Amanda Posgai

Transient B-Cell Depletion with Anti-CD20 in Combination with Proinsulin DNA Vaccine or Oral Insulin: Immunologic Effects and Efficacy in NOD Mice

A recent type 1 diabetes (T1D) clinical trial of rituximab (a B cell-depleting anti-CD20 antibody) achieved some therapeutic benefit in preserving C-peptide for a period of approximately nine months in patients with recently diagnosed diabetes. Our previous data in the NOD mouse demonstrated that co-administration of antigen (insulin) with anti-CD3 antibody (a T cell-directed immunomodulator) offers better protection than either entity alone, indicating that novel combination therapies that include a T1D-related autoantigen are possible. To accelerate the identification and development of novel combination therapies that can be advanced into the clinic, we have evaluated the combination of a mouse anti-CD20 antibody with either oral insulin or a proinsulin-expressing DNA vaccine. Anti-CD20 alone, given once or on 4 consecutive days, produced transient B cell depletion but did not prevent or reverse T1D in the NOD mouse. Oral insulin alone (twice weekly for 6 weeks) was also ineffective, while proinsulin DNA (weekly for up to 12 weeks) showed a trend toward modest efficacy. Combination of anti-CD20 with oral insulin was ineffective in reversing diabetes in NOD mice whose glycemia was controlled with SC insulin pellets; these experiments were performed in three independent labs. Combination of anti-CD20 with proinsulin DNA was also ineffective in diabetes reversal, but did show modest efficacy in diabetes prevention (p = 0.04). In the prevention studies, anti-CD20 plus proinsulin resulted in modest increases in Tregs in pancreatic lymph nodes and elevated levels of proinsulin-specific CD4+ T-cells that produced IL-4. Thus, combination therapy with anti-CD20 and either oral insulin or proinsulin does not protect hyperglycemic NOD mice, but the combination with proinsulin offers limited efficacy in T1D prevention, potentially by augmentation of proinsulin-specific IL-4 production.

Anti-Thymocyte Globulin + G-CSF Combination Therapy Leads to Sustained Immunomodulatory and Metabolic Effects in a Subset of Responders with Established Type 1 Diabetes

Low-dose antithymocyte globulin (ATG) plus pegylated granulocyte colony-stimulating factor (G-CSF) preserves β-cell function for at least 12 months in type 1 diabetes. Herein, we describe metabolic and immunological parameters 24 months following treatment. Patients with established type 1 diabetes (duration 4–24 months) were randomized to ATG and pegylated G-CSF (ATG+G-CSF) (N = 17) or placebo (N = 8). Primary outcomes included C-peptide area under the curve (AUC) following a mixed-meal tolerance test (MMTT) and flow cytometry. “Responders” (12-month C-peptide ≥ baseline), “super responders” (24-month C-peptide ≥ baseline), and “nonresponders” (12-month C-peptide < baseline) were evaluated for biomarkers of outcome. At 24 months, MMTT-stimulated AUC C-peptide was not significantly different in ATG+G-CSF (0.49 nmol/L/min) versus placebo (0.29 nmol/L/min). Subjects treated with ATG+G-CSF demonstrated reduced CD4+ T cells and CD4+/CD8+ T-cell ratio and increased CD16+CD56hi natural killer cells (NK), CD4+ effector memory T cells (Tem), CD4+PD-1+ central memory T cells (Tcm), Tcm PD-1 expression, and neutrophils. FOXP3+Helios+ regulatory T cells (Treg) were elevated in ATG+G-CSF subjects at 6, 12, and 18 but not 24 months. Immunophenotyping identified differential HLA-DR expression on monocytes and NK and altered CXCR3 and PD-1 expression on T-cell subsets. As such, a group of metabolic and immunological responders was identified. A phase II study of ATG+G-CSF in patients with new-onset type 1 diabetes is ongoing and may support ATG+G-CSF as a prevention strategy in high-risk subjects.

A Preclinical Consortium Approach for Assessing the Efficacy of Combined Anti-CD3 Plus IL-1 Blockade in Reversing New-Onset Autoimmune Diabetes in NOD Mice

There is an ongoing need to develop strategic combinations of therapeutic agents to prevent type 1 diabetes (T1D) or to preserve islet β-cell mass in new-onset disease. Although clinical trials using candidate therapeutics are commonly based on preclinical studies, concern is growing regarding the reproducibility as well as the potential clinical translation of reported results using animal models of human disorders. In response, the National Institutes of Health Immune Tolerance Network and JDRF established a multicenter consortium of academic institutions designed to assess the efficacy and intergroup reproducibility of clinically applicable immunotherapies for reversing new-onset disease in the NOD mouse model of T1D. Predicated on prior studies, this consortium conducted coordinated, prospective studies, using joint standard operating procedures, fixed criteria for study entry, and common reagents, to optimize combined anti-CD3 treatment plus interleukin-1 (IL-1) blockade to reverse new-onset disease in NOD mice. We did not find that IL-1 blockade with anti–IL-1β monoclonal antibody or IL-1trap provided additional benefit for reversing new-onset disease compared with anti-CD3 treatment alone. These results demonstrate the value of larger, multicenter preclinical studies for vetting and prioritizing therapeutics for future clinical use.

