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Autoimmune diseases such as rheumatoid arthritis are caused by immune system recognition of self-proteins and subsequent production of effector T cells that recognize and attack healthy tissue. Therapies for these diseases typically utilize broad immune suppression, which can be effective, but which also come with an elevated risk of susceptibility to infection and cancer. T cell recognition of antigens is driven by binding of T cell receptors to peptides displayed on major histocompatibility complex proteins (MHCs) on the cell surface of antigen-presenting cells. Technology for recombinant production of the extracellular domains of MHC proteins and loading with peptides to produce pMHCs has provided reagents for detection of T cell populations, and with the potential for therapeutic intervention. However, production of pMHCs in large quantities remains a challenge and a translational path needs to be established. Here, we demonstrate a fusion protein strategy enabling large-scale production of pMHCs. A peptide corresponding to amino acids 259–273 of collagen II was fused to the N-terminus of the MHC_II beta chain, and the alpha and beta chains were each fused to human IgG4 Fc domains and co-expressed. A tag was incorporated to enable site-specific conjugation. The cytotoxic drug payload, MMAF, was conjugated to the pMHC and potent, peptide-specific killing of T cells that recognize the collagen pMHC was demonstrated with tetramerized pMHC-MMAF conjugates. Finally, these pMHCs were incorporated into MMAF-loaded 3DNA nanomaterials in order to provide a biocompatible platform. Loading and pMHC density were optimized, and peptide-specific T cell killing was demonstrated. These experiments highlight the potential of a pMHC fusion protein-targeted, drug-loaded nanomaterial approach for selective delivery of therapeutics to disease-relevant T cells and new treatment options for autoimmune disease.

Immunological tolerance represents a mechanism by which cells of the host remain protected from the immune system. Breaking of immunological tolerance can result in a variety of autoimmune diseases such as rheumatoid arthritis, diabetes, and multiple sclerosis. The reasons for tolerance breaking down and autoimmune processes arising are largely unknown but of obvious interest for therapeutic intervention of these diseases. Although reversal of the tolerant state is generally unwanted, there are instances where this may be of benefit to the host. In particular, one way a cancerous cell escapes being targeted by the immune system is through tolerance mechanisms that in effect turn off the reactivity of T lymphocytes that can respond to tumor-associated peptides. Thus tolerance represents a major obstacle in developing effective immunotherapy against tumors. The molecules that are involved in regulating immunological tolerance are then of interest as they may be great targets for positively or negatively manipulating the tolerance process.

Autoimmune diseases such as rheumatoid arthritis are caused by immune system recognition of self-proteins and subsequent production of effector T cells that recognize and attack healthy tissue. Therapies for these diseases typically utilize broad immune suppression, which can be effective, but which also come with an elevated risk of susceptibility to infection and cancer. T cell recognition of antigens is driven by binding of T cell receptors to peptides displayed on major histocompatibility complex proteins (MHCs) on the cell surface of antigen-presenting cells. Technology for recombinant production of the extracellular domains of MHC proteins and loading with peptides to produce pMHCs has provided reagents for detection of T cell populations, and with the potential for therapeutic intervention. However, production of pMHCs in large quantities remains a challenge and a translational path needs to be established. Here, we demonstrate a fusion protein strategy enabling large-scale production of pMHCs. A peptide corresponding to amino acids 259-273 of collagen II was fused to the N-terminus of the MHC_II beta chain, and the alpha and beta chains were each fused to human IgG4 Fc domains and co-expressed. A tag was incorporated to enable site-specific conjugation. The cytotoxic drug payload, MMAF, was conjugated to the pMHC and potent, peptide-specific killing of T cells that recognize the collagen pMHC was demonstrated with tetramerized pMHC-MMAF conjugates. Finally, these pMHCs were incorporated into MMAF-loaded 3DNA nanomaterials in order to provide a biocompatible platform. Loading and pMHC density were optimized, and peptide-specific T cell killing was demonstrated. These experiments highlight the potential of a pMHC fusion protein-targeted, drug-loaded nanomaterial approach for selective delivery of therapeutics to disease-relevant T cells and new treatment options for autoimmune disease.

Protein kinase Cθ (PKCθ) regulates a key step in the activation of T cells. On the basis of its mechanism of action, inhibition of this kinase is hypothesized to serve as an effective therapy for autoimmune diseases such as rheumatoid arthritis (RA), inflammatory bowel disease (IBD), and psoriasis. Herein, the discovery of a small molecule PKCθ inhibitor is described, starting from a fragment hit 1 and advancing to compound 41 through the use of structure-based drug design. Compound 41 demonstrates excellent in vitro activity, good oral pharmacokinetics, and efficacy in both an acute in vivo mechanistic model and a chronic in vivo disease model but suffers from tolerability issues upon chronic dosing.

