjugation system, we targeted the hybrid insulin peptide (HIP) 2.5HIP toward thymic resident Langerin-positive dendritic cells to enhance thymic presentation during the early neonatal period. Our results indicated that anti–Langerin-2.5HIP delivery can enhance T-cell central tolerance toward cognate thymocytes in NOD.BDC2.5 mice. Strikingly, a single dose treatment with anti–Langerin-2.5HIP during the neonatal period delayed diabetes onset in NOD mice, indicating the potential of antibody-mediated delivery of autoimmune neoantigens during early stages of life as a therapeutic option in the prevention of autoimmune diseases.

Thymic presentation of self-antigens is critical for establishing a functional yet self-tolerant T cell population. Hybrid peptides formed through transpeptidation within pancreatic beta cell lysosomes have been proposed as a new class of autoantigens in Type 1 Diabetes (T1D). While the production of hybrid peptides in the thymus has not been explored, due to the nature of their generation, it is thought to be highly unlikely. Therefore, hybrid peptide-reactive thymocytes may preferentially escape thymic selection and contribute significantly to T1D progression. Using an antibody-peptide conjugation system, we targeted the 2.5HIP hybrid peptide towards thymic resident Langerin+ dendritic cells to enhance thymic presentation during the early neonatal period. Our results indicated that anti-Langerin-2.5HIP delivery can enhance T cell central tolerance toward cognate thymocytes in NOD.BDC2.5 mice. Strikingly, a single dose treatment with anti-Langerin-2.5HIP during neonatal period delayed diabetes onset in NOD mice, indicating the potential of antibody-mediated delivery of autoimmune neo-antigens during early stages of life as a therapeutic option in the prevention of autoimmune diseases.

The contribution of low affinity T cells to autoimmunity in the context of polyclonal T cell responses is understudied due to the limitations in their capture by tetrameric reagents and low level of activation in response to antigenic stimulation. As a result, low affinity T cells are often disregarded as non-antigen specific cells irrelevant to the immune response. Our study aimed to assess how the level of self-antigen reactivity shapes T cell lineage and effector responses in the context of spontaneous tissue specific autoimmunity observed in NOD mice. Using multi-color flow cytometry in combination with Nur77(GFP) reporter of TCR signaling we identified a dormant population of T cells that infiltrated the pancreatic islets of pre-diabetic NOD mice, which exhibited reduced level of self-tissue reactivity based on expression of CD5 and Nur77(GFP). We showed that these CD5(low) T cells had a unique TCR repertoire, exhibited low activation and minimal effector function; however, induced rapid diabetes upon transfer. The CD4(+)CD5(low) T cell population displayed transcriptional signature of central memory T cells, consistent with the ability to acquire effector function post-transfer. Transcriptional profile of CD5(low) T cells was similar to T cells expressing a low affinity TCR, indicating TCR affinity to be the important factor in shaping CD5(low) T cell phenotype and function at the tissue site. Overall, our study suggests that autoimmune tissue can maintain a reservoir of undifferentiated central memory-like autoreactive T cells with pathogenic effector potential that might be an important source for effector T cells during long-term chronic autoimmunity.

Accumulating evidence supports a critical role for posttranslationally modified (PTM) islet neoantigens in type 1 diabetes. However, our understanding regarding thymic development and peripheral activation of PTM autoantigen-reactive T cells is still limited. Using HLA-DR4 humanized mice, we observed that deamidation of GAD65115–127 generates a more immunogenic epitope that recruits T cells with promiscuous recognition of both the deamidated and native epitopes and reduced frequency of regulatory T cells. Using humanized HLA/T-cell receptor (TCR) mice, we observed that TCRs reactive to the native or deamidated GAD65115–127 led to efficient development of CD4+ effector T cells; however, regulatory T-cell development was reduced in mice expressing the PTM-reactive TCR, which was partially restored with exogenous PTM peptide. Upon priming, both the native-specific and the deamidated-specific T cells accumulated in pancreatic islets, suggesting that both specificities can recognize endogenous GAD65 and contribute to anti–β-cell responses. Collectively, our observations in polyclonal and single TCR systems suggest that while effector T-cell responses can exhibit cross-reactivity between native and deamidated GAD65 epitopes, regulatory T-cell development is reduced in response to the deamidated epitope, pointing to regulatory T-cell development as a key mechanism for loss of tolerance to PTM antigenic targets.