Expansion of Human Tregs from Cryopreserved Umbilical Cord Blood for GMP-Compliant Autologous Adoptive Cell Transfer Therapy

Umbilical cord blood is a traditional and convenient source of cells for hematopoietic stem cell transplantation. Thymic regulatory T cells (Tregs) are also present in cord blood, and there is growing interest in the use of autologous Tregs to provide a low-risk, fully human leukocyte antigen (HLA)-matched cell product for treating autoimmune diseases, such as type 1 diabetes. Here, we describe a good manufacturing practice (GMP)-compatible Treg expansion protocol using fluorescence-activated cell sorting, resulting in a mean 2,092-fold expansion of Tregs over a 16-day culture for a median yield of 1.26 × 109 Tregs from single-donor cryopreserved units. The resulting Tregs passed prior clinical trial release criteria for Treg purity and sterility, including additional rigorous assessments of FOXP3 and Helios expression and epigenetic analysis of the FOXP3 Treg-specific demethylated region (TSDR). Compared with expanded adult peripheral blood Tregs, expanded cord blood Tregs remained more naive, as assessed by continued expression of CD45RA, produced reduced IFN-γ following activation, and effectively inhibited responder T cell proliferation. Immunosequencing of the T cell receptor revealed a remarkably diverse receptor repertoire within cord blood Tregs that was maintained following in vitro expansion. These data support the feasibility of generating GMP-compliant Tregs from cord blood for adoptive cell transfer therapies and highlight potential advantages in terms of safety, phenotypic stability, autoantigen specificity, and tissue distribution.

Combination therapy reverses hyperglycemia in NOD mice with established type 1 diabetes

An increasing number of therapies have proven effective at reversing hyperglycemia in the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D), yet situations of successful translation to human T1D are limited. This may be partly due to evaluating the effect of treating immediately at diagnosis in mice, which may not be reflective of the advanced disease state in humans at disease onset. In this study, we treated NOD mice with new-onset as well as established disease using various combinations of four drugs: antithymocyte globulin (ATG), granulocyte-colony stimulating factor (G-CSF), a dipeptidyl peptidase IV inhibitor (DPP-4i), and a proton pump inhibitor (PPI). Therapy with all four drugs induced remission in 83% of new-onset mice and, remarkably, in 50% of NOD mice with established disease. Also noteworthy, disease remission occurred irrespective of initial blood glucose values and mechanistically was characterized by enhanced immunoregulation involving alterations in CD4+ T cells, CD8+ T cells, and natural killer cells. This combination therapy also allowed for effective treatment at reduced drug doses (compared with effective monotherapy), thereby minimizing potential adverse effects while retaining efficacy. This combination of approved drugs demonstrates a novel ability to reverse T1D, thereby warranting translational consideration.

Plant-based vaccines for oral delivery of type 1 diabetes-related autoantigens: Evaluating oral tolerance mechanisms and disease prevention in NOD mice

Autoantigen-specific immunological tolerance represents a central objective for prevention of type 1 diabetes (T1D). Previous studies demonstrated mucosal antigen administration results in expansion of Foxp3+ and LAP+ regulatory T cells (Tregs), suggesting oral delivery of self-antigens might represent an effective means for modulating autoimmune disease. Early preclinical experiments using the non-obese diabetic (NOD) mouse model reported mucosal administration of T1D-related autoantigens [proinsulin or glutamic acid decarboxylase 65 (GAD)] delayed T1D onset, but published data are conflicting regarding dose, treatment duration, requirement for combinatorial agents, and extent of efficacy. Recently, dogma was challenged in a report demonstrating oral insulin does not prevent T1D in NOD mice, possibly due to antigen digestion prior to mucosal immune exposure. We used transplastomic plants expressing proinsulin and GAD to protect the autoantigens from degradation in an oral vaccine and tested the optimal combination, dose, and treatment duration for the prevention of T1D in NOD mice. Our data suggest oral autoantigen therapy alone does not effectively influence disease incidence or result in antigen-specific tolerance assessed by IL-10 measurement and Treg frequency. A more aggressive approach involving tolerogenic cytokine administration and/or lymphocyte depletion prior to oral antigen-specific immunotherapy will likely be required to impart durable therapeutic efficacy.

