Modulation of hyaluronan synthases and involvement of T cell-derived hyaluronan in autoimmune responses to transplanted islets.

The extracellular matrix glycosaminoglycan hyaluronan (HA) accumulates in human and mouse islets during the onset of autoimmune type 1 diabetes (T1D). HA plays a critical role in T1D pathogenesis, as spontaneous disease is blocked in mice fed the HA synthesis inhibitor 4-methylumbelliferone (4MU). The present study demonstrates the involvement of HA in T cell-mediated autoimmune responses to transplanted islets and in in vivo and in vitro T cell activation. Scaffolded islet implants (SIs) loaded with RIP-mOVA mouse islets expressing chicken ovalbumin (OVA) on their ß cells were grafted into T and B cell-deficient RIP-mOVA mice, which subsequently received CD4+ T cells from DO11.10 transgenic mice bearing OVA peptide-specific T cell receptors (TcRs), followed by injection of OVA peptide to induce an immune response to the OVA-expressing islets. By affinity histochemistry (AHC), HA was greatly increased in grafted islets with T cell infiltrates (compared to islets grafted into mice lacking T cells) and a portion of this HA co-localized with the infiltrating T cells. Transferred T cells underwent HA synthase (HAS) isoform switching - T cells isolated from the SI grafts strongly upregulated HAS1 and HAS2 mRNAs and downregulated HAS3 mRNA, in contrast to T cells from graft-draining mesenteric lymph nodes, which expressed HAS3 mRNA only. Expression of HAS1 and HAS2 proteins by T cells in SI infiltrates was confirmed by immunohistochemistry (IHC). DO11.10 mice fed 4MU had suppressed in vivo T cell immune priming (measured as a reduced recall response to OVA peptide) compared to T cells from control mice fed a normal diet. In co-cultures of naïve DO11.10 T cells and OVA peptide-loaded antigen-presenting cells (APCs), pre-exposure of the T cells (but not pre-exposure of APCs) to 4MU inhibited early T cell activation (CD69 expression). In addition, T cells exposed to 4MU during activation in vitro with anti-CD3/CD28 antibodies had inhibited phosphorylation of the CD3ζ subunit of the TcR, a very early event in TcR signaling. Collectively, our results demonstrate that T cell-derived HA plays a significant role in T cell immune responses, and that expression of T cell HAS isoforms changes in a locale-specific manner during in vivo priming and functional phases of the T cell response.

Hyaluronan synthesis inhibition impairs antigen presentation and delays transplantation rejection.

A coat of pericellular hyaluronan surrounds mature dendritic cells (DC) and contributes to cell-cell interactions. We asked whether 4-methylumbelliferone (4MU), an oral inhibitor of HA synthesis, could inhibit antigen presentation. We find that 4MU treatment reduces pericellular hyaluronan, destabilizes interactions between DC and T-cells, and prevents T-cell proliferation in vitro and in vivo. These effects were observed only when 4MU was added prior to initial antigen presentation but not later, consistent with 4MU-mediated inhibition of de novo antigenic responses. Building on these findings, we find that 4MU delays rejection of allogeneic pancreatic islet transplant and allogeneic cardiac transplants in mice and suppresses allogeneic T-cell activation in human mixed lymphocyte reactions. We conclude that 4MU, an approved drug, may have benefit as an adjunctive agent to delay transplantation rejection.

Adipocyte-Derived Versican and Macrophage-Derived Biglycan Control Adipose Tissue Inflammation in Obesity.

Obesity is characterized by adipose tissue inflammation. Because proteoglycans regulate inflammation, here we investigate their role in adipose tissue inflammation in obesity. We find that adipose tissue versican and biglycan increase in obesity. Versican is produced mainly by adipocytes and biglycan by adipose tissue macrophages. Both proteoglycans are also present in adipose tissue from obese human subjects undergoing gastric bypass surgery. Deletion of adipocyte-specific versican or macrophage-specific biglycan in mice reduces macrophage accumulation and chemokine and cytokine expression, although only adipocyte-specific versican deletion leads to sustained improvement in glucose tolerance. Macrophage-derived biglycan activates inflammatory genes in adipocytes. Versican expression increases in cultured adipocytes exposed to excess glucose, and adipocyte-conditioned medium stimulates inflammation in resident peritoneal macrophages, in part because of a versican breakdown product, versikine. These findings provide insights into the role of adipocyte- and macrophage-derived proteoglycans in adipose tissue inflammation in obesity.

