In this study, we examined the potential of enhanced CTLA-4 ligation by β-cell antigen presenting DCs in modulating autoimmunity in a spontaneous model of T1D. We show that treatment using pancreatic β-cell antigen-pulsed DCs coated with agonistic anti-CTLA-4 Ab can significantly delay T1D in NOD mice. This delay in hyperglycemia appears to be dependent on the suppressor function of Tregs induced and/or expanded upon enhanced engagement of CTLA-4 on effector T cells. Our observations demonstrate the potential of approaches to enhance the strength of CTLA-4 specific ligand on professional APCs to induce T cell tolerance and modulation of autoimmunity in T1D.
Susceptibility to T1D and other autoimmune diseases has been linked to CTLA-4 gene polymorphism (2
). In addition, expression levels of soluble and ligand-independent forms of CTLA-4 have been implicated for the lack of peripheral tolerance to self-antigens (17
). Previous studies have also suggested that T1D susceptibility is associated with an insufficient T cell regulation through CTLA-4 due to the expression of variants of CTLA-4, ligand strength on APCs and defects in T cell activation (17
). On the other hand, studies have shown that peripheral and ex vivo
generated DCs of T1D patients and NOD mice are defective and, perhaps, deficient in their ability to provide effective help to natural Tregs (40
). Our studies demonstrating that the natural Treg function can be enhanced in autoimmune models by modulating DC function supported this notion (43
). Based on these observations, we hypothesized that enhancing the CTLA-4 engagement strength through increasing its selective ligand intensity on pancreatic β-cell antigen presenting APCs would have a modulatory effect on autoreactive T cells. Our recent study using immunization models showing robust adaptive Treg induction upon treatment with antigen pulsed agonistic anti-CTLA-4 Ab coated DCs (16
) further supported this notion. Therefore, to realize the clinical applicability of this approach, we examined whether enhancing the CTLA-4 specific ligand strength on pancreatic β-cell antigen presenting DCs using an agonistic Ab can modulate spontaneously occurring autoimmunity and T1D in NOD mice.
In spite of the widely reported abnormalities in DC function and CTLA-4 signaling in NOD mice, our observations show that antigen presentation by anti-CTLA-4 Ab coated NOD mouse DCs can suppress the proliferation of β-cell antigen specific T cells from the spleen cells of diabetic mice. Enhanced CTLA-4 engagement during β-cell antigen presentation not only resulted in the production of significant amounts suppressor cytokines such as of IL-10 and TGF-β1, but also suppressed IL-2 and IFN-γ responses. These findings indicated that the T cells are activated upon antigen recognition, but the cytokine profile is skewed as a result of enhanced CTLA-4 engagement. Importantly, activation of T cells by natural ligands expressed on antigen presenting DCs is critical for providing β-cell antigen specific signal to achieve the primary goal of selectively modulating autoreactive T cell function.
Although several immunodominant self-antigenic peptides are identified in T1D, it is widely believed that autoimmune response in T1D might be directed against a wide array of β-cell associated proteins. Therefore, use of one or a few known antigenic peptides for controlling T1D may have a limited therapeutic value. Nevertheless, the ability of Ag-pulsed anti-CTLA-4 DCs to induce significant amounts of suppressor cytokines and concomitantly suppress effector cytokine production in T cells from diabetic mice in vitro prompted us to examine whether these DCs can delay the onset and/or treat hyperglycemia in pre-diabetic and diabetic NOD mice. A significant delay in the onset of hyperglycemia observed in pre-diabetic mice that received Ag-pulsed anti-CTLA-4-Ab DCs suggests that the autoimmune response is suppressed in these mice, which prolongs the normal functioning of remaining β-cells. However, suppression of autoimmunity using Ag-pulsed anti-CTLA-4-Ab DCs alone does not appear to be sufficient to reverse established hyperglycemia perhaps due to the loss of a majority of the functional islets prior to the initiation of the treatment. Combinational approaches such as therapy to enhance β-cell mass and function along with DC directed CTLA-4 engagement might hold the potential for reversing already established hyperglycemia.