Accumulating evidence supports a critical role for post-translationally modified (PTM) islet neoantigens in type 1 diabetes. However, our understanding regarding thymic development and peripheral activation of PTM autoantigen reactive T cells is still limited. Using HLA-DR4 humanized mice, we observed that deamidation of GAD65115-127 generates a more immunogenic epitope that recruits T cells with promiscuous recognition of both the deamidated and native epitopes, and reduced frequency of regulatory T cells. Using humanized HLA/TCR mice we observed that TCRs reactive to the native or deamidated GAD65115-127 led to efficient development of CD4+ effector T cells; however, regulatory T cell development was reduced in mice expressing the PTM reactive TCR, which was partially restored with exogenous PTM peptide. Upon priming, both the native specific and the deamidated specific T cells accumulated in pancreatic islets, suggesting that both specificities can recognize endogenous GAD65 and contribute to anti-beta-cell responses. Collectively, our observations in polyclonal and single TCR systems suggest that while effector T cell responses can exhibit cross-reactivity between native and deamidated GAD65 epitopes, regulatory T cell development is reduced in response to the deamidated epitope, pointing to Treg development as a key mechanism for loss of tolerance to PTM antigenic targets.

Carbon nitride quantum dots (CNQDs) were embedded in the sodium carboxymethyl cellulose (CMC) matrix to form CNQDs-CMC film to explore the room temperature phosphorescence (RTP) of CNQDs, which suppress the non-radiative relaxation process due to the internal hydrogen bonding interactions between CMC and CNQDs. Then, a simple, inexpensive, background-free miniature device integrating with CNQDs-CMC film and smartphone was fabricated for rapid and quantitative detection of melamine (MEL). In the present of MEL, the yellow RTP color of the CNQDs-CMC film was quenched and photographed by the smartphone. The Color Recognizer APP in the smartphone recognized the red (R) value for quantitative detection of MEL. Thus, digital image colorimetry (DIC) determination of MEL was achieved due to the visible RTP color change of CNQDs-CMC film. The smartphone-based miniature device provided a promising platform for the on-site monitoring analytes in the complex matrix including food safety, environmental screening, health monitoring, and disease prevention.

T cells specific for post-translationally modified (PTM) islet antigens have been implicated as critical players in the development of type 1 diabetes. T cells reactive to the deamidated version of glutamic acid decarboxylase 65 (GAD65) 115-127 epitope accumulate in peripheral blood of patients as they progress to disease. However, knowledge regarding the development, activation, differentiation and islet recruitment of PTM reactive T cells is limited. Using retroviral gene delivery, we have developed a humanized TCR/HLA mouse model to assess the in vivo function of human TCRs reactive to either the wild type (GADWT) or the deamidated (GAD120E) GAD65115-127 epitope. Both TCRs supported efficient thymic selection; however, fewer regulatory T cells developed in mice expressing the PTM reactive TCR, which was partially rescued by the thymic delivery of GAD120E peptide. Immunization of immune sufficient polyclonal DR4.NOD mice with GAD120E/CFA elicited a stronger response compared to GADWT/CFA, confirming that tolerance to the wild type autoantigen is more robust. Interestingly, while islet infiltration in GAD120E single TCR mice was dependent on priming with the cognate PTM peptide, GADWT-TCR T cells could be induced to infiltrate islets after priming with either GADWT or GAD120E peptide, despite the vastly different functional affinity (EC50, GADWT: 0.047μM vs. GAD120E: ≥1.4μM). These findings suggest that the development and expansion of GAD65 PTM reactive CD4+ T cells are less restricted in peripheral lymphoid organs, but their recruitment to the tissue site is dependent on the availability of PTM antigen. Disclosure Y. Jing: None. Y. Kong: None. J. McGinty: None. G. Blahnik-Fagan: None. M. Bettini: None. E.A. James: None. M. Bettini: None. Funding National Institutes of Health (R01AI125301)

Regulatory T cells (Tregs) are critical in preventing autoimmunity and express a unique TCR repertoire with high affinity to self-antigens. However, whether Treg and effector T cell (Teff) TCR repertoires are similarly divergent in the context of autoimmunity, and whether Tregs are able to maintain TCR affinity advantage has not been clearly defined. Insulin tetramer positive Tregs isolated from pancreatic islets of NOD mice showed a higher level of self-reactivity (CD5) and TCR signaling (Nur77-GFP) than Teffs specific to the same epitope. We isolated and sequenced insulin tetramer+ Teffs and Tregs from islets of NOD mice expressing a fixed TCRα of an insulin specific TCR. Surprisingly, significant overlap was identified between Treg and Teff TCR repertoires, suggesting that similar TCRs can support autoimmune Treg and Teff development and recruitment into the autoimmune response. Both Tregs and Teffs exhibited TCRs with a broad range of reactivity to insulin epitope and Treg TCRs did not show increased reactivity. The disconnect between Nur77-GFP and TCR functional affinity suggests a Treg cell-intrinsic capacity to compete for antigen or to enhance downstream signaling. When re-expressed in vivo using the TCR retrogenic method, Treg derived TCRs showed lower Treg generation and collectively higher diabetogenicity. With comparable Treg generation, low affinity Treg TCR showed significantly lower protection against diabetes than high affinity Treg TCR. These findings indicate that in tissue specific autoimmunity Tregs can express a TCR repertoire with a broad range of functionality and have significant overlap with Teffs of the same specificity, which might negatively affect their suppressive capacity. This work was supported by the NIH (AI125301-01A1) and The Robert and Janice McNair Foundation.