Avidity and Bystander Suppressive Capacity of Human Regulatory T Cells Expressing De Novo Autoreactive T-Cell Receptors in Type 1 Diabetes

The ability to alter antigen specificity by T-cell receptor (TCR) or chimeric antigen receptor (CAR) gene transfer has facilitated personalized cellular immune therapies in cancer. Inversely, this approach can be harnessed in autoimmune settings to attenuate inflammation by redirecting the specificity of regulatory T cells (Tregs). Herein, we demonstrate efficient protocols for lentiviral gene transfer of TCRs that recognize type 1 diabetes-related autoantigens with the goal of tissue-targeted induction of antigen-specific tolerance to halt β-cell destruction. We generated human Tregs expressing a high-affinity GAD555–567-reactive TCR (clone R164), as well as the lower affinity clone 4.13 specific for the same peptide. We demonstrated that de novo Treg avatars potently suppress antigen-specific and bystander responder T-cell (Tresp) proliferation in vitro in a process that requires Treg activation (P < 0.001 versus unactivated Tregs). When Tresp were also glutamic acid decarboxylase (GAD)-reactive, the high-affinity R164 Tregs exhibited increased suppression (P < 0.01) with lower Tresp-division index (P < 0.01) than the lower affinity 4.13 Tregs. These data demonstrate the feasibility of rapid expansion of antigen-specific Tregs for applications in attenuating β-cell autoimmunity and emphasize further opportunities for engineering cellular specificities, affinities, and phenotypes to tailor Treg activity in adoptive cell therapies for the treatment of type 1 diabetes.

Mobilization without immune depletion fails to restore immunological tolerance or preserve beta cell function in recent onset type 1 diabetes

Granulocyte colony‐stimulating factor (G‐CSF) has been used to restore immune competence following chemoablative cancer therapy and to promote immunological tolerance in certain settings of autoimmunity. Therefore, we tested the potential of G‐CSF to impact type 1 diabetes (T1D) progression in patients with recent‐onset disease [n = 14; n = 7 (placebo)] and assessed safety, efficacy and mechanistic effects on the immune system. We hypothesized that pegylated G‐CSF (6 mg administered subcutaneously every 2 weeks for 12 weeks) would promote regulatory T cell (Treg) mobilization to a degree capable of restoring immunological tolerance, thus preventing further decline in C‐peptide production. Although treatment was well tolerated, G‐CSF monotherapy did not affect C‐peptide production, glycated haemoglobin (HbA1c) or insulin dose. Mechanistically, G‐CSF treatment increased circulating neutrophils during the 12‐week course of therapy (P < 0·01) but did not alter Treg frequencies. No effects were observed for CD4+ : CD8+ T cell ratio or the ratio of naive : memory (CD45RA+/CD45RO+) CD4+ T cells. As expected, manageable bone pain was common in subjects receiving G‐CSF, but notably, no severe adverse events such as splenomegaly occurred. This study supports the continued exploration of G‐CSF and other mobilizing agents in subjects with T1D, but only when combined with immunodepleting agents where synergistic mechanisms of action have previously demonstrated efficacy towards the preservation of C‐peptide.

Raising Awareness: The Need to Promote Allocation of Pancreata From Rare Nondiabetic Donors With Pancreatic Islet Autoimmunity to Type 1 Diabetes Research

It is key to recognize that our knowledge of T1D pathogenesis and etiological factors is severely limited by scarce access to the pancreata of patients. To address this shortcoming, the JDRF supported the institution of the Network for Pancreatic Organ Donors with Diabetes (nPOD; http:// www.JDRFnPOD.org). Working with U.S. organ procurement organizations (OPOs), nPOD has recovered pancreata from >100 organ donors with T1D since 2007. The nPOD promotes collaborative research by providing specimens to qualified investigators worldwide as well as scientific leadership and coordination (1). Many novel discoveries have been made through the study of nPOD pancreata (1) about key elements of autoimmune pathogenesis and additional mechanisms of disease, including chronic inflammation associated with beta cell stress and dysfunction, alterations of key extracellular matrix components in the islets and alteration of the exocrine tissue. Growing evidence shows that the disease is more chronic than previously thought because pathological signs and insulin-positive beta cells persist for many years, sometimes decades, after clinical diagnosis.

Acute Versus Progressive Onset of Diabetes in NOD Mice: Potential Implications for Therapeutic Interventions in Type 1 Diabetes

Most natural history models for type 1 diabetes (T1D) propose that overt hyperglycemia results after a progressive loss of insulin-secreting β-cell mass and/or function. To experimentally address this concept, we prospectively determined morning blood glucose measurements every other day in multiple cohorts (total n = 660) of female NOD/ShiLtJ mice starting at 8 weeks of age until diabetes onset or 26 weeks of age. Consistent with this notion, a majority of mice that developed diabetes (354 of 489 [72%]) displayed a progressive increase in blood glucose with transient excursions >200 mg/dL, followed by acute and persistent hyperglycemia at diabetes onset. However, 135 of the 489 (28%) diabetic animals demonstrated normal glucose values followed by acute (i.e., sudden) hyperglycemia. Interestingly, diabetes onset occurred earlier in mice with acute versus progressive disease onset (15.37 ± 0.3207 vs. 17.44 ± 0.2073 weeks of age, P < 0.0001). Moreover, the pattern of onset (i.e., progressive vs. acute) dramatically influenced the ability to achieve reversal of T1D by immunotherapeutic intervention, with increased effectiveness observed in situations of a progressive deterioration in euglycemia. These studies highlight a novel natural history aspect in this animal model, one that may provide important guidance for the selection of subjects participating in human trials seeking disease reversal.