Controlled release of monoclonal antibodies from poly-l-lysine-coated alginate spheres within a scaffolded implant mitigates autoimmune responses to transplanted islets and limits systemic antibody toxicity.

Immunomodulatory monoclonal antibodies (IM-mAbs) are a cornerstone of modern immunotherapy; however, when administered systemically (i.e., via injection), these agents can generate a variety of negative side effects. For many diseases, systemic delivery of IM-mAbs is the most effective mode of treatment, but in instances where the cellular target occupies a limited, well-defined space (e.g., solid tumors or cellularized implants) local, controlled release of IM-mAbs might be desirable. Antibodies are highly sensitive to a variety of environmental conditions, which limit the kinds of polymers suitable for antibody retention and controlled release. The present study evaluates the release of antibodies from biocompatible, 2-mm diameter alginate spheres coated with poly-l-lysine and a thin outer layer of alginate (APA spheres). In vitro, rates of antibody release (including IM-mAbs) could be incrementally decreased and made linear by incrementally increasing the quantity of poly-l-lysine deposited on the alginate, with linear release lasting in one scenario for at least 46 days. To evaluate the bioactivity in vivo of IM-mAbs, APA spheres loaded with either anti-CD3ε or anti-CD95 mAb were incorporated into scaffolded islet implant (SI) test-beds and the SIs implanted into a mouse model of autoimmune (type 1) diabetes. Release of mAbs within the implanted SIs resulted in reduced autoimmune responses to both transplanted and native islets. Notably, mice implanted with APA spheres loaded with quantities of anti-CD95 mAb that would be lethal if given systemically showed immunomodulation with no toxic side effects. Collectively, our results indicate that APA spheres are a relatively simple means to evaluate the effects of local, controlled release of IM-mAbs in a way that preserves mAb function and limits systemic toxicity.

Memory T cells specific to citrullinated α-enolase are enriched in the rheumatic joint.

ACPA-positive rheumatoid arthritis (RA) is associated with distinct HLA-DR alleles and immune responses to many citrullinated self-antigens. Herein we investigated the T cell epitope confined within α-enolase326-340 in the context of HLA-DRB1*04:01 and assessed the corresponding CD4+ T cells in both the circulation and in the rheumatic joint. Comparative crystallographic analyses were performed for the native and citrullinated α-enolase326-340 peptides in complex with HLA-DRB1*04:01. HLA-tetramers assembled with either the native or citrullinated peptide were used for ex vivo and in vitro assessment of α-enolase-specific T cells in peripheral blood, synovial fluid and synovial tissue by flow cytometry. The native and modified peptides take a completely conserved structural conformation within the peptide-binding cleft of HLA-DRB1*04:01. The citrulline residue-327 was located N-terminally, protruding towards TCRs. The frequencies of T cells recognizing native eno326-340 were similar in synovial fluid and peripheral blood, while in contrast, the frequency of T cells recognizing cit-eno326-340 was significantly elevated in synovial fluid compared to peripheral blood (3.6-fold, p = 0.0150). Additionally, citrulline-specific T cells with a memory phenotype were also significantly increased (1.6-fold, p = 0.0052) in synovial fluid compared to peripheral blood. The native T cell epitope confined within α-enolase326-340 does not appear to lead to complete negative selection of cognate CD4+ T cells. In RA patient samples, only T cells recognizing the citrullinated version of α-enolase326-340 were found at elevated frequencies implicating that neo-antigen formation is critical for breach of tolerance.

Local, Controlled Release In Vivo of Vascular Endothelial Growth Factor Within a Subcutaneous Scaffolded Islet Implant Reduces Early Islet Necrosis and Improves Performance of the Graft.