A fluorometric and colorimetric dual-mode sensing platform based on graphitic carbon nitrite quantum dots (g-CNQDs) and Fe (II)-bathophenanthroline complex (BPS-Fe2+) was designed to the sensitive detection of nitrite (NO2−) in sausage and water. In this system, the fluorescence of g-CNQDs was quenched by BPS-Fe2+ complex due to the inner filter effect (IFE). When NO2− was present, Fe2+ was oxidized by nitrite to form BPS-Fe3+ complex with BPS, leading to the recovery of the fluorescence from g-CNQDs. Therefore, we constructed a “turn-off-on” fluorescence probe for detection of NO2−. Moreover, with the increase of NO2− concentration, the color of the solution changed from red to colorless, so the UV–vis measurements and on-site visual detection were realized. The method is capable of detecting NO2− in the concentration range of 2.32–34.8 μM with good selectivity and high sensitivity. In addition, the method has the potential to determine NO2− in water samples and sausage samples.

The ability to express and study a single T cell receptor (TCR) in vivo is an important aspect of both basic and translational immunological research. Traditionally, this was achieved by using TCR transgenic mice. In the past decade, a more efficient approach for single TCR expression was developed. This relatively rapid and accessible method utilizes retrovirus-mediated stem cell-based gene transfer and is commonly referred to as the TCR retrogenic approach. In this approach, hematopoietic bone marrow precursors are transduced with retroviral vector carrying both alpha and beta chains of a T cell receptor. After successful transduction, bone marrow is injected into recipient mice, in which T cell development is driven by expression of the vector-encoded TCR. This article details the materials and methods required to generate TCR retrogenic mice. It is divided into three sections and provides detailed methods for generation of stable retroviral producer cell lines, isolation and optimal transduction of hematopoietic bone marrow cells, and subsequent analysis of TCR retrogenic T cells. A detailed example of such analysis is provided. The current protocol is a culmination of many years of optimization and is the most efficient approach to date. Bone marrow transduction and transfer into recipient mice can now be achieved in a short period of four days. The protocol can be followed in most laboratories with standard biomedical equipment, and is supported by a troubleshooting guide that covers potential pitfalls and unexpected results. © 2019 by John Wiley & Sons, Inc.

Type 1 Diabetes (T1D) is a T cell-mediated autoimmune disease characterized by the immune destruction of pancreatic β cells. The search for natural equivalents to the mimetopes that can stimulate diabetogenic T cell clones from the Non-Obese Diabetic (NOD) mouse model of T1D led to the realization that hybrid peptides (HP) formed by the joining of β cell granule protein-derived peptide fragments are highly stimulatory to some clones. These hybrid peptides were later identified in vivo and proposed as a new class of post-translationally modified autoantigens mediating this disease. Recent data suggest high concentrations of these proteins in β cell lysosomes during granule turnover can lead to the hybridization of peptide fragments which may then be presented on MHC molecules. In the thymus, medullary thymic epithelial cells (mTECs) mediate T cell tolerance through promiscuous gene expression, but may not mediate HP-reactive T cell tolerance. We hypothesize that HP-reactive T cells preferentially escape the thymus and contribute significantly to T1D progression. We attempted to enhance T cell central tolerance to 2.5HIP, a hybrid insulin peptide formed from Proinsulin and Chromogranin A, the only two antigens known to be critical for T1D progression in the NOD mice, to better understand its impact on T1D incidence. Using an antibody-peptide conjugation system, we targeted 2.5HIP to a subset of thymic resident DCs to enhance presentation of 2.5HIP in early neonatal thymus. In neonatal BDC2.5 TCR-transgenic mice we found that a single dose of antibody-2.5HIP enhanced antigen-specific thymocyte apoptosis and regulatory T cell development. Importantly, a single dose in wild-type neonates resulted in a significant delay of diabetes onset.