We previously demonstrated that selective inhibition of protein kinase Cθ (PKCθ) with triazinone 1 resulted in dose-dependent reduction of paw swelling in a mouse model of arthritis.1,2 However, a high concentration was required for efficacy, thus providing only a minimal safety window. Herein we describe a strategy to deliver safer compounds based on the hypothesis that optimization of potency in concert with good oral pharmacokinetic (PK) properties would enable in vivo efficacy at reduced exposures, resulting in an improved safety window. Ultimately, transformation of 1 yielded analogues that demonstrated excellent potency and PK properties and fully inhibited IL-2 production in an acute model. In spite of good exposure, twice-a-day treatment with 17l in the glucose-6-phosphate isomerase chronic in vivo mouse model of arthritis yielded only moderate efficacy. On the basis of the exposure achieved, we conclude that PKCθ inhibition alone is insufficient for complete efficacy in this rodent arthritis model.

Background: Potent immunomodulatory effects have been reported for mesenchymal stem/stromal cells (MSCs), multipotent adult progenitor cells (MAPCs), and fibroblasts. However, side-by-side comparisons of these cells specifically regarding immunophenotype, gene expression, and suppression of proliferation of CD4(+) and CD8(+) lymphocyte populations have not been reported., Methods: We developed MAPC and MSC lines from rhesus macaque bone marrow and fibroblast cell lines from rhesus dermis and assessed phenotypes based upon differentiation potential, flow cytometric analysis of immunophenotype, and quantitative RT-PCR analysis of gene expression. Using allogeneic lymphocyte proliferation assays, we compared the in vitro immunomodulatory potency of each cell type., Results and Conclusions: Extensive phenotypic similarities exist among each cell type, although immunosuppressive potencies are distinct. MAPCs are most potent, and fibroblasts are the least potent cell type. All three cell types demonstrated immunomodulatory capacity such that each may have potential therapeutic applications such as in organ transplantation, where reduced local immune response is desirable., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Tregs play an important role in controlling immune responses, particularly autoimmunity. In NOD mouse model, an excellent model for autoimmune diabetes, transfer of Tregs was shown to prevent diabetes, whereas depletion of Tregs in vivo enhanced disease progression, suggesting that Treg dysfunction contributes to the pathogenesis of diabetes. However, the mechanisms leading to Treg dysfunction and their role in diabetes progression has remained unclear. In this study we assessed quantitative and qualitative changes in Tregs during the development of autoimmune diabetes in NOD. We compared female NOD with males that have similar predisposition to but a lower incidence of diabetes and found that Treg numbers remained unchanged between 6 to 16 weeks of age in both groups. Although female Tregs produced lower TGF-β compared to male, regulatory function of female Tregs was only marginally inferior to male upon GAD65 autoantigen stimulation. GAD65-reactive female Teffectors were more responsive and progressively became refractory to regulation compared to male effectors, in part due to lower expression of TGF-β RII, accounting for reduced sensitivity to Tregs. Moreover, we unexpectedly found that TGF-β suppressed IFN-γ production to GAD65 antigen in male, not in female responders. These data suggest that TGF-β plays a major role in Teff resistance to regulation and Treg dysfunction, and may account for autoimmune diabetes. Our study implies that development of a successful supplemental Treg therapy for halting autoimmunity may require further understanding of Teff responses to regulation in order to generate highly effective Tregs.

