Fig 2: Correlational analyses at 6 months between L-PHA MFI and serum 25(OH)D levels. Association of serum 25(OH)D levels in both arms with the L-PHA MFI of (A) CD3+, (B) CD4+, (C) CD8+ T cells, (D) CD19+ B cells, (E) CD56dim, and (F) CD56bright NK cells. Spearman’s Rho (n = 38).

Fig 3: Development of total-drug-bound receptor occupancy (RO) assay to detect RG7769 on peripheral blood derived immune cells. (A) CD3-CD28 activated peripheral blood mononuclear cells (PBMCs) were incubated with increasing concentrations of RG7769 (100 pg/ml?100 µg/ml). PD-1 expression on CD3+ T cells was analyzed using anti-PD-1, clone EH12.1, by flow cytometry in a representative donor. (B) Pooled normalized relative CD3+ T cell PD-1 expression from two independent experiments assessing anti-PD-1 clones EH12.1, NAT105 and J105. (C) Schematic depicting RG7769 engaging T cells via high-affinity anti-PD-1 and low-affinity anti-TIM3 binding on target cells. RG7769 Fc harbors the P329 IgG1 mutation (“PGLALA”) which can be specifically detected via anti-PGLALA-PE (M-1.7.24). (D) PBMCs from n = 3 different donors incubated with pembrolizumab (IgG4 or PGLALA), rituximab (IgG1) or respective isotype controls (Iso IgG1, Iso IgG4, DP47-PGLALA), and detected via anti-PGLALA-PE. Pembrolizumab staining is shown for CD3+ T cells [Color figure can be viewed at wileyonlinelibrary.com]

Fig 4: Activation-induced ATP and cell death. (A) T cells from type 1 diabetes patients (T?1D) have lower basal cellular ATP content compare to healthy controls (HC) but cellular ATP ?was? significantly increased after in vitro activation. Enriched total T cells were stimulated in vitro with plate-bound anti-CD3 and anti-CD28 or isotype antibodies for 24 hours. ATP content was detected in cell lysate and corrected by protein amount. The trend remains that T cells from type 1 diabetes patients have lower ATP content even though in vitro activation induces a significant increase of ATP in this group. (B) T cells from T1D patients and HC showed the same level of activation-induced apoptosis. After 24-hour activation with plate-bound anti-CD3 and anti-CD28, T cell apoptosis was identified by staining with Annexin-V and Propidium Iodide. Activation-induced apoptosis was calculated as mentioned in method session. (C) T cells with MHP showed the same activation-induced apoptosis as normal ??m T cells. (D) Activation-induced apoptosis was not correlated with T cell ??m. *P < 0.05, unpaired t test with Welch’s correction. **P < 0.01 Wilcoxon matched pair test for (A) and Mann-Whitney test for (B).

Fig 5: TGF-ß1 mediates decreased NK cell cytotoxicity. (A) NK cells purified from PBMCs of JORRP patients were preincubated with LY364947 for 3 days in the presence of 100 U/ml IL-2, then stimulated with K562 target cells for an additional 6 h at the ratio = 1:1. (A, B) Representative paragraph and percentage analysis of K562 cell apoptosis (the percentage of Annexin V on K562 cells) was determined by flow cytometry. Blank, K562 cells alone; NC, NK cells only; DMSO, NK cells + DMSO; LY364947, NK cells + LY364947. (C, D) Representative histograms and percentage analysis of CD107a on NK cells. (E) NKp30, NKG2D, and NKp46 expression on NK cells. MFI, mean fluorescent intensity. (F, G) PBMCs from HCs, PBMCs and TILs were isolated from JORRP patients. Representative paragraph and percentage analysis of CXCR6 gated on CD56+CD3- cells. TIL, tumor-infiltrating lymphocyte. Data are presented as median with interquartile range. Each dot represents a single individual. *P < 0.05; ***P < 0.001; ****P < 0.0001.