Islet transplantation remains the only alternative to daily insulin therapy for control of type 1 diabetes (T1D) in humans. To avoid the drawbacks of intrahepatic islet transplantation, we are developing a scaffolded islet implant to transplant islets into nonhepatic sites. The implant test bed, sized for mice, consists of a limited (2-mm) thickness, large-pore polymeric sponge scaffold perforated with peripheral cavities that contain islets suspended in a collagen hydrogel. A central cavity in the scaffold holds a 2-mm diameter alginate sphere for controlled release of the angiogenic cytokine vascular endothelial growth factor ( VEGF). Host microvessels readily penetrate the scaffold and collagen gel to vascularize the islets. Here, we evaluate the performance of the implant in a subcutaneous (SC) graft site. Implants incorporating 500 syngeneic islets reversed streptozotocin-induced diabetes in mice approximately 30 d after SC placement. Controlled release of a modest quantity (20 ng) of VEGF within the implant significantly reduced the time to normoglycemia compared to control implants lacking VEGF. Investigation of underlying causes for this effect revealed that inclusion of 20 ng of VEGF in the implants significantly reduced central necrosis of islets 24 h after grafting and increased implant vascularization (measured 12 d after grafting). Collectively, our results demonstrate (1) that the scaffolded islet implant design can reverse diabetes in SC sites in the absence of prevascularization of the graft site and (2) that relatively low quantities of VEGF, delivered by controlled release within the implant, can be a useful approach to limit islet stress after grafting.

A Novel Approach of Identifying Immunodominant Self and Viral Antigen Cross-Reactive T Cells and Defining the Epitopes They Recognize.

Infection and vaccination can lead to activation of autoreactive T cells, including the activation of cross-reactive T cells. However, detecting these cross-reactive T cells and identifying the non-self and self-antigen epitopes is difficult. The current study demonstrates the utility of a novel approach that effectively accomplishes both. We utilized surface expression of CD38 on newly activated CD4 memory T cells as a strategy to identify type 1 diabetes associated autoreactive T cells activated by influenza vaccination in healthy subjects. We identified an influenza A matrix protein (MP) specific CD4+ T cell clone that cross-recognizes an immunodominant epitope from Glutamic Acid Decarboxylase 65 (GAD65) protein. The sequences of the MP and GAD65 peptides are rather distinct, with only 2 identical amino acids within the HLA-DR binding region. This result suggests that activation of autoreactive T cells by microbial infection under certain physiological conditions can occur amongst peptides with minimum amino acid sequence homology. This novel strategy also provides a new research pathway in which to examine activation of autoreactive CD4+ T cells after vaccination or natural infection.

Modified High-Molecular-Weight Hyaluronan Promotes Allergen-Specific Immune Tolerance.

The extracellular matrix in asthmatic lungs contains abundant low-molecular-weight hyaluronan, and this is known to promote antigen presentation and allergic responses. Conversely, high-molecular-weight hyaluronan (HMW-HA), typical of uninflamed tissues, is known to suppress inflammation. We investigated whether HMW-HA can be adapted to promote tolerance to airway allergens. HMW-HA was thiolated to prevent its catabolism and was tethered to allergens via thiol linkages. This platform, which we call "XHA," delivers antigenic payloads in the context of antiinflammatory costimulation. Allergen/XHA was administered intranasally to mice that had been sensitized previously to these allergens. XHA prevents allergic airway inflammation in mice sensitized previously to either ovalbumin or cockroach proteins. Allergen/XHA treatment reduced inflammatory cell counts, airway hyperresponsiveness, allergen-specific IgE, and T helper type 2 cell cytokine production in comparison with allergen alone. These effects were allergen specific and IL-10 dependent. They were durable for weeks after the last challenge, providing a substantial advantage over the current desensitization protocols. Mechanistically, XHA promoted CD44-dependent inhibition of nuclear factor-κB signaling, diminished dendritic cell maturation, and reduced the induction of allergen-specific CD4 T-helper responses. XHA and other potential strategies that target CD44 are promising alternatives for the treatment of asthma and allergic sinusitis.

Genetically modified human CD4(+) T cells can be evaluated in vivo without lethal graft-versus-host disease.

