Cellular aggregates formed between CAR T cells and circulating tumors accumulate in the lungs. (A) Endogenous B cells limit CAR T cell accumulation in the bone marrow. RAG2^−/− or C57BL/6 mice were injected with a mixture of CAR⁺ and CAR⁻ GFP⁺ T cells (containing 60% CAR⁺ T cells). One group of mice was treated 3 d before with 50 µg anti-CD20 mAb i.v. The percentage of CAR⁺ T cells (as detected by hCD34 expression) among GFP⁺ cells was assessed in the bone marrow 3 d after T cell transfer using flow cytometry. Red horizontal bars represent mean values. Representative of two independent experiments (n = 3 mice per group). Unpaired t test was used for statistical analysis. **, P < 0.01; ***, P < 0.001; ns, not significant. (B) CAR T cells form clusters with B cells. Representative two-photon images of the lungs 15 min after CAR T cell injection in CD19-RFP recipients showing the presence of large cellular aggregates containing CAR T cells (green) and B cells (red). Representative of two independent experiments. Scale bar represents 20 µm. (C) RAG2^−/− mice were conditioned by irradiation (4 Gy) and injected (or not) with Eμ-myc cells. After 2 wk, 10 × 10⁶ CAR T cells and 10 × 10⁶ control T cells were coinjected i.v., and their trafficking to the bone marrow was assessed 4 h later by flow cytometry. Four mice per group were analyzed in two independent experiments. Mann–Whitney test was used for statistical analysis. *, P < 0.05. (D and E) CAR T cells form clusters with tumor cells. Mice were conditioned by irradiation (4 Gy) and injected with Eμ-myc-DEVD cells. After 10 d, 10 × 10⁶ CAR T cells were injected i.v. We used untreated RAG2^−/− mice as control recipients that did not contain any CD19⁺ targets. (D) Representative two-photon images of the lungs showing that CAR T cells form cell aggregates in tumor-bearing mice, but not in control RAG2^−/− recipients. Representative of two independent experiments. (E) Quantification of cluster volume in the indicated recipients. The graph shows the percentage of clusters calculated to be >10,000 µm³. (F) CAR T cells and circulating tumor cells formed clusters that accumulated in the lungs. RAG2^−/− mice were adoptively transferred with a 1:1 mixture of dye-labeled CAR T cells and control untransduced T cells and 5 min later with tumor cells i.v. Two-photon imaging of the explanted lungs was performed 15 min later. Representative images showing that CAR T cells (red) and tumor cells (blue) in the circulation formed cellular aggregates detected within minutes in the lungs. Control T cells were not clustered and are shown in orange. Vessels (gray) were visualized by i.v. injection of a PE-conjugated anti-CD31 mAb. Representative of 12 images in two independent experiments. Scale bar represents 20 µm.