The Effect of Inhibitory Signals on the Priming of Drug Hapten–Specific T Cells That Express Distinct Vβ Receptors

Proliferation of SMX-NO–primed naive T cells, with or without coinhibitory receptor-blocking Abs. Naive T cells from healthy donors were cultured with mature autologous monocyte-derived DCs in the presence of SMX-NO (50 μM) for one 1 wk, with or without PD-L1–blocking Abs (5 μg/ml) (A), CTLA4-blocking Abs (5 μg/ml) (B), or TIM-3–blocking Abs (7.5 μg/ml) (C). T cells were harvested and restimulated with fresh mature DCs and SMX-NO (20–50 μM) for 48 h. [³H]Thymidine was added and incubated for an additional 16 h. Data from three representative donors show the mean ± SD of triplicate cultures. Statistical significance denotes a significant increase in the proliferative response compared with medium-only–treated wells within that condition (i.e., TIM-3–blocked cells only). Statistical significance between conditions denotes a significant increase in the proliferative response compared with “no block” wells at a particular drug concentration after normalization of all data values to account for differing basal stimulation. *p ≤ 0.05, **p ≤ 0.005, ***p < 0.001.

Proliferation of SMX-NO–exposed memory T cells, with or without coinhibitory receptor-blocking Abs. Memory T cells from healthy donors were cultured with mature autologous monocyte-derived DCs in the presence of SMX-NO (50 μM) for one 1 wk, with or without PD-L1–blocking Abs (5 μg/ml) (A), CTLA4-blocking Abs (5 μg/ml) (B), or TIM-3–blocking Abs (7.5 μg/ml) (C). T cells were harvested and restimulated with fresh mature DCs and SMX-NO (20–50 μM) for 48 h. [³H]Thymidine was added and incubated for an additional 16 h. Data from three representative donors show the mean ± SD of triplicate cultures. Statistical significance denotes a significant increase in the proliferative response compared with medium-only–treated wells within that condition (i.e., TIM-3–blocked cells only). Statistical significance between conditions denotes a significant increase in the proliferative response compared with “no block” wells at a particular drug concentration after normalization of all data values to account for differing basal stimulation. *p ≤ 0.05, **p ≤ 0.005, ***p < 0.001.

Coinhibitory receptor expression during the SMX-NO–specific activation of CD4⁺ and CD8⁺ naive and memory T cells from healthy donors. Expression of coinhibitory receptors on naive [PD-1 (A), CTLA4 (B), TIM-3 (C)] or memory T cells [PD-1 (D), CTLA4 (E), TIM-3 (F)] from healthy donors during stimulation and during restimulation with SMX-NO (50 μM) using mature autologous DCs. Samples of T cells were harvested at various time points and labeled with CD4, CD8, and PD-1/CTLA4/TIM-3 fluorochrome-bound Abs. Percentages indicate the proportion of cells that stain positive for each coinhibitory receptor.

Expression of CTLA4 and TIM-3 on dividing and nondividing T cells derived from priming healthy donor naive T cells to SMX-NO. Naive T cells from healthy donors were cultured with mature autologous DCs in the presence of SMX-NO (50 μM) for 1 wk. T cells were stained with CFSE and restimulated with fresh mature DCs and SMX-NO (50 μM) for 72 h. T cells were incubated with CTLA4-allophycocyanin or TIM-3–PE, and with CD4- and CD8-specific fluorochromes. Expression of CTLA4 (A) and TIM-3 (B) on CD4⁺ or CD8⁺ T cell populations separately (shaded graph, expression on dividing T cells; black line, expression on nondividing T cells). Percentages indicate the percentage of each population gated positive for each coinhibitory receptor. (C) Comparative expression on both dividing and nondividing T cell populations of the respective isotype (IgG1) control Abs for CTLA4 or TIM-3 (dark gray shaded graph, unstained expression; light gray shaded graph, expression of isotype control; black line, expression of CTLA4 or TIM-3).

Expression–activity relationship of CTLA4 and TIM-3 on SMX-NO–responsive T cell clones. (A) Expression of PD-1, CTLA4, and TIM-3 on 40 representative T cell clones derived from priming healthy donor naive T cells to SMX-NO. (B) The proliferative response of T cell clones expressing varied levels of CTLA4 or TIM-3 in response to SMX-NO stimulation, as measured by [³H]thymidine incorporation. The MFI derived from flow cytometry, indicating the relative expression of CTLA4 or TIM-3, is shown for each clone at the top of the graphs. (C) Expression of CTLA4 and TIM-3 on representative T cell clones over 240 h, with or without SMX-NO stimulation (40 μM).

Activation of SMX-NO–responsive T cell clones, with or without coinhibitory receptor blockade. Secretion of IFN-γ, IL-13, and granzyme B (A) and the proliferative response of T cell clones in response to SMX-NO stimulation (B), with or without anti-CTLA4 (10 μg/ml) or anti–TIM-3 (7.5 μg/ml) treatment. Cytokine secretion was measured by ELISPOT, and proliferative analysis was by [³H]thymidine incorporation. Statistical significance between conditions denotes a significant increase/decrease in proliferative response or cytokine release.

Suppressive effect of autologous CD25⁺ Tregs on the SMX-NO–induced activation of naive and memory T cells from healthy donors. Naive (A) or memory (B) T cells were cultured with mature DCs and SMX-NO (50 μM) for 1 wk, with or without autologous CD25⁺ Tregs (2–6 × 10⁵ per well). T cells were harvested and restimulated with fresh mature DCs and SMX-NO (20–50 μM) for 48 h before measurement of proliferative capacity through the incorporation of [³H]thymidine. (C) Expression of PD-1, PD-L1, PD-L2, CTLA4, and TIM-3 on autologous Tregs was analyzed by flow cytometry to identify the propensity for coinhibitory pathways to aid Treg-mediated suppression of SMX-NO–induced T cell responses. Statistical significance between conditions denotes a significant increase/decrease in proliferative response. *p ≤ 0.05, **p ≤ 0.005, ***p < 0.001.