Adoptive cell immunotherapy for human diseases, including the use of T cells modified to express an anti-tumour T-cell receptor (TCR) or chimeric antigen receptor, is showing promise as an effective treatment modality. Further advances would be accelerated by the availability of a mouse model that would permit human T-cell engineering protocols and proposed genetic modifications to be evaluated in vivo. NOD-scid IL2rγ(null) (NSG) mice accept the engraftment of mature human T cells; however, long-term evaluation of transferred cells has been hampered by the xenogeneic graft-versus-host disease (GVHD) that occurs soon after cell transfer. We modified human primary CD4(+) T cells by lentiviral transduction to express a human TCR that recognizes a pancreatic beta cell-derived peptide in the context of HLA-DR4. The TCR-transduced cells were transferred to NSG mice engineered to express HLA-DR4 and to be deficient for murine class II MHC molecules. CD4(+) T-cell-depleted peripheral blood mononuclear cells were also transferred to facilitate engraftment. The transduced cells exhibited long-term survival (up to 3 months post-transfer) and lethal GVHD was not observed. This favourable outcome was dependent upon the pre-transfer T-cell transduction and culture conditions, which influenced both the kinetics of engraftment and the development of GVHD. This approach should now permit human T-cell transduction protocols and genetic modifications to be evaluated in vivo, and it should also facilitate the development of human disease models that incorporate human T cells.

Structural and Nonstructural Viral Proteins Are Targets of T-Helper Immune Response against Human Respiratory Syncytial Virus.

Proper antiviral humoral and cellular immune responses require previous recognition of viral antigenic peptides that are bound to HLA class II molecules, which are exposed on the surface of antigen-presenting cells. The helper immune response is critical for the control and the clearance of human respiratory syncytial virus (HRSV) infection, a virus with severe health risk in infected pediatric, immunocompromised, and elderly populations. In this study, using a mass spectrometry analysis of complex HLA class II-bound peptide pools that were isolated from large amounts of HRSV-infected cells, 19 naturally processed HLA-DR ligands, most of them included in a complex nested set of peptides, were identified. Both the immunoprevalence and the immunodominance of the HLA class II response to HRSV were focused on one nonstructural (NS1) and two structural (matrix and mainly fusion) proteins of the infective virus. These findings have clear implications for analysis of the helper immune response as well as for antiviral vaccine design.

Inhibition of hyaluronan synthesis restores immune tolerance during autoimmune insulitis.

We recently reported that abundant deposits of the extracellular matrix polysaccharide hyaluronan (HA) are characteristic of autoimmune insulitis in patients with type 1 diabetes (T1D), but the relevance of these deposits to disease was unclear. Here, we have demonstrated that HA is critical for the pathogenesis of autoimmune diabetes. Using the DO11.10xRIPmOVA mouse model of T1D, we determined that HA deposits are temporally and anatomically associated with the development of insulitis. Moreover, treatment with an inhibitor of HA synthesis, 4-methylumbelliferone (4-MU), halted progression to diabetes even after the onset of insulitis. Similar effects were seen in the NOD mouse model, and in these mice, 1 week of treatment was sufficient to prevent subsequent diabetes. 4-MU reduced HA accumulation, constrained effector T cells to nondestructive insulitis, and increased numbers of intraislet FOXP3+ Tregs. Consistent with the observed effects of 4-MU treatment, Treg differentiation was inhibited by HA and anti-CD44 antibodies and rescued by 4-MU in an ERK1/2-dependent manner. These data may explain how peripheral immune tolerance is impaired in tissues under autoimmune attack, including islets in T1D. We propose that 4-MU, already an approved drug used to treat biliary spasm, could be repurposed to prevent, and possibly treat, T1D in at-risk individuals.

Citrulline-specific Th1 cells are increased in rheumatoid arthritis and their frequency is influenced by disease duration and therapy.

OBJECTIVE: Rheumatoid arthritis (RA) is thought to be a T cell-mediated disease, based on its strong association with HLA class II alleles, clinical responsiveness to T cell-directed therapies, and the presence of CD4+ T cells in rheumatoid joints. The presence of anti-citrullinated protein antibodies (ACPAs) in RA serum and the association of these antibodies with HLA-DR4 alleles implicate citrulline-specific autoreactive T cells in the development and progression of RA. The goal of this study was to determine the characteristics and specificity of autoreactive T cell responses in RA. METHODS: We developed a panel of HLA-DRB1*04:01 tetramers, selecting citrullinated peptides from synovial antigens and verifying their immunogenicity in DRB1*04:01-transgenic mice. Seven tetramers were used to examine the ex vivo frequency and surface phenotype of citrulline-specific (Cit-specific) T cells in patients with RA and healthy subjects with DRB1*04:01 haplotypes, using a magnetic enrichment procedure. RESULTS: Cit-specific T cells were detectable in peripheral blood samples from both healthy subjects and RA patients. In comparison to healthy subjects, RA patients had significantly higher frequencies of Cit-specific T cells, and a greater proportion of these cells displayed a Th1 memory phenotype. Among RA patients, the frequency of Cit-specific T cells was highest within the first 5 years after diagnosis of RA and was decreased in patients taking biologic agents, irrespective of disease duration. CONCLUSION: These findings link the presence of ACPAs in RA with Th1 cells specific for citrullinated epitopes and provide tools for disease-specific immunomonitoring of autoreactive T cells.

