Wendy W. J. Unger

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.

Induction of Treg by monocyte-derived DC modulated by vitamin D3 or dexamethasone: differential role for PD-L1.

Specific therapy with modulated DC may restore immunological tolerance, thereby obviating the need for chronic immunosuppression in transplantation or autoimmunity. In this study we compared the tolerizing capacity of dexamethasone (Dex)‐ and 1α,25‐dihydroxyvitamin D3 (VD3)‐modulated DC. Treatment of monocytes with either VD3 or Dex resulted in DC with stable, semi‐mature phenotypes compared with standard DC, with intermediate levels of co‐stimulatory and MHC class II molecules, which remained unaltered after subsequent pro‐inflammatory stimulation. IL‐12p70 secretion was lost by VD3‐ and Dex‐DC, whereas IL‐10 secretion was unaffected. VD3‐DC distinctly produced large amounts of TNF‐α. Both VD3‐ and Dex‐DC possessed the capacity to convert CD4 T cells into IL‐10‐secreting Treg potently suppressing the proliferation of responder T cells. However, only Treg induced by VD3‐DC exhibited antigen specificity. VD3‐, but not Dex‐, DC expressed significant high levels of PD‐L1 (programmed death‐1 ligand), upon activation. Blockade of PD‐L1 during priming redirected T cells to produce IFN‐γ instead of IL‐10 and abolished acquisition of regulatory capacity. Our findings demonstrate that both VD3‐ and Dex‐DC possess durable but differential tolerogenic features, acting via different mechanisms. Both are potentially useful to specifically down‐regulate unwanted immune responses and induce immune tolerance. These modulated DC appear suitable as adjuvant in antigen‐specific clinical vaccination intervention strategies.

Simultaneous detection of circulating autoreactive CD8+ T-cells specific for different islet cell-associated epitopes using combinatorial MHC multimers.

OBJECTIVE Type 1 diabetes results from selective T-cell–mediated destruction of the insulin-producing β-cells in the pancreas. In this process, islet epitope–specific CD8+ T-cells play a pivotal role. Thus, monitoring of multiple islet–specific CD8+ T-cells may prove to be valuable for measuring disease activity, progression, and intervention. Yet, conventional detection techniques (ELISPOT and HLA tetramers) require many cells and are relatively insensitive. RESEARCH DESIGN AND METHODS Here, we used a combinatorial quantum dot major histocompatibility complex multimer technique to simultaneously monitor the presence of HLA-A2 restricted insulin B10–18, prepro-insulin (PPI)15–24, islet antigen (IA)-2797–805, GAD65114–123, islet-specific glucose-6-phosphatase catalytic subunit–related protein (IGRP)265–273, and prepro islet amyloid polypeptide (ppIAPP)5–13–specific CD8+ T-cells in recent-onset diabetic patients, their siblings, healthy control subjects, and islet cell transplantation recipients. RESULTS Using this kit, islet autoreactive CD8+ T-cells recognizing insulin B10–18, IA-2797–805, and IGRP265–273 were shown to be frequently detectable in recent-onset diabetic patients but rarely in healthy control subjects; PPI15–24 proved to be the most sensitive epitope. Applying the “Diab-Q-kit” to samples of islet cell transplantation recipients allowed detection of changes of autoreactive T-cell frequencies against multiple islet cell–derived epitopes that were associated with disease activity and correlated with clinical outcome. CONCLUSIONS A kit was developed that allows simultaneous detection of CD8+ T-cells reactive to multiple HLA-A2–restricted β-cell epitopes requiring limited amounts of blood, without a need for in vitro culture, that is applicable on stored blood samples.