Relentless destruction of β-cells through apoptosis and sustained endogenous antigen presentation in the pancreatic microenvironment leads to continued β-cell destruction under inflammatory conditions and contribute to the progression of T1D (47
). Therefore, it is possible that β-cell antigens are continuously released from dying or dead cells. If sufficient amounts of β cell antigens are released then treatment with DCs that are not loaded with antigenic peptide might also be able to produce similar protective effect by capturing, processing, and presenting endogenous β-cell antigens to T cells. However, our observations show that β-cell antigen-pulsed DCs, but not DCs that were not pulsed with antigen, modulated the disease outcome (either aggravation or suppression) indicating that mature DCs do not capture sufficient amounts of β-cell antigens in vivo
and present to pathogenic T cells. Therefore, loading the DCs with antigenic peptide prior to anti-CTLA-4 Ab coating and injection is critical for achieving a significant therapeutic effect.
T cells from NOD mice treated with Ag-pulsed anti-CTLA-4-Ab DCs produced significant amounts of IL-10 and TGF-β1 suggesting that these cytokines and T cells may be responsible for protecting remaining functional β cells in pre-diabetic mice. These T cells appeared to be hypo-proliferative in nature as indicated by their inability to proliferate significantly upon challenge with β-cell antigenic peptides. While the suppressor cytokine response by T cells exposed to Ag-pulsed anti-CTLA-4-Ab DCs was significantly higher, the effector cytokine (IFN-γ) response was lower when compared to T cells from control-Ab DC recipients. This suggests that enhanced CTLA-4 engagement upon antigen presentation can skew the T cell response from pathogenic towards suppressor type.
The DCs are recognized as the only type of APCs that are capable of activating naïve T cells against a particular antigen. Although upon inoculation Ag-pulsed anti-CTLA-4-Ab DCs may primarily target existing memory T cells, it is possible that they will also activate naïve T cells through β-cell antigen presentation. Therefore, we assume that while Ag-pulsed control-Ab DCs can induce new pathogenic T cells from naïve cells along with expansion of existing memory T cells, Ag-pulsed anti-CTLA-4-Ab DCs may induce and/or expand antigen specific T cells with suppressor phenotype. This may partially explain why significantly higher number of T cells from control DC recipient mice compared to untreated control mice proliferated upon challenge with β-cell antigen. More severe insulitis in Ag-pulsed control-Ab DC recipient mice compared to untreated mice is also indicative of this effect. This is in contrast to the induction of hypo-proliferative T cells with suppressor phenotype that could delay the progression of insulitis upon treatment with Ag-pulsed anti-CTLA-4-Ab DCs.
Our earlier study using animals immunized with foreign- or self-antigen has shown that profound numbers of adaptive Tregs with Foxp3 expression and surface bound TGF-β1 are induced upon treatment using cognate Ag-pulsed anti-CTLA-4-Ab DCs (16
). In contrast, current study using spontaneous autoimmune diabetic model showed no significant difference in the frequency of T cells with surface bound TGF-β1 in test and control groups of mice. Further, Treg induced and/or expanded in the spontaneous NOD model does not appear to be as robust as those we have found in the immunization induced disease model. This difference might be attributable to a relatively fewer antigen specific T cells in the former model. Further, in the current study, we used only a limited number of self-antigenic peptides and therefore T cells with specificities towards these peptides, which may represent only a small proportion of the pathogenic T cell repertoire present in this spontaneous disease model, were affected. Yet another explanation for the difference in the frequencies of Tregs in an immunization model versus a spontaneous model upon treatment using Ag-pulsed anti-CTLA-4-Ab DCs could be the difference in the amount of IL-2 and/or TGF-β1 produced in the lymphoid microenvironment during enhanced CTLA-4 engagement. As suggested by our in vitro
experiments, both IL-2 and TGF-β1 can promote enhanced CTLA-4 engagement-mediated induction of T cells with regulatory properties. Since the overall levels of IL-2 and TGF-β1 in an immunization model are expected to be higher due to the presence of large numbers of antigen specific T cells compared to a spontaneous model, we believe that these cytokines may be contributing to a robust Treg response in the former model.
