Figure 6. High Affinity FRβ CAR T-cells specifically eliminate FRβ⁺ myeloid lineage cells without toxicity against HSCs.

(a) Number of total and lineage specific colonies from CFU assay following 4h co-culture of 2000 CD34⁺ and 2000 CAR⁺ T cells. Error bars represent mean ± SD of duplicate wells. One representative of 4 independent experiments is shown. (b) Phenotype of CD34⁽⁻⁾ adult BM following 5h co-culture with CAR T cells. Untreated samples were cultured in the absence of T cells. Upper panels - frequency of CD33 and CD19 expression in total live, CD3⁽⁻⁾ target cells surviving co-culture with the indicated T cells. Lower panels - FRβ expression in total live, CD3⁽⁻⁾CD33⁺ myeloid lineage BM target cells surviving co-culture with the indicated T cells. (Gray histogram– isotype, black histogram – HA-FRβ IgG). One representative of 3 independent experiments is shown. (c) Percent lysis of isolated CD14⁺ peripheral blood monocytes following 4h co-culture with CAR T cells at 25:1, 5:1, or 1:1 E:T ratios. Error bars represent mean ± SD of 6 replicate wells. 5 independent healthy monocyte donors were assessed. CFU– colony forming unit, GEMM–granulocyte/erythrocyte/monocyte/megakaryocyte, GM–granulocyte/monocyte, G–granulocyte, M–monocyte, BFU-E– erythroid blast forming unit, BM– bone marrow, SD– standard deviation, SEM– standard error, LA – low affinity FRβ, HA – high affinity FRβ.