TLR Triggering on Tolerogenic Dendritic Cells Results in TLR2 Up-Regulation and a Reduced Proinflammatory Immune Program

Tolerogenic dendritic cells (TDC) offer a promising therapeutic potential to ameliorate autoimmune diseases. Reported to inhibit adaptive immune responses, little is known about their innate immunity receptor repertoire. In this study, we compared three types of human TDC (IL-10-DC, dexamethasone (DX)-DC, and 1,25(OH)2D3-DC) by their TLR expression and response to a set of TLR ligands. TDC are endowed with the same TLR set as standard monocyte-derived dendritic cells but respond differentially to the TLR stimuli Pam3CSK4, polyinosinic-polycytidylic acid, LPS, and flagellin. TDC expressed low or no IL-12-related cytokines and remarkably elevated IL-10 levels. Interestingly, only TDC up-regulated the expression of TLR2 upon stimulation. This boosted the tolerogenic potential of these cells, because IL-10 production was up-regulated in TLR2-stimulated, LPS-primed DX-DC, whereas IL-12 and TNF-α secretion remained low. When comparing the TDC subsets, DX-DC and 1,25(OH)2D3-DC up-regulated TLR2 irrespective of the TLR triggered, whereas in IL-10-DC this effect was only mediated by LPS. Likewise, DX-DC and 1,25(OH)2D3-DC exhibited impaired ability to mature, reduced allostimulatory properties, and hampered capacity to induce Th1 differentiation. Therefore, both DX-DC and 1,25(OH)2D3-DC display the strongest tolerogenic and anti-inflammatory features and might be most suitable tools for the treatment of autoimmune diseases.

Islet-Specific CTL Cloned from a Type 1 Diabetes Patient Cause Beta-Cell Destruction after Engraftment into HLA-A2 Transgenic NOD/SCID/IL2RG Null Mice

Despite increasing evidence that autoreactive CD8 T-cells are involved in both the initiation of type 1 diabetes (T1D) and the destruction of beta-cells, direct evidence for their destructive role in-vivo is lacking. To address a destructive role for autoreactive CD8 T-cells in human disease, we assessed the pathogenicity of a CD8 T-cell clone derived from a T1D donor and specific for an HLA-A2-restricted epitope of islet-specific glucose-6-phosphatase catalytic-subunit related protein (IGRP). HLA-A2/IGRP tetramer staining revealed a higher frequency of IGRP-specific CD8 T-cells in the peripheral blood of recent onset human individuals than of healthy donors. IGRP265–273-specific CD8 T-cells that were cloned from the peripheral blood of a recent onset T1D individual were shown to secrete IFNγ and Granzyme B after antigen-specific activation and lyse HLA-A2-expressing murine islets in-vitro. Lytic capacity was also demonstrated in-vivo by specific killing of peptide-pulsed target cells. Using the HLA-A2 NOD-scid IL2rγnull mouse model, HLA-A2-restricted IGRP-specific CD8 T-cells induced a destructive insulitis. Together, this is the first evidence that human HLA-restricted autoreactive CD8 T-cells target HLA-expressing beta-cells in-vivo, demonstrating the translational value of humanized mice to study mechanisms of disease and therapeutic intervention strategies.

Discovery of low-affinity preproinsulin epitopes and detection of autoreactive CD8 T-cells using combinatorial MHC multimers.

Autoreactive cytotoxic CD8 T-cells (CTLs) play a key pathogenic role in the destruction of insulin-producing beta-cells resulting in type 1 diabetes. However, knowledge regarding their targets is limited, restricting the ability to monitor the course of the disease and immune interventions. In a multi-step discovery process to identify novel CTL epitopes in human preproinsulin (PPI), PPI was digested with purified human proteasomes, and resulting COOH-fragments aligned with algorithm-predicted HLA-binding peptides to yield nine potential HLA-A1, -A2, -A3 or -B7-restricted candidates. An UV-exchange method allowed the generation of a repertoire of multimers including low-affinity HLA-binding peptides. These were labeled with quantum dot-fluorochromes and encoded in a combinatorial fashion, allowing parallel and sensitive detection of specific, low-avidity T-cells. Significantly increased frequencies of T-cells against four novel PPI epitopes (PPI(4-13)/B7, PPI(29-38)/A2, PPI(76-84)/A3 and PPI(79-88)/A3) were detected in stored blood of patients with recent onset diabetes but not in controls. Changes in frequencies of circulating CD8 T-cells against these novel epitopes were detected in blood of islet graft recipients at different time points after transplantation, which correlated with clinical outcome. In conclusion, our novel strategy involving a sensitive multiplex detection technology and requiring minimal volumes of stored blood represents a major improvement in the direct ex-vivo characterization and enumeration of immune cells in the pathogenesis of type 1 diabetes.