Protein engineering has been successfully applied in protein drug discovery. Using this technology, we previously have constructed a fusion protein by linking the globular domain of adiponectin to the C-terminus of a glucagon-like peptide-1 (GLP-1) analog. Herein, to further improve its bioactivity, we reconstructed this fusion protein by introducing linker peptides of different length and flexibility. The reconstructed fusion proteins were overexpressed in Escherichia coli and purified using nickel affinity chromatography. Their agonist activity towards receptors of GLP-1 and adiponectin were assessed in vitro by using luciferase assay and AMP-activated protein kinase (AMPK) immunoblotting, respectively. The effects of the selected fusion protein on glucose and lipid metabolism were evaluated in mice. The fusion protein reconstructed using a linker peptide of AMGPSSGAPGGGGS showed high potency in activating GLP-1 receptor and triggering AMPK phosphorylation via activating the adiponectin receptor. Remarkably, the optimized fusion protein was highly effective in lowering blood glucose and lipids in mice. Collectively, these findings demonstrate that the bioactivity of this GLP-1 fusion protein can be significantly promoted by linker engineering, and indicate that the optimized GLP-1 fusion protein is a promising lead structure for anti-diabetic drug discovery.

Previously we constructed a fusion protein based on GLP-1 and globular adiponectin but unfortunately its yield was low because it was mainly expressed as inclusion bodies. Herein to optimize the soluble expression of this fusion protein we tried several fusion tag systems. Fusion tags, including GST-, Trx- and MBP-tag, greatly improved the soluble expression of the fusion protein. However, these tag-fusion proteins were aggregation-prone as judged by Native PAGE and gel filtration chromatography, and this aggregation reduced the specificity of enterokinase-mediated enzyme cleavage which was essential to remove the fusion tags. To improve the specificity of protein cleavage, we employed on-column cleavage for downstream purification. Finally using optimized expression followed by on-column cleavage, we obtained the product fusion protein with a yield of 1.2 mg per g wet bacterial cells which was 8-fold higher than before. This method improved the yield and simplified the process, and as a convenient method it can also be used for the preparation of other aggregation-prone proteins.

T cell infiltration and accumulation in pancreatic islets during autoimmune diabetes is largely dependent on TCR specificity for beta cell antigens. However, it is not fully clear whether the level of self-reactivity is an important determinant of T cell function in autoimmunity. We observed that CD5 expression is proportional to the level of self-reactivity based on Nur77-GFP reporter of TCR signaling and insulin tetramer staining of islet infiltrating T cells. Using RNAseq profiling, TCR sequencing, and cell transfer diabetes model we show that CD5 segregates functionally and transcriptionally distinct effector T cells. Transfer of CD5hi islet infiltrating T cells resulted in accelerated diabetes development compared to CD5lo cells (8 weeks vs. 11 weeks post transfer), suggesting that both T cell populations are able to re-infiltrate the pancreas and cause beta cell destruction, but CD5hi T cells exhibit increased pathogenicity. RNAseq analysis showed an increase in expression of Lag3 and FR4 in CD5hi cells, consistent with their increased activation and subsequent induction of regulatory and tolerogenic pathways. However, cell intrinsic negative regulators did not suppress IFNg production in CD5hi T cells, which was significantly higher compared to CD5lo cells. Our findings show that the strength of TCR reactivity to beta cell antigens is an important determinant of T cell pathogenicity in autoimmune diabetes.

Regulatory T cells (Tregs) have been shown to play a critical protective role in type 1 diabetes. However, TCR specificity of islet-infiltrating Tregs and its influence on T cell function during autoimmune diabetes is largely unknown. It has been shown that Tregs have a higher affinity for self and possess a unique TCR repertoire. Accordingly, we observed increased TCR signaling in islet-infiltrating insulin-tetramer labeled Tregs compared to effector T cells (Teffs), as measured by the Nur77-GFP reporter of TCR activation. In order to obtain the profile of Treg and Teff TCR repertoires specific for a single epitope, we isolated and sequenced insulin tetramer binding Teffs and Tregs from islets of NOD mice expressing a fixed alpha chain of an insulin specific TCR. Surprisingly, TCR sequence analysis showed relatively high similarity (Morisita-Horn index: 0.27) between Treg and Teff TCR repertoires, where a substantial portion (46.4%) of Tregs expressed shared TCRs. Collectively, our data suggest that Treg and Teff specific for a single epitope can express the same TCRs, which they exploit to exert different effector or suppressive functions.