Richard Ellis

CTLs are targeted to kill β cells in patients with type 1 diabetes through recognition of a glucose-regulated preproinsulin epitope

The final pathway of beta cell destruction leading to insulin deficiency, hyperglycemia, and clinical type 1 diabetes is unknown. Here we show that circulating CTLs can kill beta cells via recognition of a glucose-regulated epitope. First, we identified 2 naturally processed epitopes from the human preproinsulin signal peptide by elution from HLA-A2 (specifically, the protein encoded by the A*0201 allele) molecules. Processing of these was unconventional, requiring neither the proteasome nor transporter associated with processing (TAP). However, both epitopes were major targets for circulating effector CD8+ T cells from HLA-A2+ patients with type 1 diabetes. Moreover, cloned preproinsulin signal peptide-specific CD8+ T cells killed human beta cells in vitro. Critically, at high glucose concentration, beta cell presentation of preproinsulin signal epitope increased, as did CTL killing. This study provides direct evidence that autoreactive CTLs are present in the circulation of patients with type 1 diabetes and that they can kill human beta cells. These results also identify a mechanism of self-antigen presentation that is under pathophysiological regulation and could expose insulin-producing beta cells to increasing cytotoxicity at the later stages of the development of clinical diabetes. Our findings suggest that autoreactive CTLs are important targets for immune-based interventions in type 1 diabetes and argue for early, aggressive insulin therapy to preserve remaining beta cells.

Plasmacytoid Dendritic Cells Are Proportionally Expanded at Diagnosis of Type 1 Diabetes and Enhance Islet Autoantigen Presentation to T-Cells Through Immune Complex Capture

OBJECTIVE—Immune-mediated destruction of β-cells resulting in type 1 diabetes involves activation of proinflammatory, islet autoreactive T-cells, a process under the control of dendritic cells of the innate immune system. We tested the hypothesis that type 1 diabetes development is associated with disturbance of blood dendritic cell subsets that could enhance islet-specific autoimmunity. RESEARCH DESIGN AND METHODS—We examined blood dendritic cells (plasmacytoid and myeloid) in 40 patients with recent-onset diabetes (median duration 28 days) and matched control subjects. We also examined the relative ability of different dendritic cell subsets to process and present soluble or immune complexed islet cell autoantigen (the islet tyrosine phosphatase IA-2) to responder CD4 T-cells. RESULTS—The balance of blood dendritic cells was profoundly disturbed at diabetes diagnosis, with a significantly elevated proportion of plasmacytoid and reduction of myeloid cells compared with control subjects. Dendritic cell subset distribution was normal in long-standing disease and in patients with type 2 diabetes. Both dendritic cell subsets processed and presented soluble IA-2 to CD4 T-cells after short-term culture, but only plasmacytoid dendritic cells enhanced (by as much as 100%) autoantigen presentation in the presence of IA-2+ autoantibody patient serum. CONCLUSIONS—The plasmacytoid subset of dendritic cells is overrepresented in the blood close to diabetes onset and shows a distinctive ability to capture islet autoantigenic immune complexes and enhance autoantigen-driven CD4 T-cell activation. This suggests a synergistic proinflammatory role for plasmacytoid dendritic cells and islet cell autoantibodies in type 1 diabetes.

Circulating, Preproinsulin Signal Peptide–Specific CD8 T Cells Restricted by the Susceptibility Molecule HLA-A24 Are Expanded at Onset of Type 1 Diabetes and Kill β-Cells

Type 1 diabetes results from T cell–mediated β-cell destruction. The HLA-A*24 class I gene confers significant risk of disease and early onset. We tested the hypothesis that HLA-A24 molecules on islet cells present preproinsulin (PPI) peptide epitopes to CD8 cytotoxic T cells (CTLs). Surrogate β-cell lines secreting proinsulin and expressing HLA-A24 were generated and their peptide ligandome examined by mass spectrometry to discover naturally processed and HLA-A24–presented PPI epitopes. A novel PPI epitope was identified and used to generate HLA-A24 tetramers and examine the frequency of PPI-specific T cells in new-onset HLA-A*24+ patients and control subjects. We identified a novel naturally processed and HLA-A24–presented PPI signal peptide epitope (PPI3–11; LWMRLLPLL). HLA-A24 tetramer analysis reveals a significant expansion of PPI3–11-specific CD8 T cells in the blood of HLA-A*24+ recent-onset patients compared with HLA-matched control subjects. Moreover, a patient-derived PPI3–11-specific CD8 T-cell clone shows a proinflammatory phenotype and kills surrogate β-cells and human HLA-A*24+ islet cells in vitro. These results indicate that the type 1 diabetes susceptibility molecule HLA-A24 presents a naturally processed PPI signal peptide epitope. PPI-specific, HLA-A24–restricted CD8 T cells are expanded in patients with recent-onset disease. Human islet cells process and present PPI3–11, rendering themselves targets for CTL-mediated killing.