Irrespective of the above described difference in the Treg frequencies, current study shows relatively higher frequencies of Foxp3+ and IL-10+ T cells in freshly isolated spleen and PnLN of Ag-pulsed anti-CTLA-4-Ab DC recipient NOD mice compared to Ag-pulsed control-Ab DC recipients. In addition, as observed in our earlier study, T cells from Ag-pulsed anti-CTLA-4-Ab DC recipient mice secreted higher amounts of IL-10 and TGF-β1 upon challenge with antigen. This suggested that enhancing CTLA-4 specific ligand strength on DCs could be an effective way of promoting self-antigen specific tolerance.
Although self-antigen specific T cell tolerance is the key feature of our DC directed CTLA-4 engagement approach, we could not achieve a lasting protection from diabetes in NOD mice using a short-term treatment that targeted a limited repertoire of T cells with known antigen specificity. Failure to achieve long-term benefit may be due to the inherent defects in the NOD immune system, repertoire spreading to include other peptide specificities, regeneration and/or re-activation of autoreative T cells. Targeting a significant portion of autoreactive T cells by pulsing the DCs with a cocktail of full-length pancreatic β-cell antigens instead of selected peptides and/or intermittent injections with these therapeutic DCs may prove effective in achieving a lasting protection from T1D. Importantly, a robust increase in functional Treg frequency that may produce long lasting protection from the disease in anti-CTLA-4 Ab DC treated mice is hindered by enhanced CTLA-4 signaling mediated suppression of IL-2 secretion by T cells. Although enhanced CTLA-4 engagement upon antigen presentation can result in the production of significant amounts of IL-10 and TGF-β1 by T cells, this cytokine milieu alone does not appear to be sufficient to promote a robust Treg response even with a suppressed level of inflammatory cytokines such as IFN-γ. Therefore based on our observations showing the ability of exogenous IL-2 to promote a profound increase in Foxp3+ and IL-10+ T cell frequencies in anti-CTLA-4 Ab DC containing cultures, we believe that therapeutic efficacy of DC directed CTLA-4 engagement approach could be profoundly enhanced by co-administration of recombinant IL-2. Future studies are important and required to address this notion.
Approaches to modulate co-stimulatory and co-inhibitory functions are considered very effective in inducing T cell tolerance for treating autoimmunity and transplant rejection. While blockade of these pathways have shown therapeutic potential in various conditions, methods to enhance signaling through the dominant T cell repressor-receptor concurrent with TCR engagement has excellent therapeutic potential due to the active nature of T cell down-regulation. In fact, enhancing CTLA-4-specific ligand strength on APCs and target cells aimed at down-regulating T cell response has been tested in both allotransplant and autoimmune models (11
). We have reported that thyroid targeted delivery of anti-CTLA-4 Ab leads to suppression of anti-thyroglobulin immune response and suppression of experimental autoimmune thyroiditis (11
). Others and we have also shown that coating allogeneic cells with anti-CTLA-4 Ab or transfecting these cells to express single chain anti-CTLA-4 Ab on the surface could substantially reduce the immune response against that alloantigen in the recipient mice and induce adaptive Tregs with the ability to produce IL-10 and/or TGF-β1 (12
). In addition, transgenic mice expressing anti-CTLA-4 agonistic Ab on B cells can delay T1D in NOD mice (T1D) (15
). Similarly, co-administration of vector constructs encoding a CTLA-4 specific ligand, B7.1wa and an islet specific protein in a DNA vaccination study has demonstrated the ability to delay hyperglycemia in NOD mice (49
). Our recent study showing that robust adaptive Treg response can be induced using Ag-pulsed anti-CTLA-4-Ab DCs in antigen-primed mice (16
) indicated that enhancing CTLA-4 specific ligand strength on APCs, DCs in particular, is an effective strategy for treating autoimmunity. Importantly, defective DC function, CTLA-4 signaling and the spontaneous onset of the disease, as in human T1D patients (17
), make NOD mouse model of T1D a unique system to demonstrate the therapeutic efficacy of this DC directed CTLA-4 engagement approach. This study also provides additional insights on the mechanism of enhanced CTLA-4 engagement mediated adaptive Treg induction and/or expansion and suggests methods to enhance the therapeutic efficacy of this approach.