mCD86-28z CAR T cells are safe and do not interfere with bacterial host defense in polymicrobial sepsis models. (A) Summary of the treatment schedule used for toxicity assessment in C57Bl/6 mice. (B) Weight curves of mice treated with CD86-28z (mocha) or GFP control T cells (forest). There were 5 to 8 mice per group. As control mCD86-28z CAR T cells were injected in non-lymphodepleted mice (gray, n = 3). (C) Percentage of transferred T cells in the blood of C57Bl/6 mice at indicated time points after adoptive T-cell transfer (ACT) measured by flow cytometry. (D) Percentage of CD86⁺ B cells (left) or CD86⁺ monocytes (right) in the blood of C57Bl/6 mice at indicated time points after ACT. (E) Simple linear regression of CD3⁺CD8⁺ GFP⁺ T cells (x-axis) and CD86⁺ B cells (y-axis) in the blood of the mice over the different time points after ACT (days 6, 13, 23). r = Pearson correlation coefficient. (F-H) Mice were sacrificed 28 days after ACT, and organs were analyzed by flow cytometry. (F) Percentage of transferred T cells in the different organs of C57Bl/6 mice. (G) Immune cell composition in organs. (H) Percentage of CD86⁺ CD11b⁺ cells in different organs. (A-H) Data are mean ± SEM of 3 to 8 mice per group. Statistical significance was calculated using 2-way ANOVA with Tukey multiple comparison correction. (I,L) Summary of the treatment schedule used to assess formation of antigen-specific T cells in C57Bl/6 mice. (J,M) Percentage of OVA-specific T cells in the blood or spleen of antibody (J; n = 4-5 mice per group) or CAR T cell-treated mice (M; n = 7-8 mice per group). SINFEKL pentamer staining was used to measure antigen-specific T cells by flow cytometry. (K,N) IFN-γ–positive T cells measured by intracellular flow cytometry after restimulating harvested splenocytes with a SINFEKL peptide. (K,N) Mice were injected with aCD86 antibody or isotype control antibody on the indicated days (K) or treated with depicted amounts of mCD86-28z CAR T cells or GFP control T cells (N). (O,R) Summary of the treatment schedule used to assess bacterial host defense in C57Bl/6 mice. (O) 4 to 5 mice per group. (R) 8 mice per group. (P,S) Sepsis severity score after injection of cecal slurry. (Q,T) Bacterial colony counts in the blood (colony formation units per microliter) of mice IP injected with cecal slurry. For all panels, data are mean ± SEM of the indicated n number. Statistical significance was calculated using 2-way ANOVA with Sidak or Tukey multiple comparison correction or unpaired t test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. ns, P > .05. i.p., intraperitoneal.