Gayatri Mukherjee

The immunotherapeutic potential of dendritic cells in type 1 diabetes

Type 1 diabetes is an autoimmune disease characterized by destruction of the pancreatic islet beta cells that is mediated primarily by T cells specific for beta cell antigens. Insulin administration prolongs the life of affected individuals, but often fails to prevent the serious complications that decrease quality of life and result in significant morbidity and mortality. Thus, new strategies for the prevention and treatment of this disease are warranted. Given the important role of dendritic cells (DCs) in the establishment of peripheral T cell tolerance, DC‐based strategies are a rational and exciting avenue of exploration. DCs employ a diverse arsenal to maintain tolerance, including the induction of T cell deletion or anergy and the generation and expansion of regulatory T cell populations. Here we review DC‐based immunotherapeutic approaches to type 1 diabetes, most of which have been employed in non‐obese diabetic (NOD) mice or other murine models of the disease. These strategies include administration of in vitro‐generated DCs, deliberate exposure of DCs to antigens before transfer and the targeting of antigens to DCs in vivo. Although remarkable results have often been obtained in these model systems, the challenge now is to translate DC‐based immunotherapeutic strategies to humans, while at the same time minimizing the potential for global immunosuppression or exacerbation of autoimmune responses. In this review, we have devoted considerable attention to antigen‐specific DC‐based approaches, as results from murine models suggest that they have the potential to result in regulatory T cell populations capable of both preventing and reversing type 1 diabetes.

Generation of β cell-specific human cytotoxic T cells by lentiviral transduction and their survival in immunodeficient human leucocyte antigen-transgenic mice

Several β cell antigens recognized by T cells in the non‐obese diabetic (NOD) mouse model of type 1 diabetes (T1D) are also T cell targets in the human disease. While numerous antigen‐specific therapies prevent diabetes in NOD mice, successful translation of rodent findings to patients has been difficult. A human leucocyte antigen (HLA)‐transgenic mouse model incorporating human β cell‐specific T cells might provide a better platform for evaluating antigen‐specific therapies. The ability to study such T cells is limited by their low frequency in peripheral blood and the difficulty in obtaining islet‐infiltrating T cells from patients. We have worked to overcome this limitation by using lentiviral transduction to ‘reprogram’ primary human CD8 T cells to express three T cell receptors (TCRs) specific for a peptide derived from the β cell antigen islet‐specific glucose‐6‐phosphatase catalytic subunit‐related protein (IGRP265–273) and recognized in the context of the human class I major histocompatibility complex (MHC) molecule HLA‐A2. The TCRs bound peptide/MHC multimers with a range of avidities, but all bound with at least 10‐fold lower avidity than the anti‐viral TCR used for comparison. One exhibited antigenic recognition promiscuity. The β cell‐specific human CD8 T cells generated by lentiviral transduction with one of the TCRs released interferon (IFN)‐γ in response to antigen and exhibited cytotoxic activity against peptide‐pulsed target cells. The cells engrafted in HLA‐A2‐transgenic NOD‐scid IL2rγnull mice and could be detected in the blood, spleen and pancreas up to 5 weeks post‐transfer, suggesting the utility of this approach for the evaluation of T cell‐modulatory therapies for T1D and other T cell‐mediated autoimmune diseases.

Hemolysin Induces Toll-like Receptor (TLR)-independent Apoptosis and Multiple TLR-associated Parallel Activation of Macrophages

Vibrio cholerae hemolysin (HlyA) displays bipartite property while supervising macrophages (MΦ). The pore-forming toxin causes profound apoptosis within 3 h of exposure and in parallel supports activation of the defying MΦ. HlyA-induced apoptosis of MΦ remains steady for 24 h, is Toll-like receptor (TLR)-independent, and is driven by caspase-9 and caspase-7, thus involving the mitochondrial or intrinsic pathway. Cell activation is carried forward by time dependent up-regulation of varying TLRs. The promiscuous TLR association of HlyA prompted investigation, which revealed the β-prism lectin domain of HlyA simulated TLR4 up-regulation by jacalin, a plant lectin homologue besides expressing CD86 and type I cytokines TNF-α and IL-12. However, HlyA cytolytic protein domain up-regulated TLR2, which controlled CD40 for continuity of cell activation. Expression of TOLLIP before TLR2 and TLR6 abrogated TLR4, CD40, and CD86. We show that the transient expression of TOLLIP leading to curbing of activation-associated capabilities is a plausible feedback mechanism of MΦ to deploy TLR2 and prolong activation involving CD40 to encounter the HlyA cytolysin domain.

Delivery of siRNAs to Dendritic Cells Using DEC205-Targeted Lipid Nanoparticles to Inhibit Immune Responses.

