CD103+ cDC1 and endogenous CD8+ T cells are necessary for improved CD40L-overexpressing CAR T cell antitumor function

While effective in specific settings, adoptive chimeric antigen receptor (CAR) T cell therapy for cancer requires further improvement and optimization. Our previous results show that CD40L-overexpressing CAR T cells mobilize endogenous immune effectors, resulting in improved antitumor immunity. However, the cell populations required for this protective effect remain to be identified. Here we show, by analyzing Batf3−/− mice lacking the CD103+ conventional dendritic cell type 1 (cDC1) subpopulation important for antigen cross-presentation, that CD40L-overexpressing CAR T cells elicit an impaired antitumor response in the absence of cDC1s. We further find that CD40L-overexpressing CAR T cells stimulate tumor-resident CD11b−CD103− double-negative (DN) cDCs to proliferate and differentiate into cDC1s in wild-type mice. Finally, re-challenge experiments show that endogenous CD8+ T cells are required for protective antitumor memory in this setting. Our findings thus demonstrate the stimulatory effect of CD40L-overexpressing CAR T cells on innate and adaptive immune cells, and provide a rationale for using CD40L-overexpressing CAR T cells to improve immunotherapy responses.


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October 2018
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Survival experiments in wild-type and Batf3-/-mice were performed 2x independently. Results were pooled. To this end, survival experiments in Figure 4 were not replicated because of a lack of long-term surviving mice.
Experiments in Figures 1 and 3, and Supplementary Figures 1, 2, and 3 were performed 2x independently. Results were pooled. Experiments in Supplementary Figure 4 requiring wild-type mice were successfully repeated in an independent experiment. Experiments in Supplementary Figure 4 requiring Batf3-/-were not repeated, due to lack of transgenic animals. Experiments in Supplementary Figure 5 were all performed 2x independently.
All attempts at replication were successful.
Tumor-bearing animals were randomized into control and treatment groups before CAR T cell injection, both for survival and non-survival experiments.
Investigators injecting CAR T cells were not blinded because same investigators prepared and injected CAR T cells. For assessment of survival and tumor burden by imaging, investigators were blinded to treatment groups. Investigators were not blinded for other experiments because same investigators euthanized, processed, and analyzed the samples. Note that full information on the approval of the study protocol must also be provided in the manuscript.

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A20 and Phoenix-ECO cells were obtained from ATCC.
Certificate of authentication was received from ATCC. No further authentication was performed.
Used cell lines were tested for mycoplasma contamination and always found to be negative.
No commonly misidentified cell line per ICLAC register was used.
All mice were housed in pathogen-free conditions and kept in a room with controlled temperature (~22 degrees C) and humidity under 12 h light/dark cycle. This study did not involve wild animals.
This study did not involve samples collected in the field.