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. 2023 Dec 11;32(1):101171. doi: 10.1016/j.omtm.2023.101171

Figure 5.

Figure 5

Negative selection results in a final CART product enriched in γδ T cells

(A) Volcano plot representation of differential gene expression between CD22 CARTpos and CARTneg (day 9). Fold changes of >2 and p < 0.05 were considered significant (dotted lines). The identities of several differentially expressed transcripts are indicated. (B) A representative dot plot showing the percentages of TCRαβneg cells within the viable cell gate is shown for one donor (left). Quantification of TCRαβneg cells in all donors is shown (right). The B-ALL patient sample is indicated with an “x.” (C) T cells expressing TCRVδ1 or TCRVδ2 were monitored by flow cytometry. Representative plots (left) and quantifications (right) are shown. (D) Expression of TCRVδ1 and TCRVδ2 were assessed in CD4+, CD8+, and CD4−CD8– compartments. A representative TCRVδ1/TCRVδ2 profile in the CD4−CD8- subset (left) and quantifications (right) are shown. (E) Representative dot plots showing the percentages of CD22 CAR-transduced cells within the TCRVδ1 and TCRVδ2 subsets are shown for one donor (left). Quantifications of the percentages of CD22 CAR-transduced cells within the αβ and γδ populations in the 6 donors, following positive and negative selection, are shown (right). (F) The percentages of γδ (TCRVδ1/TCRVδ2) T cells within the total CAR+ population (left) as well as the percentages of γδ T cells within the CD4−CD8−CAR+ population (right) are presented. (G) T cells were isolated from elutriated lymphocytes using either CD3/CD28 Dynabeads (positive enrichment) or a non-GMP negative T cell selection (EasySep) as compared with CD4/CD8-selected cells. Quantifications of the percentages of γδ T cells within the CD3 populations are indicated (n = 3). (H) Schematic showing that only αβ T cells are retained by CD4/CD8 positive selection whereas γδ T cells are also retained during negative selection (image created with biorender.com). ∗p < 0.05. ∗∗p < 0.01, ∗∗∗p < 0.001, ratio paired t test (B, C, and F); ns > 0.05, ∗p < 0.05, ∗∗p < 0.01, two-way ANOVA with multiple comparisons (E); ∗∗∗∗p < 0.0001, one-way ANOVA with multiple comparison (G).