Low-Dose Anti-Thymocyte Globulin Preserves C-Peptide, Reduces HbA1c, and Increases Regulatory to Conventional T-Cell Ratios in New-Onset Type 1 Diabetes: Two-Year Clinical Trial Data

Abstract

A three-arm, randomized, double-masked, placebo-controlled phase 2b trial performed by the Type 1 Diabetes TrialNet Study Group previously demonstrated that low-dose anti-thymocyte globulin (ATG) (2.5 mg/kg) preserved β-cell function and reduced HbA_1c for 1 year in new-onset type 1 diabetes. Subjects (N = 89) were randomized to 1) ATG and pegylated granulocyte colony-stimulating factor (GCSF), 2) ATG alone, or 3) placebo. Herein, we report 2-year area under the curve (AUC) C-peptide and HbA_1c, prespecified secondary end points, and potential immunologic correlates. The 2-year mean mixed-meal tolerance test-stimulated AUC C-peptide, analyzed by ANCOVA adjusting for baseline C-peptide, age, and sex (n = 82) with significance defined as one-sided P < 0.025, was significantly higher in subjects treated with ATG versus placebo (P = 0.00005) but not ATG/GCSF versus placebo (P = 0.032). HbA_1c was significantly reduced at 2 years in subjects treated with ATG (P = 0.011) and ATG/GCSF (P = 0.022) versus placebo. Flow cytometry analyses demonstrated reduced circulating CD4:CD8 ratio, increased regulatory T-cell:conventional CD4 T-cell ratios, and increased PD-1⁺CD4⁺ T cells following low-dose ATG and ATG/GCSF. Low-dose ATG partially preserved β-cell function and reduced HbA_1c 2 years after therapy in new-onset type 1 diabetes. Future studies should determine whether low-dose ATG might prevent or delay the onset of type 1 diabetes.

Trial registration: ClinicalTrials.gov NCT02215200.

Figure 1

Consolidated Standards of Reporting Trials diagram. One hundred thirteen patients were screened for eligibility, of whom 89 were randomized and 82 completed the secondary outcome measure at 2 years. One randomized subject withdrew consent before receiving any study drug. All remaining subjects received ATG and placebo infusions as specified in the protocol. This included two subjects who received reduced doses per protocol specifications (one ATG and one placebo). mos., months.

Figure 2

Effects of low-dose ATG and low-dose ATG/GCSF on C-peptide (A), mixed-model predicted C-peptide (B), HbA_1c (C), and insulin (D). C-peptide AUC mean over time by treatment group (A). Analysis at the 2-year end point: ATG alone vs. placebo P = 0.0005 and ATG/GCSF vs. placebo P = 0.032. Mixed-model predicted population mean of the C-peptide AUC mean by treatment over time (B). Two-year decline in mean C-peptide AUC mean: placebo −0.635 nmol/L, ATG −0.337 nmol/L, and ATG/GCSF −0.446 nmol/L. HbA_1c over time by treatment group (C). Analysis at 2-year end point: ATG alone vs. placebo P = 0.011 and ATG/GCSF vs. placebo P = 0.022. Insulin use over time by treatment group (D). No significant differences at 2 years. A, C, and D show adjusted means and 95% CIs at each time point; B shows the mixed-model predicted population mean of the C-peptide AUC mean by treatment. Two-year decline was −0.635 nmol/L, −0.337 nmol/L, and −0.446 nmol/L in placebo-, ATG-, and ATG/GCSF-treated subjects, respectively.

Figure 3

CD4:CD8 T-cell ratio declined with low-dose ATG and ATG/GCSF treatment. Absolute counts were generated at the 2-week time point by multiplying lymphocyte complete blood cell counts with flow cytometry–detected CD4⁺ (A) or CD8⁺ (B) T-cell percentages within the lymphocyte gate. Significant differences in CD4⁺ cells were identified between each of the treatment arms and placebo (P < 0.001 for both pairwise comparisons). No significant differences were observed for CD8⁺ cells. Longitudinal CD4:CD8 T-cell ratios (C) were determined using frequencies in the lymphocyte gate. Average measures with SDs by treatment arm are shown. Dotted lines in C denote initiation of ATG and last dose of GCSF. The CD4:CD8 T-cell ratios were significantly different at the postbaseline time points for those treated with ATG or ATG/GCSF vs. placebo (all P < 0.001).

Figure 4

Low-dose ATG reduced CD4⁺ Tconv and increased Treg:Tconv ratios. A and B: Absolute counts of Tconv (CD127^highFOXP3^neg) and CD4⁺ Tregs (CD127^low FOXP3^high) for samples collected at the 2-week time point. C: Treg:Tconv ratios were calculated using values from A and B. D and E: Treg phenotyping is reported as % CD45RO⁺ memory cells and % TIGIT⁺ cells within the Treg gate. In C and E, average measures with SDs are shown; dotted lines denote initiation of ATG and last dose of GCSF. Treg:CD4 Tconv counts shown for placebo vs. ATG arm (2 weeks P < 0.001, 12 weeks P = 0.001, 24 weeks P = 0.05) and placebo vs. ATG/GCSF arm (2 weeks P = 0.003, 12 weeks P = 0.004, 24 weeks P = 0.018). Percent memory of Tregs was not significantly different between ATG and placebo but was significantly different between placebo and ATG/GCSF at 24 weeks (P = 0.04). Percent TIGIT⁺ Tregs shown for the ATG/GCSF arm vs. placebo (2 weeks P = 0.28, 12 weeks P = 0.009, 24 weeks P = 0.017) and the ATG arm vs. placebo (2 weeks P = 0.07, 12 weeks P = 0.017, 24 weeks P = 0.07).

Figure 5

Low-dose ATG enhances PD-1 expression on CD4⁺ but not CD8⁺ T cells. Percent of cells expressing PD-1 within the CD4⁺ (A) or CD8⁺ (B) T-cell gates are shown, with average measures at the longitudinal time points (0, 2, 12, and 24 weeks) by treatment arm with SDs. Dotted lines denote initiation of ATG and last dose of GCSF. Pairwise comparisons were performed at each time point for placebo vs. ATG (all time points P < 0.001) and vs. ATG/GCSF (all time points P < 0.01).