Afshin Shameli

Reversal of Autoimmunity by Boosting Memory-like Autoregulatory T Cells

Blunting autoreactivity without compromising immunity remains an elusive goal in the treatment of autoimmunity. We show that progression to autoimmune diabetes results in the conversion of naive low-avidity autoreactive CD8(+) T cells into memory-like autoregulatory cells that can be expanded in vivo with nanoparticles coated with disease-relevant peptide-major histocompatibility complexes (pMHC-NP). Treatment of NOD mice with monospecific pMHC-NPs expanded cognate autoregulatory T cells, suppressed the recruitment of noncognate specificities, prevented disease in prediabetic mice, and restored normoglycemia in diabetic animals. pMHC-NP therapy was inconsequential in mice engineered to bear an immune system unresponsive to the corresponding epitope, owing to absence of epitope-experienced autoregulatory T cells. pMHC-NP-expanded autoregulatory T cells suppressed local presentation of autoantigens in an interferon-gamma-, indoleamine 2,3-dioxygenase-, and perforin-dependent manner. Nanoparticles coated with human diabetes-relevant pHLA complexes restored normoglycemia in a humanized model of diabetes. These observations expose a paradigm in the pathogenesis of autoimmunity amenable for therapeutic intervention.

Adenosine deamination sustains dendritic cell activation in inflammation.

Adenosine is a suppressive agent that protects the host from excessive tissue injury associated with strong inflammation. In tissue stress, higher levels of adenosine signal through adenosine receptors to exert strong anti-inflammatory effects, and thus protect host cells. Existing evidence also suggests that elevated adenosine potently down-regulates the activation of lymphocytes during inflammation. This notion, however, is in contrast with another basic observation that the immune system is highly activated precisely under the same circumstances against pathogens. In this study, we show that inflammatory responses of dendritic cells (DCs) are highly sensitive to adenosine suppression. However, they intrinsically carry high adenosine deaminase activity, which in turn degrades and removes adenosine from the surroundings, cutting off DCs from the suppression. This regulatory mechanism is important in DC responses to pathogen-associated molecular patterns and their activation of T cells. Our findings suggest a mechanism that DCs maintain their hyperreactive state in inflammation despite the general state of suppression, and reveal a regulatory role of adenosine deaminase in DC innate immune responses.

TLR9 Blockade Inhibits Activation of Diabetogenic CD8+ T Cells and Delays Autoimmune Diabetes

Diabetogenic CD8+ T cells are primed in the pancreatic lymph nodes (PLNs) by dendritic cells (DCs) carrying islet cell Ags. TLR signaling modifies DC function. The goal of this study was to determine the effect of TLR9 signaling on diabetogenic CD8+ T cell activation and the course of type 1 diabetes. We explored the effects of CpG oligonucleotide, TLR9 antagonists, and genetic TLR9 deficiency on the activation of diabetogenic CD8+ T cells. NOD bone marrow-derived DCs pulsed with freeze-thawed insulinoma cells in the presence of TLR9 agonist CpG and CD40 agonist induced diabetogenic CD8+ T cell activation. The addition of TLR9 antagonist oligodeoxynucleotide or chloroquine inhibited bone marrow-derived DCs activation and CD8+ T cell priming in response to CpG. CpG alone or with CD40 agonist induced CTL activity that triggered diabetes development in 8.3-TCR transgenic NOD mice. Oligodeoxynucleotide treatment of 8.3-TCR transgenic NOD mice delayed spontaneous diabetes development. Chloroquine treatment delayed the spontaneous onset of diabetes in NOD mice, coincident with the decreased activation of PLN DCs. TLR9−/− NOD mice had delayed onset of diabetes compared with TLR9−/+ NOD littermates. TLR9−/− NOD mice had lower levels of IFN-α in PLNs and decreased frequencies of plasmacytoid DCs and diabetogenic CD8+ T cells compared with NOD mice. We propose that TLR9 activation contributes to the spontaneous onset of diabetes in NOD mice by increasing IFN-α and promoting diabetogenic CD8 T cell activation.

The Proline-Rich Sequence of CD3ε as an Amplifier of Low-Avidity TCR Signaling

Engagement of peptide-MHC by the TCR induces a conformational change in CD3ε that exposes a proline-rich sequence (PRS) and recruits the cytoskeletal adaptor Nck. This event, which precedes phosphorylation of the CD3ε ITAM, has been implicated in synapse formation and T cell function. However, there is compelling evidence that responsiveness to TCR ligation is CD3ε PRS independent. In this study, we show that the CD3ε PRS is necessary for peptide-MHC-induced phosphorylation of CD3ε and for recruitment of protein kinase Cθ to the immune synapse in differentiated CD8+ T lymphocytes. However, whereas these two events are dispensable for functional T cell responsiveness to high-avidity ligands, they are required for responsiveness to low-avidity ones. Thus, in at least certain T cell clonotypes, the CD3ε PRS amplifies weak TCR signals by promoting synapse formation and CD3ε phosphorylation.

