Supplemental Methods CD107a assay For each T cell culture that was tested, two tubes were prepared. One tube contained BCMA-K562 cells, and the other tube contained NGFR-K562 cells. Both tubes contained CAR-transduced T cells, 1 ml of AIM-V medium+5% human AB serum, a titrated concentration of an anti-cd107a antibody (ebioscience, clone ebioh4a3), and 1 L of Golgi Stop (monesin, BD). All tubes were incubated at 37 C for 4 hours and then stained for CD3, CD4, and. Intracellular cytokine staining assay (ICCS) For each type of T cell culture that was tested, two tubes were prepared. One tube contained BCMA-K562 cells, and the other tube contained NGFR-K562 cells. Both tubes contained effector cells, 1 ml of AIM-V medium+5% human AB serum, and 1 L of Golgi Plug (BD). All tubes were incubated at 37 C for 6 hours. The cells were surface stained with anti-cd3, anti-cd4, and anti-. The cells were permeabilized, and intracellular staining was conducted for IFN (BD, clone B27), IL-2 (BD, clone MQ1-17H12), and (BD, clone MAb11) by following the instructions of the Cytofix/Cytoperm kit (BD). Interferon BCMA + or BCMA-negative target cells were combined with CAR-transduced T cells in duplicate wells of a 96 well round bottom plate in AIM-V medium+5% human serum. The plates were incubated at 37 C for 18-20 hours. Following the incubation, ELISAs for IFN were performed by using standard methods (Pierce). When BCMA protein was added to assess the effect of soluble BCMA on CAR function, graded concentrations of the extracelluar portion of BCMA, amino acids 1-54, were added (Novus Biologicals). Human BCMA was detected in the serum of tumor-bearing mice by using the human BCMA/SF17 Duoset ELISA kit (R&D) as recommended by the manufacturer. Proliferation assays
Cocultures were set up in 24-well plates. Target cells included in cocultures were either 0.5x10 6 irradiated BCMA-K562 cells or 0.5x10 6 irradiated NGFR-K562 cells. The cocultures also included 1x10 6 T cells from cultures that had been transduced with either anti-bcma2 or SP6. The T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE, Invitrogen) as previously described (1). The medium used in the cocultures was AIM V+5% human AB serum. IL-2 was not added to the medium. Four days after initiation, the live cells in each coculture were counted with trypan blue for dead cell exclusion, and flow cytometry was performed as described under CAR detection on transduced T cells by anti-fab antibody staining. 1. Mannering SI, Morris JS, Jensen KP, Purcell AW, Honeyman MC, Van Endert PM, et al. A sensitive method for detecting proliferation of rare autoantigen- specific human T cells. Journal of Immunological Methods. 2003;283:173-83.
Supplemental Figure 1 anti-bcma2 SP6 BCMA-K562 NGFR-K562 BCMA-K562 NGFR-K562 anti-bcma2 SP6 BCMA-K562 NGFR-K562 BCMA-K562 NGFR-K562 Anti-bcma2 transduced T cells from Myeloma Patient 4 produced and IFN in a BCMA-specific manner. SP6-transduced T cells produced only background levels of cytokines when stimulated with either BCMA-K562 cells or NGFR-K562 cells. Plots are gated on CD3 + T cells.
Supplemental Figure 2 A B C BCMA protein in solution does not block recognition of BCMA + target cells by anti-bcma2-transduced T cells in vitro. A, Anti-bcma2-transduced T cells were cocultured overnight with BCMA-K562 cells in medium containing the indicated concentrations of BCMA protein. B, Anti-bcma2-transduced T cells were cocultured overnight with RPMI8226 cells in medium containing the indicated concentrations of BCMA protein. C, Anti-bcma2-transduced T cells without any other cells were cultured overnight with 150 ng/ml, 25 ng/ml, or 0 ng/ml of BCMA protein as indicated. This elicited only very low levels of IFN production. Note that the y-axis of C is different from the Y axes of A and B. Anti-bcma2-transduced T cells were also cocultured overnight with the BCMA-negative cell line NGFR-K562 as a negative control.
Supplemental Figure 3 Human BCMA protein was detected by ELISA in the serum of NSG mice with RPMI8226 tumors, but human BCMA protein was not detected in the serum of a control mouse that did not have a tumor. Tumor 1-3 refers to 3 mice with RPMI8226 tumors, and control refers to a strain-matched mouse without a tumor.