Comparison of Regulatory T Cells in Hemodialysis Patients and Healthy Controls: Implications for Cell Therapy in Transplantation

BACKGROUND AND OBJECTIVES Cell-based therapy with natural (CD4(+)CD25(hi)CD127(lo)) regulatory T cells to induce transplant tolerance is now technically feasible. However, regulatory T cells from hemodialysis patients awaiting transplantation may be functionally/numerically defective. Human regulatory T cells are also heterogeneous, and some are able to convert to proinflammatory Th17 cells. This study addresses the suitability of regulatory T cells from hemodialysis patients for cell-based therapy in preparation for the first clinical trials in renal transplant recipients (the ONE Study). DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Healthy controls and age- and sex-matched hemodialysis patients without recent illness/autoimmune disease on established, complication-free hemodialysis for a minimum of 6 months were recruited. Circulating regulatory T cells were studied by flow cytometry to compare the regulatory T cell subpopulations. Regulatory T cells from members of each group were compared for suppressive function and plasticity (IL-17-producing capacity) before and after in vitro expansion with and without Rapamycin, using standard assays. RESULTS Both groups had similar total regulatory T cells and subpopulations I and III. In each subpopulation, regulatory T cells expressed similar levels of the function-associated markers CD27, CD39, HLA-DR, and FOXP3. Hemodialysis regulatory T cells were less suppressive, expanded poorly compared with healthy control regulatory T cells, and produced IL-17 in the absence of Rapamycin. However, Rapamycin efficiently expanded hemodialysis regulatory T cells to a functional and stable cell product. CONCLUSIONS Rapamycin-based expansion protocols should enable clinical trials of cell-based immunotherapy for the induction of tolerance to renal allografts using hemodialysis regulatory T cells.

Phenotypic Complexity of the Human Regulatory T Cell Compartment Revealed by Mass Cytometry

Regulatory T cells (Tregs) are an essential component of the cellular immune response, occupying a key role in maintaining immunological tolerance and present an attractive therapeutic target in a range of immunopathologies. Comprehensive analysis of the human Treg compartment has been restricted due to technical limitations. The advent of mass cytometry enables simultaneous assessment of vastly increased phenotypic parameters at single-cell resolution. In this study, we used mass cytometry to examine the complexity of human Tregs using an extensive panel of surface markers associated with Treg function and phenotype. We applied unsupervised clustering analysis, revealing 22 distinct subpopulations of Tregs, representing previously identified and novel subpopulations. Our data represent the most in-depth phenotypic description of the human Treg compartment at single-cell resolution and show a hitherto unrecognized degree of phenotypic complexity among cells of the regulatory lineage.

Acquisition of MHC:Peptide Complexes by Dendritic Cells Contributes to the Generation of Antiviral CD8+ T Cell Immunity In Vivo

There is an increasing body of evidence suggesting that the transfer of preformed MHC class I:peptide complexes between a virus-infected cell and an uninfected APC, termed cross-dressing, represents an important mechanism of Ag presentation to CD8+ T cells in host defense. However, although it has been shown that memory CD8+ T cells can be activated by uninfected dendritic cells (DCs) cross-dressed by Ag from virus-infected parenchymal cells, it is unknown whether conditions exist during virus infection in which naive CD8+ T cells are primed and differentiate to cytolytic effectors through cross-dressing, and indeed which DC subset would be responsible. In this study, we determine whether the transfer of MHC class I:peptide complexes between infected and uninfected murine DC plays a role in CD8+ T cell priming to viral Ags in vivo. We show that MHC class I:peptide complexes from peptide-pulsed or virus-infected DCs are indeed acquired by splenic CD8α− DCs in vivo. Furthermore, the acquired MHC class I:peptide complexes are functional in that they induced Ag-specific CD8+ T cell effectors with cytolytic function. As CD8α− DCs are poor cross-presenters, this may represent the main mechanism by which CD8α− DCs present exogenously encountered Ag to CD8+ T cells. The sharing of Ag as preformed MHC class I:peptide complexes between infected and uninfected DCs without the restraints of Ag processing may have evolved to accurately amplify the response and also engage multiple DC subsets critical in the generation of strong antiviral immunity.