Anti-CD25 antibodies are used as an induction therapy in islet allotransplantation for type 1 diabetes. Although previous reports suggested that anti-CD25 treatment may lead to depletion of CD4+CD25+ regulatory T cells (Tregs) and questioned its use in tolerance-promoting protocols for transplantation, the effect of anti-CD25 antibodies on the frequency and function of Tregs remains unclear. We examined the effect of anti-CD25 antibody, daclizumab, in vivo on Tregs in islet allograft recipients enrolled in a single-center study and monitored post-transplant. Our data shows that the reduction in CD25+ Treg cells observed post-transplant is due to masking of CD25 receptor by daclizumab and not due to depletion. In addition, using Treg marker, FoxP3, we show that anti-CD25+ ATG treatment leads to an increase in FoxP3+ Tregs post-transplant. These data suggest that anti-CD25-based therapy has beneficial effects on Tregs and combined with ATG may be a promising therapy for autoimmunity and transplantation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Despite advances in islet transplantation, challenges remain in monitoring for anti-islet immune responses. Soluble CD30 (sCD30) has been investigated as a predictor of acute rejection in kidney, lung, and heart transplantation as well as in a single study in human islet cell recipients. In this study, sCD30 levels were retrospectively assessed in 19 allograft recipients treated with three different immunosuppression induction therapies. Soluble CD30 levels were assessed at pre-transplant; early post-transplant (day 4-day 7); one-month post-transplant; and late post-transplant (day 90-day 120) and then correlated with eventual graft outcomes at 1-year follow-up. Results showed no correlation between mean serum sCD30 levels at any point in time pre- or post-transplant and graft function at 1-year follow-up. However, analysis demonstrated that mean sCD30 levels at day 28 or day 90-day 120 decreased from pre-transplant levels in recipients with long-term islet allograft function compared to recipients with partial or non-graft function (a decrease of 43.6+/-25.6% compared to 16.7+/-35.2%, p<0.05). In another finding, immunosuppression with the ATG protocol led to a greater reduction in sCD30 levels post-transplant overall. A larger reduction post-transplant correlated with full graft function. The results demonstrate that a relative reduction in sCD30 levels post-transplant may be applicable as a biomarker to monitor graft function in islet allograft recipients. Additionally, knowledge of the impact of various immunosuppression protocols on the timing and extent of changes in post-transplant sCD30 levels could aid in patient-specific tailoring of immunosuppression., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: The major obstacle faced by patients with type 1 diabetes who undergo islet transplantation is a gradual decline in insulin independence. This decline may reflect alloimmune rejection, autoimmune recurrence and toxicity of drugs such as rapamycin to islet beta cells. Thus, there is a pressing need to refine immunosuppressive protocols in order to reduce toxicity to islet grafts and yet prevent rejection. Recent studies demonstrated that TGF-beta1 is a critical cytokine for the regulation of immune responses. In naive T cells, TGF-beta1 induces FoxP3(+) regulatory T cells and thus could promote transplant tolerance. In this study, in vitro experiments were performed to determine whether TGF-beta1 could synergize with low-dose rapamycin and inhibit T cell activation and production of inflammatory cytokines, as well as enhance FoxP3 expression for potential application in islet transplantation., Methods: Human peripheral blood mononuclear cells were stimulated with either anti-CD3/CD28 or anti-CD3 during TGF-beta1 and rapamycin treatment., Results: TGF-beta1 inhibited T cell proliferation induced with anti-CD3 stimulation, but not with anti-CD3/CD28 stimulation. The combination of these reagents produced a synergistic inhibition of T cell proliferation induced with both anti-CD3/CD28 and anti-CD3 stimulations. Moreover, TGF-beta1 and rapamycin significantly suppressed cytokine production and induced regulatory T cells by upregulating FoxP3 expression., Conclusions: These results suggest that the combination of TGF-beta1 and low-dose rapamycin can potently inhibit T cell responses in vivo and would be beneficial in supporting islet graft survival by limiting toxicity and preventing immune rejection., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Cell-based diabetes therapy requires an abundant cell source. Here, we report reversal of diabetes for more than 100 d in cynomolgus macaques after intraportal transplantation of cultured islets from genetically unmodified pigs without Gal-specific antibody manipulation. Immunotherapy with CD25-specific and CD154-specific monoclonal antibodies, FTY720 (or tacrolimus), everolimus and leflunomide suppressed indirect activation of T cells, elicitation of non-Gal pig-specific IgG antibody, intragraft expression of proinflammatory cytokines and invasion of infiltrating mononuclear cells into islets.

Autoreactive T cells play a major role in the development of insulin-dependent diabetes mellitus, suggesting that costimulatory molecules that regulate T cell responses might be essential for disease progression. In NOD mice, CD28/B7 and CD40/CD40 ligand (L) interactions control the onset of diabetes from 2 to 4 weeks of age, but blocking these molecules has little effect after this time. Hence, it is possible that other ligand/receptor pairs control a later phase of disease. We now show that OX40 is expressed on CD4 and CD8 T cells several weeks prior to islet destruction, which is initiated around weeks 12-14, and that OX40L is present on dendritic cells in both secondary lymphoid organs and the pancreas from 11 to 13 weeks of age. Blocking OX40L at 6, 9, or 15 weeks after birth had little effect on disease; however, inhibiting OX40/OX40L interactions at week 12, or continuous treatment from week 12 onwards, significantly reduced the incidence of diabetes. Histological examination showed that islet destruction was prevented and insulitis reduced by targeting OX40L. These studies show that OX40/OX40L interactions form a late checkpoint in diabetes development and suggest that these molecules are realistic targets for therapeutic intervention.