Antigen-specific immunomodulation for type 1 diabetes by novel recombinant antibodies directed against diabetes-associates auto-reactive T cell epitope.

The trimolecular complex composed of autoreactive T-cell receptor, MHC class II, and an autoantigenic peptide plays a central role in the activation of pathogenic Islet-specific CD4+ T cells in type 1 diabetes (T1D). We isolated and characterized novel antibodies against autoreactive T-cell epitopes associated with T1D. Our antibodies mimic the specificity of the T-cell receptor (TCR), while binding MHC class II/peptide complexes in an autoantigen peptide specific, MHC-restricted manner. The isolated TCR-like antibodies were directed against the minimal T-cell epitope GAD-555-567 in the context of the HLA-DR4-diabetic-associated molecule. A representative high-affinity TCR-like antibody clone (G3H8) enabled the detection of intra- and extra-cellular DR4/GAD-555-567 complexes in antigen presenting cells. I561M single mutation at the central position (P5) of the GAD-555-567 peptide abolished the binding of G3H8 to the DR4/GAD complex, demonstrating its high fine TCR-like specificity. The G3H8 TCR-like antibody significantly inhibited GAD-555-567 specific, DR4 restricted T-cell response in vitro and in vivo in HLA-DR4 transgenic mice. Our findings constitute a proof-of-concept for the utility of TCR-like antibodies as antigen-specific immunomodulation agents for regulating pathogenic T-cells and suggest that TCR-like antibodies targeting autoreactive MHC class II epitopes are valuable research tools that enable studies related to antigen presentation as well as novel therapeutic agents that may be used to modulate autoimmune disorders such as T1D.

IL-10 induction from implants delivering pancreatic islets and hyaluronan.

Local induction of pro-tolerogenic cytokines, such as IL-10, is an appealing strategy to help facilitate transplantation of islets and other tissues. Here, we describe a pair of implantable devices that capitalize on our recent finding that hyaluronan (HA) promotes IL-10 production by activated T cells. The first device is an injectable hydrogel made of crosslinked HA and heparan sulfate loaded with anti-CD3/anti-CD28 antibodies and IL-2. T cells embedded within this hydrogel prior to polymerization go on to produce IL-10 in vivo. The second device is a bioengineered implant consisting of a polyvinyl alcohol sponge scaffold, supportive collagen hydrogel, and alginate spheres mediating sustained release of HA in fluid form. Pancreatic islets that expressed ovalbumin (OVA) antigen were implanted within this device for 14 days into immunodeficient mice that received OVA-specific DO.11.10 T cells and a subsequent immunization with OVA peptide. Splenocytes harvested from these mice produced IL-10 upon re-challenge with OVA or anti-CD3 antibodies. Both of these devices represent model systems that will be used, in future studies, to further evaluate IL-10 induction by HA, with the objective of improving the survival and function of transplanted islets in the setting of autoimmune (type 1) diabetes.

Reversal of diabetes in mice with a bioengineered islet implant incorporating a type I collagen hydrogel and sustained release of vascular endothelial growth factor.

We have developed a bioengineered implant (BI) to evaluate strategies to promote graft survival and function in models of islet transplantation in mice. The BI, sized for implantation within a fold of intestinal mesentery, consists of a disk-shaped, polyvinyl alcohol sponge infused with a type I collagen hydrogel that contains dispersed donor islets. To promote islet vascularization, the BI incorporates a spherical alginate hydrogel for sustained release of vascular endothelial growth factor (VEGF). BIs that contained 450-500 islets from syngeneic (C57Bl/6) donors and 20 ng of VEGF reversed streptozotocin (STZ)-induced diabetes in 100% of mice (8/8), whereas BIs that contained an equivalent number of islets, but which lacked VEGF, reversed STZ-induced diabetes in only 62.5% of mice (5/8). Between these "+VEGF" and "-VEGF" groups, the time to achieve normoglycemia (8-18 days after implantation) did not differ statistically; however, transitory, postoperative hypoglycemia was markedly reduced in the +VEGF group relative to the -VEGF group. Notably, none of the mice that achieved normoglycemia in these two groups required exogenous insulin therapy once the BIs began to fully regulate levels of blood glucose. Moreover, the transplanted mice responded to glucose challenge in a near-normal manner, as compared to the responses of healthy, nondiabetic (control) mice that had not received STZ. In future studies, the BIs described here will serve as platforms to evaluate the capability of immunomodulatory compounds, delivered locally within the BI, to prevent or reverse diabetes in the setting of autoimmune (type 1) diabetes.