Due to their ability to knock down the expression of any gene, siRNAs have been heralded as ideal candidates for treating a wide variety of diseases, including those involving “undruggable” targets. However, the therapeutic potential of siRNAs remains severely limited by a lack of effective delivery vehicles. Recently, lipid nanoparticles (LNPs) containing ionizable cationic lipids have been developed for hepatic siRNA delivery. However, their suitability for delivery to other cell types has not been determined. We have modified LNPs for preferential targeting to dendritic cells (DCs), central regulators of immune responses. To achieve directed delivery, we coated LNPs with a single-chain antibody (scFv; DEC-LNPs), specific to murine DEC205, which is highly expressed on distinct DC subsets. Here we show that injection of siRNAs encapsulated in DEC-LNPs are preferentially delivered to DEC205+ DCs. Gene knockdown following uptake of DEC-LNPs containing siRNAs specific for the costimulatory molecules CD40, CD80, and CD86 dramatically decreases gene expression levels. We demonstrate the functionality of this knockdown with a mixed lymphocyte response (MLR). Overall, we report that injection of LNPs modified to restrict their uptake to a distinct cell population can confer profound gene knockdown, sufficient to inhibit powerful immune responses like the MLR.

Compensatory Mechanisms Allow Undersized Anchor-deficient Class I MHC Ligands to Mediate Pathogenic Autoreactive T Cell Responses

Self-reactive T cells must escape thymic negative selection to mediate pathogenic autoimmunity. In the NOD mouse model of autoimmune diabetes, several β cell–cytotoxic CD8 T cell populations are known, with the most aggressive of these represented by AI4, a T cell clone with promiscuous Ag-recognition characteristics. We identified a long-elusive β cell–specific ligand for AI4 as an unusually short H-2Db–binding 7-mer peptide lacking a C-terminal anchor residue and derived from the insulin A chain (InsA14–20). Crystallography reveals that compensatory mechanisms permit peptides lacking a C-terminal anchor to bind sufficiently to the MHC to enable destructive T cell responses, yet allow cognate T cells to avoid negative selection. InsA14–20 shares two solvent-exposed residues with previously identified AI4 ligands, providing a structural explanation for AI4’s promiscuity. Detection of AI4-like T cells, using mimotopes of InsA14–20 with improved H-2Db–binding characteristics, establishes the AI4-like T cell population as a consistent feature of the islet infiltrates of NOD mice. Our work establishes undersized peptides as previously unrecognized targets of autoreactive CD8 T cells and presents a strategy for their further exploration as Ags in autoimmune disease.

Structural and functional characterization of a single-chain peptide–MHC molecule that modulates both naive and activated CD8+ T cells

Peptide–MHC (pMHC) multimers, in addition to being tools for tracking and quantifying antigen-specific T cells, can mediate downstream signaling after T-cell receptor engagement. In the absence of costimulation, this can lead to anergy or apoptosis of cognate T cells, a property that could be exploited in the setting of autoimmune disease. Most studies with class I pMHC multimers used noncovalently linked peptides, which can allow unwanted CD8+ T-cell activation as a result of peptide transfer to cellular MHC molecules. To circumvent this problem, and given the role of self-reactive CD8+ T cells in the development of type 1 diabetes, we designed a single-chain pMHC complex (scKd.IGRP) by using the class I MHC molecule H-2Kd and a covalently linked peptide derived from islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP206–214), a well established autoantigen in NOD mice. X-ray diffraction studies revealed that the peptide is presented in the groove of the MHC molecule in canonical fashion, and it was also demonstrated that scKd.IGRP tetramers bound specifically to cognate CD8+ T cells. Tetramer binding induced death of naive T cells and in vitro- and in vivo-differentiated cytotoxic T lymphocytes, and tetramer-treated cytotoxic T lymphocytes showed a diminished IFN-γ response to antigen stimulation. Tetramer accessibility to disease-relevant T cells in vivo was also demonstrated. Our study suggests the potential of single-chain pMHC tetramers as possible therapeutic agents in autoimmune disease. Their ability to affect the fate of naive and activated CD8+ T cells makes them a potential intervention strategy in early and late stages of disease.

