Figure 5: Transient CD28 blockade limits inflammatory cytokines in the MM BME without affecting survival of hBCMAmBBmζ CAR T cell treated mice.

(A) Diagram of experimental setup. MM.1S bearing NSG mice were treated with hBCMABBζ CAR T cells ± 200μg abatacept on days −1, 1, 3, 5, 7 (5B and 5C) or 5TGM1^hBCMA bearing RAG2^−/− mice were treated with CD28^fl/fl or CD28^iKO hBCMAmBBmζ CAR T cells (5D and 5E) and euthanized 7 days post adoptive transfer for hind limb BM harvest.

(B) Myeloma burden assessed by flow cytometry for human CD138⁺ cells in the bone marrow of MM.1S bearing mice treated as described in 5A. Bars represent mean ± SD, dots indicate individual mice. **p<0.01 by one-way ANOVA, ns = not significant.

(C) Heatmap representation of human cytokine levels in the MM BME 7 days after treatment of MM.1S bearing mice with hBCMABBζ CAR T cells ± abatacept. Bilateral hind limbs were harvested, and BM was flushed into 15 µL PBS for multiplexed cytokine analysis. Log₂ transformed cytokine concentrations represent the mean of 3–6 mice per group.

(D) Heatmap representation of murine cytokine levels in the MM BME 7 days after treatment of 5TGM1^hBCMA bearing mice with hBCMAmBBmζ CAR T cells ± abatacept. Bilateral hind limbs were harvested, and BM was flushed into 15 µL PBS for multiplexed cytokine analysis. Log₂ transformed cytokine concentrations represent the mean of 2–3 mice per group.

(E) Bar graphs showing concentrations of murine IP-10 (left) and IL-12 (right) in the MM BME 7 days after treatment of 5TGM1^hBCMA bearing mice with CD28^fl/fl hBCMAmBBmζ CAR T cells ± abatacept versus CD28^iKO hBCMAmBBmζ CAR T cells. Bars represent mean ± SD of 2–3 biological replicates. **p<0.01, ***p<0.001 by one-way ANOVA.

(F) Diagram of experimental setup. MM.1S bearing NSG mice were treated with hBCMABBζ CAR T cells ± 200μg abatacept on days −1, 1, 3, 5, 7 and tumor burden was monitored by bioluminescent imaging 2x/week until endpoint.

(G) Kaplan – Meier analysis of survival of hBCMABBζ CAR T ± transient abatacept or mock transduced T cell treated MM.1S-luc bearing mice (n = 4–5 mice per CAR T cell treated group). Median survival of hBCMABBζ CAR T treated mice was >100 days post CAR T cell infusion vs. 97 days for hBCMABBζ CAR T + transient abatacept treated mice. Statistical significance was determined by log-rank Mantel-Cox test.