Deep phenotyping of Tregs identifies an immune signature for idiopathic aplastic anemia and predicts response to treatment

Idiopathic aplastic anemia (AA) is an immune-mediated and serious form of bone marrow failure. Akin to other autoimmune diseases, we have previously shown that in AA regulatory T cells (Tregs) are reduced in number and function. The aim of this study was to further characterize Treg subpopulations in AA and investigate the potential correlation between specific Treg subsets and response to immunosuppressive therapy (IST) as well as their in vitro expandability for potential clinical use. Using mass cytometry and an unbiased multidimensional analytical approach, we identified 2 specific human Treg subpopulations (Treg A and Treg B) with distinct phenotypes, gene expression, expandability, and function. Treg B predominates in IST responder patients, has a memory/activated phenotype (with higher expression of CD95, CCR4, and CD45RO within FOXP3(hi), CD127(lo) Tregs), expresses the interleukin-2 (IL-2)/STAT5 pathway and cell-cycle commitment genes. Furthermore, in vitro-expanded Tregs become functional and take on the characteristics of Treg B. Collectively, this study identifies human Treg subpopulations that can be used as predictive biomarkers for response to IST in AA and potentially other autoimmune diseases. We also show that Tregs from AA patients are IL-2-sensitive and expandable in vitro, suggesting novel therapeutic approaches such as low-dose IL-2 therapy and/or expanded autologous Tregs and meriting further exploration.

Hla class II molecules on haplotypes associated with type 1 diabetes exhibit similar patterns of binding affinities for coxsackievirus P2C peptides

Enteroviruses such as coxsackievirus B4 (CVB4) are proposed as possible environmental triggers or accelerants of the autoimmune process that leads to type 1 diabetes mellitus. One putative mechanism to account for this association is mimicry between virus components and islet autoantigens. Particular interest has focused on the CVB4 non‐structural protein P2C, which we previously showed to be a major target of the effector memory anti‐CVB4 CD4 T‐cell response, and which harbours a region of sequence similarity with the islet autoantigen, glutamic acid decarboxylase (GAD65). Since several distinct human leucocyte antigen (HLA) Class II molecules are associated with development of type 1 diabetes, we hypothesized that for functional mimicry to be important, any potential region(s) of mimicry in P2C should bind to each of these susceptibility molecules. In the present study therefore we examined the affinity of 20‐mer overlapping P2C peptides for soluble HLA‐DR4, ‐DR3, ‐DQ2 and ‐DQ8. We identified one discrete region of P2C with high binding affinities for all of these HLA Class II molecules. Moreover, the binding affinity of P2C peptides was significantly correlated between HLA molecules present on the same susceptibility haplotype (e.g. DR4 and DQ8, P =0·0076; DR3 and DQ2 P = 0·002). We conclude that possession of these haplotypes favours restricted presentation of viral epitopes, and speculate that this could promote the potential for mimicry between microbial proteins and islet autoantigens.

Spatiotemporal segregation of human marginal zone and memory B cell populations in lymphoid tissue

Human memory B cells and marginal zone (MZ) B cells share common features such as the expression of CD27 and somatic mutations in their IGHV and BCL6 genes, but the relationship between them is controversial. Here, we show phenotypic progression within lymphoid tissues as MZ B cells emerge from the mature naïve B cell pool via a precursor CD27−CD45RBMEM55+ population distant from memory cells. By imaging mass cytometry, we find that MZ B cells and memory B cells occupy different microanatomical niches in organised gut lymphoid tissues. Both populations disseminate widely between distant lymphoid tissues and blood, and both diversify their IGHV repertoire in gut germinal centres (GC), but nevertheless remain largely clonally separate. MZ B cells are therefore not developmentally contiguous with or analogous to classical memory B cells despite their shared ability to transit through GC, where somatic mutations are acquired.Human memory and marginal zone B cells share some features including CD27 expression and somatic hypermutation, but their lineage relationship is still unclear. Here the authors use mass cytometry and sequential clustering methods to show that, despite their shared features, memory and marginal zone B cells represent distinct lineage choices.