Identification and functional characterization of T cells reactive to citrullinated vimentin in HLA-DRB1*0401-positive humanized mice and rheumatoid arthritis patients.

OBJECTIVE: Antibodies toward the citrullinated form of the synovial antigen vimentin are specific for rheumatoid arthritis (RA) and are associated with HLA-DRB1*0401. This suggests that T cells specific for peptides derived from citrullinated vimentin presented in the context of HLA-DRB1*0401 may contribute to the etiopathogenesis of RA. The aim of this study was to identify immunodominant epitopes from citrullinated vimentin presented by HLA-DRB1*0401 and to characterize the resulting T cell responses. METHODS: We first predicted an HLA-binding T cell epitope from citrullinated vimentin based on the binding motif of HLA-DRB1*0401 and then confirmed its affinity. A class II major histocompatibility complex (MHC) tetramer loaded with the citrullinated form of vimentin aa 59-78 (cit-vimentin aa 59-78) was constructed and used to screen for specific T cells in HLA-DRB1*0401-transgenic mice, patients with RA, and healthy control subjects. Additionally, the cytokine output following cit-vimentin aa 59-78 challenge was analyzed in patients and healthy control subjects by multicolor flow cytometry and Luminex-based analysis. RESULTS: The citrullinated form of vimentin aa 59-78 bound to HLA-DRB1*0401, but the native form could not. Subsequently, cit-vimentin aa 59-78-specific T cells were detected in immunized mice and in the periphery of both HLA-DR*0401-positive healthy control subjects and HLA-DR*0401-positive patients with RA, using class II MHC tetramers, CD154 up-regulation, and intracellular cytokine measurements. As demonstrated in cell culture supernatants, the production of cytokines (predominantly interferon-γ) in response to cit-vimentin aa 59-78 was significantly higher in patients compared with controls. CONCLUSION: Here, we describe a posttranslational modification of an RA candidate autoantigen toward which HLA-DRB1*0401-restricted T cells can be detected in both patients with RA and healthy controls but for which a proinflammatory response is observed uniquely in patients with RA.

ECM components guide IL-10 producing regulatory T-cell (TR1) induction from effector memory T-cell precursors.

We describe a role for ECM as a biosensor for inflammatory microenvironments that plays a critical role in peripheral immune tolerance. We show that hyaluronan (HA) promotes induction of Foxp3- IL-10-producing regulatory T cells (TR1) from conventional T-cell precursors in both murine and human systems. This is, to our knowledge, the first description of an ECM component inducing regulatory T cells. Intact HA, characteristic of healing tissues, promotes induction of TR1 capable of abrogating disease in an IL-10-dependent mouse colitis model whereas fragmentary HA, typical of inflamed tissues, does not, indicating a decisive role for tissue integrity in this system. The TR1 precursor cells in this system are CD4(+)CD62L(-)FoxP3(-), suggesting that effector memory cells assume a regulatory phenotype when they encounter their cognate antigen in the context of intact HA. Matrix integrity cues might thereby play a central role in maintaining peripheral tolerance. This TR1 induction is mediated by CD44 cross-linking and signaling through p38 and ERK1/2. This induction is suppressed, also in a CD44-dependent manner, by osteopontin, a component of chronically inflamed ECM, indicating that CD44 signaling serves as a nexus for fate decisions regarding TR1 induction. Finally, we demonstrate that TR1 induction signals can be recapitulated using synthetic matrices. These results reveal important roles for the matrix microenvironment in immune regulation and suggest unique strategies for immunomodulation.

Peptide-MHC cellular microarray with innovative data analysis system for simultaneously detecting multiple CD4 T-cell responses.