Human Clonal CD8 Autoreactivity to an IGRP Islet Epitope Shared between Mice and Men

Abstract:u2002 Type 1 diabetes (T1D) is a multifactorial disease characterized by the infiltration and subsequent destruction of the pancreatic insulin‐producing β cells by autoreactive T cells. CD8+ T cells play an essential role in this β cell destruction. However, little is known about the target antigens of CD8+ T cells in human T1D patients. The aim of this study was to assess whether an epitope derived from the islet‐specific glucose‐6‐phosphatase catalytic subunit–related protein (IGRP), IGRP265‐273, which has recently been identified as a target in non‐obese diabetic (NOD) mice and is fully homologous to the human epitope, is a target of human diabetogenic CD8+ T cells. We isolated a human CD8 T cell clone against this epitope, which confirms that this IGRP epitope is shared across species.

Glycan-based DC-SIGN targeting to enhance antigen cross-presentation in anticancer vaccines

In vivo dendritic-cell targeting constitutes a promising strategy for anticancer vaccination. Here, we discuss the usage of multivalent DC-SIGN-targeting glycan platforms that allow for the efficient routing of antigens to the endo-lysosomal pathway as well as to a yet uncharacterized cross-presentation mechanism inducing CD4+ and CD8+ T-cell responses.

Tumor sialylation impedes T cell mediated anti-tumor responses while promoting tumor associated-regulatory T cells

The increased presence of sialylated glycans on the tumor surface has been linked to poor prognosis, yet the effects on tumor-specific T cell immunity are hardly studied. We here show that hypersialylation of B16 melanoma substantially influences tumor growth by preventing the formation of effector T cells and facilitating the presence of high regulatory T cell (Treg) frequencies. Knock-down of the sialic acid transporter created “sialic acid low” tumors, that grew slower in-vivo than hypersialylated tumors, altered the Treg/Teffector balance, favoring immunological tumor control. The enhanced effector T cell response in developing “sialic acid low” tumors was preceded by and dependent on an increased influx and activity of Natural Killer (NK) cells. Thus, tumor hypersialylation orchestrates immune escape at the level of NK and Teff/Treg balance within the tumor microenvironment, herewith dampening tumor-specific T cell control. Reducing sialylation provides a therapeutic option to render tumors permissive to immune attack.

Original ArticleIn situ Delivery of Antigen to DC-SIGN+CD14+ Dermal Dendritic Cells Results in Enhanced CD8+ T-Cell Responses

CD14(+) dendritic cells (DCs) present in the dermis of human skin represent a large subset of dermal DCs (dDCs) that are considered macrophage-like cells with poor antigen (cross)-presenting capacity and limited migratory potential to the lymph nodes. CD14(+) dDC highly express DC-specific ICAM-3-grabbing non-integrin (DC-SIGN), a receptor containing potent endocytic capacity, facilitating intracellular routing of antigens to major histocompatibility complex I and II (MHC-I andII) loading compartments for the presentation to antigen-specific CD8(+) and CD4(+) T cells. Here we show using a human skin explant model that the in situ targeting of antigens to DC-SIGN using glycan-modified liposomes enhances the antigen-presenting capacity of CD14(+) dDCs. Intradermal vaccination of liposomes modified with the DC-SIGN-targeting glycan Lewis(X), containing melanoma antigens (MART-1 or Gp100), accumulated in CD14(+) dDCs and resulted in enhanced Gp100- or MART-1-specific CD8(+) T-cell responses. Simultaneous intradermal injection of the cytokines GM-CSF and IL-4 as adjuvant enhanced the migration of the skin DCs and increased the expression of DC-SIGN on the CD14(+) and CD1a(+) dDCs. These data demonstrate that human CD14(+) dDCs exhibit potent cross-presenting capacity when targeted in situ through DC-SIGN.