The persistence of functional CD8 T cell responses is dependent on checkpoints established during priming. Although naive CD8 cells can proliferate with a short period of stimulation, CD4 help, inflammation, and/or high peptide affinity are necessary for the survival of CTL and for effective priming. Using OX40-deficient CD8 cells specific for a defined Ag, and agonist and antagonist OX40 reagents, we show that OX40/OX40 ligand interactions can determine the extent of expansion of CD8 T cells during responses to conventional protein Ag and can provide sufficient signals to confer CTL-mediated protection against tumor growth. OX40 signaling primarily functions to maintain CTL survival during the initial rounds of cell division after Ag encounter. Thus, OX40 is one of the costimulatory molecules that can contribute signals to regulate the accumulation of Ag-reactive CD8 cells during immune responses.

Aging is associated with an increased susceptibility to infectious agents and correlates with a decreased ability to mount an immune response. It has been postulated that the major defect is related to a reduced capacity of an aged T cell to proliferate and to survive after encounter with Ag. This is similar to the phenotype associated with T cell tolerance in young adults. In this study, we determined whether targeting 4-1BB (CD137), a member of the TNFR family implicated in providing expansion and survival signals to T cells, can rescue defective priming in aged and tolerized animals. Agonist Abs to 4-1BB injected in vivo were capable of preventing CD4 T cell tolerance to soluble peptide in young mice. Moreover, anti-4-1BB rescued defective priming of aged TCR transgenic CD4 T cells responding to peptide Ag in a young host, and as importantly, anti-4-1BB completely restored T cell priming to protein Ag in nontransgenic aged mice. These studies demonstrate that 4-1BB, and potentially other costimulatory members of the TNFR family, are targets for therapies aimed at augmenting weak T cell responses in elderly immunocompromised individuals.

4-1BB is expressed on activated T cells. We analyzed the role of 4-1BB during the CD8 T cell response of OT-I TCR-transgenic T cells to ovalbumin. In vitro, blocking 4-1BB during peptide presentation reduced proliferation of naive CD8 T cells, but did not affect the generation of CTL. Using an in vivo adoptive transfer model, clonal expansion of CD8 T cells to whole protein in adjuvant was significantly reduced when 4-1BB was blocked, with 50-70% fewer CD8 T cells accumulating. This was due to a reduction in T cell division and to enhanced apoptosis of CD8 T cells that had undergone many divisions. T cells generated in the absence of 4-1BB were impaired in their ability to secrete IFN-gamma whereas CTL activity of the T cells that survived was unaffected. These findings demonstrate that 4-1BB contributes to clonal expansion, survival, and development of Tc1 cells when protein antigen is encountered by primary CD8 T cells in an inflammatory environment in vivo.

Immunological tolerance represents a mechanism by which cells of the host remain protected from the immune system. Breaking of immunological tolerance can result in a variety of autoimmune diseases such as rheumatoid arthritis, diabetes, and multiple sclerosis. The reasons for tolerance breaking down and autoimmune processes arising are largely unknown but of obvious interest for therapeutic intervention of these diseases. Although reversal of the tolerant state is generally unwanted, there are instances where this may be of benefit to the host. In particular, one way a cancerous cell escapes being targeted by the immune system is through tolerance mechanisms that in effect turn off the reactivity of T lymphocytes that can respond to tumor-associated peptides. Thus tolerance represents a major obstacle in developing effective immunotherapy against tumors. The molecules that are involved in regulating immunological tolerance are then of interest as they may be great targets for positively or negatively manipulating the tolerance process.

Peripheral T-cell tolerance is a mechanism to limit autoimmunity, but represents a major obstacle in diseases such as cancer. Tolerance is due to limited accumulation of antigen-specific T cells accompanied by functional hypo-responsiveness, and is induced by antigen encounter in a non-inflammatory environment. In contrast to advances in preventing induction of T-cell tolerance, there has been little progress in defining targets to reverse established tolerance. Here we show that signals from a single dose of an agonistic antibody against OX40 (CD134, a member of the tumor necrosis-factor family of receptors) can break an existing state of tolerance in the CD4+ T-cell compartment. OX40 signals promote T-cell expansion after the hypo-responsive phenotype is induced and restore normal functionality. These data highlight the potent costimulatory capacity of OX40, and indicate OX40 as a target for therapeutic intervention in a variety of related diseases.