In situ recognition of autoantigen as an essential gatekeeper in autoimmune CD8+ T cell inflammation

A current paradigm states that non-antigen-specific inflammatory cues attract noncognate, bystander T cell specificities to sites of infection and autoimmune inflammation. Here we show that cues emanating from a tissue undergoing spontaneous autoimmune inflammation cannot recruit naive or activated bystander T cell specificities in the absence of local expression of cognate antigen. We monitored the recruitment of CD8+ T cells specific for the prevalent diabetogenic epitope islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)206–214 in gene-targeted nonobese diabetic (NOD) mice expressing a T cell “invisible” IGRP206–214 sequence. These mice developed islet inflammation and diabetes with normal incidence and kinetics, but their inflammatory lesions could recruit neither naive (endogenous or exogenous) nor ex vivo-activated IGRP206–214-reactive CD8+ T cells. Conversely, IGRP206–214-reactive, but not nonautoreactive CD8+ T cells rapidly homed to and accumulated in the inflamed islets of wild-type NOD mice. Our results indicate that CD8+ T cell recruitment to a site of autoimmune inflammation results from an active process that is strictly dependent on local display of cognate pMHC and suggest that CD8+ T cells contained in extralymphoid autoimmune lesions are largely autoreactive.

High Levels of Adenosine Deaminase on Dendritic Cells Promote Autoreactive T Cell Activation and Diabetes in Nonobese Diabetic Mice

Adenosine has been established as an important regulator of immune activation. It signals through P1 adenosine receptors to suppress activation of T cells and professional APCs. Adenosine deaminase (ADA) counters this effect by catabolizing adenosine. This regulatory mechanism has not been tested in a disease model in vivo. Questions also remain as to which cell types are most sensitive to this regulation and whether its dysregulation contributes to any autoimmune conditions. We approached this issue using the NOD model. We report that ADA is upregulated in NOD dendritic cells, which results in their exuberant and spontaneous activation. This, in turn, triggers autoimmune T cell activation. NOD DCs deficient in ADA expression have a greatly reduced capacity to trigger type I diabetes. We also provide evidence that although many cell types, particularly T cells, have been implicated as the suppression targets by adenosine in an in vitro setting, DCs also seem to be affected by this regulatory mechanism. Therefore, this report illustrates a role of ADA in autoimmunity and suggests a potential target for therapeutic intervention.

IL-2 promotes the function of memory-like autoregulatory CD8+ T cells but suppresses their development via FoxP3+ Treg cells.

IL‐2 plays a critical role in both effector T‐cell development and FoxP3+CD4+ Treg‐cell homeostasis. A reduction in Il2 transcription results in impaired FoxP3+CD4+ Treg‐cell recruitment and function, and accounts for the association between murine Il2 and type 1 diabetes (T1D). The progression of T1D elicits a disease‐countering negative feedback regulatory loop that involves the differentiation of low‐avidity autoreactive CD8+ T cells into memory‐like autoregulatory T cells in a CD4+ Th‐dependent manner. Since these auto‐regulatory T cells express IL‐2Rβ (CD122), we hypothesized that their development might also be regulated by IL‐2. Here, we investigate the effects of differences in IL‐2 expression on this autoregulatory subset. We show that decreased IL‐2 production impairs the regulatory capacity of memory‐like autoregulatory CD8+CD122+ T cells. Surprisingly, we also find that a reduction in IL‐2 production capacity increases memory autoregulatory CD8+ T‐cell formation indirectly, by decreasing the development and function of FoxP3+ Treg cells in nonobese diabetic mice. These results illustrate a complex homeostatic interplay between IL‐2, CD4+ Th cells, FoxP3+CD4+ Treg cells and autoregulatory CD8+ T‐cell memory whereby IL‐2 controls the function of both Treg‐cell subsets, but IL‐2‐potentiation of FoxP3+CD4+ Treg‐cell function results in the suppression of CD4+ Th‐cell activation and autoregulatory memory CD8+ T‐cell formation.

