Extended Data Fig. 3: Flow cytometry reveals predominant memory T-cell differentiation.

(a) Flow cytometry analysis of lymphocyte populations in PB. Upper Panel: Lymphocytes were identified based on forward scatter (FS) and side scatter (SS) parameters, and subsequently divided into CD5+ and CD5- populations. Middle Panel: Analysis of the CD5+ population, and Lower Panel: Analysis of the CD5- population. In both the middle and lower panels, naïve and memory T cells were identified by CD45RA and CD45RO expression, respectively. Staining for CCR7 and CD62L allowed further subclassification. (b) Flow cytometry analysis of bone marrow 9 months post-CAR-T infusion, at the time of cutaneous lesion development, and prior to dexamethasone treatment. Upper left: Lymphocytes are identified based on side scatter (SS) and CD45 expression. Lower left: CD5+ BCMA-CAR- T cells. Upper right: CD5+ BCMA-CAR+ T cells. Lower right: CD5- BCMA-CAR+ T cells. Expression profiles of TRBC1 and TRBC2, as well as CD4 and CD8, are shown for each population. The percentage of expression for each marker and the corresponding fluorochrome are indicated in the graph. (c) Sorting strategy for flow cytometry-based cell sorting of CAR+CD5+, CAR+CD5-, and CAR- CD5+ T-cell populations. Singlets were selected based on the SS, and viable T-cells based on the expression of CD3+ and Calcein+. From the single Calcein+CD3+ T-cells, the three respective populations were sorted based on the expression of CAR and CD5.