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.

In diabetic patients and susceptible mice, insulin is a targeted autoantigen. Insulin B chain 9-23 (B:9-23) autoreactive CD4 T cells are key for initiating autoimmune diabetes in NOD mice; however, little is known regarding their origin and function. To this end, B:9-23-specific, BDC12-4.1 T-cell receptor (TCR) transgenic (Tg) mice were studied, of which, despite expressing a single TCR on the recombination activating gene-deficient background, only a fraction develops diabetes in an asynchronous manner. BDC12-4.1 CD4 T cells convert into effector (Teff) and Foxp3(+)-expressing adaptive regulatory T cells (aTregs) soon after leaving the thymus as a result of antigen recognition and homeostatic proliferation. The generation of aTreg causes the heterogeneous diabetes onset, since crossing onto the scurfy (Foxp3) mutation, BDC12-4.1 TCR Tg mice develop accelerated and fully penetrant diabetes. Similarly, adoptive transfer and bone marrow transplantation experiments showed differential diabetes kinetics based on Foxp3(+) aTreg's presence in the BDC12-4.1 donors. A single-specificity, insulin-reactive TCR escapes thymic deletion and simultaneously converts into aTreg and Teff, establishing an equilibrium that determines diabetes penetrance. These results are of particular importance for understanding disease pathogenesis. They suggest that once central tolerance is bypassed, autoreactive cells arriving in the periphery do not by default follow solely a pathogenic fate upon activation.

Neoadjuvant therapy with ipilimumab in combination with high dose IFNα was evaluated in patients with locally/regionally advanced melanoma in a previously reported clinical trial [NCT01608594]. In this study, peripheral immune cell profiling was performed in order to investigate the underlying mechanisms of tumor immune susceptibility and resistance. Peripheral blood mononuclear cells (PBMCs) from treated patients (N = 28) were collected at baseline and then at 6-weeks, 3-months and 12-months. High complexity (14-color) flow cytometry, designed to detect key immunological biomarkers was used to evaluate the frequencies of immune cell subsets. Statistical significance was determined using R-package employing Kruskal's test. We found that higher levels of Th1 cells at baseline (defined as CD45RA- CCR6- CXCR3+ CCR4-) correlated with the preoperative radiological response (p = 0.007) while higher Th2 cells (defined as CD45RA- CCR6- CXCR3- CCR4+) were associated with progressive disease (p = 0.009). A multimarker score consisting of higher levels of Th1 cells and CD8+ central memory T-cells was associated with pathologic complete response (pCR) (p = 0.041) at surgical resection. On the other hand, high TIM3 expression on T-cells correlated with gross viable tumor (p = 0.047). With regard to immune related toxicity, higher levels of phenotypically naive (defined as CCR7+CD45RA+) and effector memory (defined as CCR7-CD45RO+) CD8+ T-cells (p = 0.014) or lower levels of Th2 cells were associated with lower toxicity (p = 0.024). Furthermore, a multimarker score consisting of higher CD19+ and CD8+ cells was associated with lower toxicity (p = 0.0014). In conclusion, our study yielded mechanistic insights related to the immune impact of CTLA4 blockade and IFNα and potential biomarkers of immune response and toxicity.

The infusion of ex vivo-expanded autologous T regulatory (Treg) cells is potentially an effective immunotherapeutic strategy against graft-versus-host disease (GvHD) and several autoimmune diseases, such as type 1 diabetes (T1D). However, in vitro differentiation of antigen-specific T cells into functional and stable Treg (iTreg) cells has proved challenging. As insulin is the major autoantigen leading to T1D, we tested the capacity of insulin-specific T-cell receptor (TCR) transgenic CD4(+) T cells of the BDC12-4.1 clone to convert into Foxp3(+) iTreg cells. We found that in vitro polarization toward Foxp3(+) iTreg was effective with a majority (>70%) of expanded cells expressing Foxp3. However, adoptive transfer of Foxp3(+) BDC12-4.1 cells did not prevent diabetes onset in immunocompetent NOD mice. Thus, in vitro polarization of insulin-specific BDC12-4.1 TCR transgenic CD4(+) T cells toward Foxp3+ cells did not provide dominant tolerance in recipient mice. These results highlight the disconnect between an in vitro acquired Foxp3(+) cell phenotype and its associated in vivo regulatory potential.

Multiple immune parameters such as frequencies of autoreactive CD4(+), CD8(+) T-cells and CD4(+)CD25(+)Foxp3(+) T-cells have been explored as biomarkers in human T1D. However, intra-individual temporal variation of these parameters has not been assessed systematically over time. We determined the variation in each of these parameters in a cohort of T1D and healthy donors (HDs), at monthly intervals for one year. Despite low intra- and inter-assay co-efficient of variation (CV), mean CVs for each of the immune parameters were 119.1% for CD4(+) T-cell-derived IFN-γ, 50.44% for autoreactive CD8(+) T-cells, and 31.24% for CD4(+)CD25(+)Foxp3(+) T-cells. Further, both HDs and T1D donors had similar CVs. The variation neither correlated with BMI, age, disease duration or insulin usage, nor were there detectable cyclical patterns of variation. However, averaging results from multiple visits for an individual provided a better estimate of the CV between visits. Based on our data we predict that by averaging values from three visits a treatment effect on these parameters with a 50% effect size could be detected with the same power using 1.8-4-fold fewer patients within a trial compared to using values from a single visit. Thus, our present data contribute to a more robust, accurate endpoint design for future clinical trials in T1D and aid in the identification of truly efficacious therapies.