Development of Memory-Like Autoregulatory CD8+ T Cells Is CD4+ T Cell Dependent

Progression of spontaneous autoimmune diabetes is associated with development of a disease-countering negative-feedback regulatory loop that involves differentiation of low-avidity autoreactive CD8+ cells into memory-like autoregulatory T cells. Such T cells blunt diabetes progression by suppressing the presentation of both cognate and noncognate Ags to pathogenic high-avidity autoreactive CD8+ T cells in the pancreas-draining lymph nodes. In this study, we show that development of autoregulatory CD8+ T cell memory is CD4+ T cell dependent. Transgenic (TG) NOD mice expressing a low-affinity autoreactive TCR were completely resistant to autoimmune diabetes, even after systemic treatment of the mice with agonistic anti-CD40 or anti–4-1BB mAbs or autoantigen-pulsed dendritic cells, strategies that dramatically accelerate diabetes development in TG NOD mice expressing a higher affinity TCR for the same autoantigenic specificity. Furthermore, whereas abrogation of RAG-2 expression, hence endogenous CD4+ T cell and B cell development, decelerated disease progression in high-affinity TCR-TG NOD mice, it converted the low-affinity TCR into a pathogenic one. In agreement with these data, polyclonal CD4+ T cells from prediabetic NOD mice promoted disease in high-affinity TCR-TG NOD.Rag2−/− mice, but inhibited it in low-affinity TCR-TG NOD.Rag2−/− mice. Thus, in chronic autoimmune responses, CD4+ Th cells contribute to both promoting and suppressing pathogenic autoimmunity.

Endoplasmic Reticulum Stress Caused by Overexpression of Islet-Specific Glucose-6-Phosphatase Catalytic Subunit-Related Protein in Pancreatic Beta-Cells

The high rate of protein synthesis in beta-cells renders them susceptible to endoplasmic reticulum (ER) stress, a condition that can be aggravated by additional imbalances in ER homeostasis and could potentially contribute to the pathogenesis of type-1 and type-2 diabetes. Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is an ER-resident protein that is specifically expressed in pancreatic beta-cells and is a major target of diabetogenic CD8(+) T cell responses in non-obese diabetic (NOD) mice. We produced transgenic mice expressing human IGRP (hIGRP) under the control of rat insulin promoter (RIP) to study epitopes in hIGRP capable of driving diabetogenic human leukocyte antigen (HLA)-restricted CD8(+) T-cell responses in hIGRP/HLA transgenic NOD mice. Surprisingly, we found that 3 out of 14 lines expressing RIP-hIGRP in a non-T1D-prone genetic background developed a form of early-onset diabetes that was dissociated from autoimmune inflammation of pancreatic islets. We show that diabetes in these 3 lines resulted from increased rates of beta-cell death because of ER stress. We hypothesize that IGRP compounds the viability of beta-cells undergoing ER stress by generating unfolded proteins in the ER lumen, and that IGRPs location in the ER accounts, in part, for its exquisite immunogenicity in T1D-prone genetic backgrounds.

Spontaneous Autoimmunity Sufficiently Potent to Induce Diabetes Mellitus Is Insufficient to Protect against Insulinoma

Intact tolerogenic mechanisms preclude effective immunity against tumors, as most tumor Ags differ little from normal host Ags. In contrast, when tolerance fails, the immune system becomes inappropriately activated against an autoantigen. We postulated that CD8+ T cells activated during autoimmunity are capable of protecting against tumors that express the targeted autoantigen. To test this hypothesis, double-transgenic 8.3-NOD-RIPTAg mice were developed (where NOD is nonobese diabetic, RIP is rat insulin promoter, and TAg is large T Ag). In this model, individuals with the RIPTAg transgene develop insulinoma; those expressing a transgenic TCR (8.3-TCR) recognizing the islet-specific glucose 6 phosphatase catalytic subunit-related protein (IGRP) harbor a peripheral immune system dominated by diabetogenic CD8+ T cells. Although tumor emergence was significantly slower in 8.3-NOD-RIPTAg mice compared with NOD-RIPTAg mice, all 8.3-NOD-RIPTAg mice eventually developed insulinoma. Tumor emergence was not secondary to clonal deletion or anergy. Ag loss and MHC down-regulation were not apparent. Endogenous 8.3-TCR CD8+ T cells were recruited to the tumor site and proliferated upon arrival to the tumor, although they were notably absent from the central parts of more advanced tumors. These results demonstrate that a breakdown of tolerance capable of causing autoimmune disease is insufficient for effective tumor immunity. Alterations in the tumor microenvironment may inhibit efficient and comprehensive delivery of CD8+ T cells to all regions of the tumor. These data suggest that any immunotherapeutic strategy for cancer must involve enhancement of a proinflammatory tumor microenvironment in addition to inhibition of tolerogenic mechanisms.