BACKGROUND: Peptide:MHC cellular microarrays have been proposed to simultaneously characterize multiple Ag-specific populations of T cells. The practice of studying immune responses to complicated pathogens with this tool demands extensive knowledge of T cell epitopes and the availability of peptide:MHC complexes for array fabrication as well as a specialized data analysis approach for result interpretation. METHODOLOGY/PRINCIPAL FINDINGS: We co-immobilized peptide:DR0401 complexes, anti-CD28, anti-CD11a and cytokine capture antibodies on the surface of chamber slides to generate a functional array that was able to detect rare Ag-specific T cell populations from previously primed in vitro T cell cultures. A novel statistical methodology was also developed to facilitate batch processing of raw array-like data into standardized endpoint scores, which linearly correlated with total Ag-specific T cell inputs. Applying these methods to analyze Influenza A viral antigen-specific T cell responses, we not only revealed the most prominent viral epitopes, but also demonstrated the heterogeneity of anti-viral cellular responses in healthy individuals. Applying these methods to examine the insulin producing beta-cell autoantigen specific T cell responses, we observed little difference between autoimmune diabetic patients and healthy individuals, suggesting a more subtle association between diabetes status and peripheral autoreactive T cells. CONCLUSIONS/SIGNIFICANCE: The data analysis system is reliable for T cell specificity and functional testing. Peptide:MHC cellular microarrays can be used to obtain multi-parametric results using limited blood samples in a variety of translational settings.

Th1 cytokines promote T-cell binding to antigen-presenting cells via enhanced hyaluronan production and accumulation at the immune synapse.

Hyaluronan (HA) production by dendritic cells (DCs) is known to promote antigen presentation and to augment T-cell activation and proliferation. We hypothesized that pericellular HA can function as intercellular 'glue' directly mediating T cell-DC binding. Using primary human cells, we observed HA-dependent binding between T cells and DCs, which was abrogated upon pre-treatment of the DCs with 4-methylumbelliferone (4-MU), an agent which blocks HA synthesis. Furthermore, T cells regulate HA production by DCs via T cell-derived cytokines in a T helper (Th) subset-specific manner, as demonstrated by the observation that cell-culture supernatants from Th1 but not Th2 clones promote HA production. Similar effects were seen upon the addition of exogenous Th1 cytokines, IL-2, interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). The critical factors which determined the extent of DC-T cell binding in this system were the nature of the pre-treatment the DCs received and their capacity to synthesize HA, as T-cell clones which were pre-treated with monensin, added to block cytokine secretion, bound equivalently irrespective of their Th subset. These data support the existence of a feedforward loop wherein T-cell cytokines influence DC production of HA, which in turn affects the extent of DC-T cell binding. We also document the presence of focal deposits of HA at the immune synapse between T-cells and APC and on dendritic processes thought to be important in antigen presentation. These data point to a pivotal role for HA in DC-T cell interactions at the IS.

Restricted autoantigen recognition associated with deletional and adaptive regulatory mechanisms.

Autoimmune diabetes (T1D) is characterized by CD4(+) T cell reactivity to a variety of islet-associated Ags. At-risk individuals, genetically predisposed to T1D, often have similar T cell reactivity, but nevertheless fail to progress to clinically overt disease. To study the immune tolerance and regulatory environment permissive for such autoreactive T cells, we expressed TCR transgenes derived from two autoreactive human T cells, 4.13 and 164, in HLA-DR4 transgenic mice on a C57BL/6-derived "diabetes-resistant" background. Both TCR are responsive to an immunodominant epitope of glutamic acid decarboxylase 65(555-567), which is identical in sequence between humans and mice, is restricted by HLA-DR4, and is a naturally processed self Ag associated with T1D. Although both TCR use the identical Valpha and Vbeta genes, differing only in CDR3, we found stark differences in the mechanisms utilized in vivo in the maintenance of immune tolerance. A combination of thymic deletion (negative selection), TCR down-regulation, and peripheral activation-induced cell death dominated the phenotype of 164 T cells, which nevertheless still maintain their Ag responsiveness in the periphery. In contrast, 4.13 T cells are much less influenced by central and deletional tolerance mechanisms, and instead display a peripheral immune deviation including differentiation into IL-10-secreting Tr1 cells. These findings indicate a distinct set of regulatory alternatives for autoreactive T cells, even within a single highly restricted HLA-peptide-TCR recognition profile.