The altering cellular components and function in tumor microenvironment during remissive and relapsed stages of anti-CD19 CAR T-cell treated lymphoma mice

Abstract

Anti-CD19 chimeric antigen receptor (CAR) T cells represent a highly promising strategy for B-cell malignancies. Despite the inspiring initial achievement, remission in a notable fraction of subjects is short-lived, and relapse remains a major challenge. Tumor microenvironment (TME) was proved to be aroused by CAR T cells; however, little is known about the dynamic characteristics of cellular components in TME especially during the different phases of disease after anti-CD19 CAR T-cell treatment. We took advantage of an immunocompetent model receiving syngeneic A20 lymphoma cells to dissect the changes in TME with or without CAR T-cell injection. We found that anti-CD19 CAR T-cell treatment attenuated the symptoms of lymphoma and significantly prolonged mice survival through eradicating systemic CD19⁺ cells. Increased myeloid subsets, including CD11c⁺ DCs and F4/80⁺ macrophages with higher MHC II and CD80 expression in bone marrow, spleen, and liver, were detected when mice reached remission after anti-CD19 CAR T treatment. Compared to mice without anti-CD19 CAR T administration, intrinsic T cells were triggered to produce more IFN-γ and TNF-α. However, some lymphoma mice relapsed by day 42 after therapy, which coincided with CAR T-cell recession, decreased myeloid cell activation and increased Treg cells. Elevated intrinsic T cells with high PD-1 and TIGIT exhaust signatures and attenuated cytotoxicity in TME were associated with the late-stage relapse of CAR T-cell treatment. In summary, the cellular compositions of TME as allies of CAR T cells may contribute to the anti-tumor efficacy at the initial stage, whereas anti-CD19 CAR T-cell disappearance and host response immunosuppression may work together to cause lymphoma relapse after an initial, near-complete elimination phase.

Keywords: CD19; chimeric antigen receptor T cell; immunosuppression; lymphoma; tumor microenvironment.

Figure 1

The general condition of anti-CD19 CAR T treated lymphoma mice. (A) The experiment design. (B) The state of the A20 induced lymphoma model. On day 32, the posture of mice injected with A20 cells combined with or without CD19 CAR-T cells were shown. (C) The body weight was tested every 2–3 days and the fold changes were analyzed. (D) Lymphoma models were evaluated, and the clinical score was recorded. Data were from two independent experiments. Two-way ANOVA was used to compare between the A20 (n = 23) and A20 + CAR-T groups (n = 18). *P < 0.01, ^% P < 0.001, ^# P < 0.0001.

Figure 2

The effect of anti-CD19 CAR-T administration. (A) The survival of lymphoma mice. The OS and PFS data were recorded on the day of the CAR-T-cell injection. (B) The response rate after anti-CD19 CAR T-cell therapy and the time of disease occurrence were shown. (C) The percentage of total CD19⁺ cells and GFP⁺CD19⁺ cells (A20) of BM and liver on day 15. The representative flow cytometry plots were shown for four individual mice. (D) The statistics data for CD19⁺ cells and A20 cell percentage (n = 8 or 12). Survival was compared using the log-rank test, and the t-test was used to compare between two groups. There were two independent experiments that were repeated. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 3

The myeloid cells were upregulated and activated in mice that achieved remission. On day 15, the BM, spleen, and liver from the lymphoma model with or without CAR-T cells were harvested. The myeloid cells were labeled with CD11b, CD11c, and F4/80. (A) The flow cytometry plot of CD11b+ mouse2 of A20 was corrected (B–D). The percentage of CD11b⁺ myeloid cells, CD11c⁺ DCs, and F4/80⁺ macrophages. Then the expression of MHC II and CD80 in different populations were stained and analyzed by flow cytometry. Two independent experiments were repeated for (B) and (C); Four independent experiments were done for (D). Data were compared using unpaired Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Intrinsic T cells were aroused in the remissive stage after anti-CD19 CAR T-cell treatment. (A) Percentage of CD3⁺ T cells and the subsets. (B, C) The cytokine expression on CD4⁺ and CD8⁺ T cells. (D, E) The representative flow data of IFN-γ and TNF-α produced CD8⁺ and CD4⁺ T cells from three mice. (F) The percentage of Treg cells in the BM, spleen, and liver were analyzed, and statistical analysis was done. (G) The representative data from SP on day 15 were shown. Cells were gated on CD4 and the expression of Foxp3 was shown. There were two or four repeated experiments. Data were mean ± SD and compared using unpaired Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5

Elevated syngeneic T cell responses were induced by CD19 CAR-T triggered tumor lysis. Splenocytes from BALB/c mice were harvested and stained with Cell Trace Far Red. Syngeneic splenocytes, CD19 CAR-T, and A20 cells were co-cultured in vitro. Flow cytometry was done to detect T-cell activation and cytokine secretion. (A) Gating strategy of flow cytometry. (B, C) The percentage of T cells and the expression of IFN-γ. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 6

Recession of the intrinsic immune response with enhanced immunosuppression of TME in relapsed mice. After anti-CD19 CAR T-cell treatment for 15 and 42 days, lymphoma mice were in the remissive and relapsed stages, respectively. (A) The percentages of A20 and anti-CD19 CAR T cells were shown. (B) The quantity and expression of activation markers on myeloid cells were detected by flow cytometry. (C) The percentage of T-cell subsets. Two independent experiments were repeated. Data were mean ± SD and compared using unpaired Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 7

Increased exhausted characteristics and decreased cytotoxicity of intrinsic CD8⁺ T cells in the TME of relapsed mice. BM and spleen were harvested from lymphoma mice during the remissive and relapsed stages after anti-CD19 CAR T-cell treatment, respectively. (A) The representative fluorescence intensities of PD-1, TIGIT, and TIM3 on intrinsic T cells were shown. (B) The statistical differences between remissive and relapsed mice were compared. (C, D) The production of IFN-γ and GranzymeB by intrinsic T-cell subsets. Each symbol represents one individual mouse. Data were mean ± SD and compared using unpaired Student’s t-test. *P < 0.05, ***P < 0.001, ****P < 0.0001, ns P > 0.05.