Responders to low-dose ATG induce CD4 T cell exhaustion in type 1 diabetes.

Low-dose anti-thymocyte globulin (ATG) transiently preserves C-peptide and lowers HbA1c in individuals with recent-onset type 1 diabetes (T1D); however, the mechanisms of action and features of response remain unclear. Here, we characterized the post-hoc immunological outcomes of ATG administration and their potential use as biomarkers of metabolic response to therapy (i.e., improved preservation of endogenous insulin production). We assessed gene and protein expression, targeted gene methylation, and cytokine concentrations in peripheral blood following treatment with ATG (n=29), ATG plus granulocyte-colony stimulating factor (ATG/G-CSF, n=28), or placebo (n=31). Treatment with low-dose ATG preserved regulatory T cells (Tregs), as measured by stable methylation of FOXP3 Treg-specific demethylation region (TSDR) and increased proportions of CD4+FOXP3+ Tregs (p<0.001) identified by flow cytometry. While treatment effects were consistent across participants, not all maintained C-peptide. Responders exhibited a transient rise in IL-6, IP-10, and TNFα (p<0.05 for all) 2 weeks post-treatment and a durable CD4 exhaustion phenotype (increased PD-1+KLRG1+CD57- on CD4+ T cells [p=0.011] and PD1+CD4 TEMRA MFI [p<0.001] at 12 weeks, following ATG and ATG/G-CSF, respectively). ATG non-responders displayed higher proportions of senescent T cells (at baseline and post-treatment) and increased methylation of EOMES (i.e., less expression of this exhaustion marker). Altogether in these exploratory analyses, Th1 inflammation-associated serum, CD4 exhaustion transcript and cellular phenotyping profiles may be useful for identifying signatures of clinical response to ATG in T1D.