DEC-205-mediated antigen targeting to steady-state dendritic cells induces deletion of diabetogenic CD8+ T cells independently of PD-1 and PD-L1

CD8⁺ T cells specific for islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) have been implicated in type 1 diabetes in both humans and non-obese diabetic (NOD) mice, in which T cells specific for IGRP₂₀₆₋₂₁₄ are highly prevalent. We sought to manipulate these pathogenic T cells by exploiting the ability of steady-state dendritic cells (DCs) to present antigens in a tolerogenic manner. The endocytic receptor DEC-205 was utilized to deliver an IGRP₂₀₆₋₂₁₄ mimotope to DCs in NOD mice, and the impact of this delivery on a polyclonal population of endogenous islet-reactive cognate T cells was determined. Assessment of islet-infiltrating CD8⁺ T cells showed a decrease in the percentage, and the absolute number, of endogenous IGRP₂₀₆₋₂₁₄-specific T cells when the mimotope was delivered to DCs, compared with delivery of a specificity control. Employing an adoptive transfer system, deletion of CD8⁺ T cells as a result of DEC-205-mediated antigen targeting was found to occur independently of programmed death-1 (PD-1) and its ligand (PD-L1), both often implicated in the regulation of peripheral T-cell tolerance. Given its promise for the manipulation of self-reactive polyclonal T cells demonstrated here, the distinctive characteristics of this antigen delivery system will be important to appreciate as its potential as an intervention for autoimmune diseases continues to be investigated.

Glucagon-reactive islet-infiltrating CD8 T cells in NOD mice

Type 1 diabetes is characterized by T‐cell‐mediated destruction of the insulin‐producing β cells in pancreatic islets. A number of islet antigens recognized by CD8 T cells that contribute to disease pathogenesis in non‐obese diabetic (NOD) mice have been identified; however, the antigenic specificities of the majority of the islet‐infiltrating cells have yet to be determined. The primary goal of the current study was to identify candidate antigens based on the level and specificity of expression of their genes in mouse islets and in the mouse β cell line MIN6. Peptides derived from the candidates were selected based on their predicted ability to bind H‐2Kd and were examined for recognition by islet‐infiltrating T cells from NOD mice. Several proteins, including those encoded by Abcc8, Atp2a2, Pcsk2, Peg3 and Scg2, were validated as antigens in this way. Interestingly, islet‐infiltrating T cells were also found to recognize peptides derived from proglucagon, whose expression in pancreatic islets is associated with α cells, which are not usually implicated in type 1 diabetes pathogenesis. However, type 1 diabetes patients have been reported to have serum autoantibodies to glucagon, and NOD mouse studies have shown a decrease in α cell mass during disease pathogenesis. Our finding of islet‐infiltrating glucagon‐specific T cells is consistent with these reports and suggests the possibility of α cell involvement in development and progression of disease.

Beyond HLA-A*0201: New HLA-Transgenic Nonobese Diabetic Mouse Models of Type 1 Diabetes Identify the Insulin C-Peptide as a Rich Source of CD8+ T Cell Epitopes

Type 1 diabetes is an autoimmune disease characterized by T cell responses to β cell Ags, including insulin. Investigations employing the NOD mouse model of the disease have revealed an essential role for β cell-specific CD8+ T cells in the pathogenic process. As CD8+ T cells specific for β cell Ags are also present in patients, these reactivities have the potential to serve as therapeutic targets or markers for autoimmune activity. NOD mice transgenic for human class I MHC molecules have previously been employed to identify T cell epitopes having important relevance to the human disease. However, most studies have focused exclusively on HLA-A*0201. To broaden the reach of epitope-based monitoring and therapeutic strategies, we have looked beyond this allele and developed NOD mice expressing human β2-microglobulin and HLA-A*1101 or HLA-B*0702, which are representative members of the A3 and B7 HLA supertypes, respectively. We have used islet-infiltrating T cells spontaneously arising in these strains to identify β cell peptides recognized in the context of the transgenic HLA molecules. This work has identified the insulin C-peptide as an abundant source of CD8+ T cell epitopes. Responses to these epitopes should be of considerable utility for immune monitoring, as they cannot reflect an immune reaction to exogenously administered insulin, which lacks the C-peptide. Because the peptides bound by one supertype member were found to bind certain other members also, the epitopes identified in this study have the potential to result in therapeutic and monitoring tools applicable to large numbers of patients and at-risk individuals.

Oligomerization of Vibrio Cholerae Hemolysin Induces CXCR3 Upregulation and Activation of B-1a Cell

The hemolysin oligomer promotes the proliferation of B-1a cells and the expression of CD25, which is indicative of cell activation, on B-1a cells. The upregulation of CD86 induced by the oligomer showed its selective bias for the B7-2 member of B7 family while the monomer failed to induce these effects. The oligomer induced the expression of CXCR3, associated with B cell activation, while the monomer induced the expression of CXCL4, a powerful angiostatic chemokine. In conclusion, we found that B-1a cells responded to the apoptogenic monomer by expressing CXCL4, whereas oligomerization of the immunogen induced CXCR3 to shift the response towards activation.