Sample FACS results are shown in Fig
Sample FACS results are shown in Fig.?1B. (K-CAR). Antitumor effects of K-CAR were demonstrated and gene in a lentiviral vector (Fig.?S2B) were used as artificial antigen-presenting cells (aAPCs) to propagate CD3+ T cells expressing K-CAR. The expression of HERV-K in K562 cells was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) (Fig.?S2C, left panel), immunoblot (Fig.?2C, right panel), and FACS using 6H5 mAb (Fig.?S2D). PU-WS13 expansion of K-CAR T cells originating from BC patients and normal female donors PBMCs from 9 BC patients (Table?2) and 12 normal female donors (NDs) were electroporated with HERV-KCD28MZ SB transposon (pSBSO) along with pCMV-SB11 transposase and cultured with IL-2 to expand CD3+ T cells expressing K-CAR. scFv expression in K-CAR T cells from various donors (BC: n = 9 and ND: n = 12) was further confirmed by RT-PCR using primers specific to the 6H5 scFv (Fig.?1A), and growth of T cells was monitored over time by microscopy (Fig.?S3A, top panel). The percentage of K-CAR T cells generated from PBMCs was determined post-electroporation by FACS using anti-CD3 and anti-Fc antibodies. Sample PU-WS13 FACS results are shown in Fig.?1B. K-CAR T cells from patient #157 had a significantly lower proliferation rate than cells from ND1 (Fig.?S3A). Nearly all T cells from BC patients as well as NDs expressed the K-CAR on day 28 post-electroporation, as measured PU-WS13 by expression of the Fc backbone (Fig.?S3B). Specific lysis of K562-HERV-K cells by K-CAR T cells obtained from two normal donors was observed, as determined by a cytotoxic T lymphocyte (CTL) assay (Fig.?S3C). Percentages of CD4+ and CD8+ T cells as well as T regulatory cells (Tregs: FOXP3 PU-WS13 and CD25 positive) were determined. A sample from a BC patient (#243) revealed a higher percentage of FOXP3 in CD4+ T cells than in CD8+ T cells (Fig.?1C, left panel), and increased percentages of CD8+ T cells were observed after CD4+ depletion (Fig.?1C, right panel). Higher percentages of both FOXP3+ and CD25+ T cells were demonstrated in K-CAR obtained from the BC patient than from ND1 control (Fig.?S3D). Higher percentages of CD4+ than CD8+ T cells were observed in BC patients (n = 7) compared with NDs (n = 12), but the differences were not significant (Fig.?1D, top panel). CD4+ cell T depletion resulted in significantly enhanced percentages of CD8+ and significantly decreased percentages of CD4+ T cells in K-CAR T cells from BC patients (Fig.?1D, bottom panel). Figure 1 . Open in a separate window Characterization of K-CAR in various donors. (A) RT-PCR was employed to detect the expression of 6H5 scFv (700?bp) using scFv specific primers. Amplified -actin was used as a loading control, and scFv plasmid was used as a positive control. Expression of 6H5 scFv was demonstrated in K-CAR T cells obtained from BC patients and NDs. No scFv expression was detected in control T cells or PBMCs. (B) Both Fc+ and CD3+ T cell populations were determined in T cells transfected with K-CAR or GFP from patient 157 (top panel) and ND1 (bottom panel) by FACS using anti-Fc and CD3 antibodies on days 7, 21 and 35 post-transfection. The isotype alone was used as control. (C) Both FOXP3+ and CD8+ or CD4+ T cells from BC patient 243 were determined by FACS (left panel). The percentage of CD8+ T cells was increased in K-CAR T cells after CD4+ depletion (right panel). (D) Lower percentages of CD8+ and higher percentages of CD4+ T cells were demonstrated in K-CAR T cells obtained from BC patients than from NDs (top panel). Significantly enhanced CD8+ (= 0.0003) and reduced CD4+ (= 0.0004) T cell populations were demonstrated in T cells from BC PU-WS13 patients (n = 7) after CD4 depletion. Figure 1. Open in a separate window (Continued) Figure 1. Open in a separate window (Continued) Figure 1. Open in a separate window (Continued) Table 2. Demographic, medical, and independent variables measured at baseline of breast cancer with K-CAR+ T cells from BC patients #157 or #108. Significantly Rabbit Polyclonal to Fyn reduced growth was observed in two BC cell lines treated with K-CAR T cells from both BC patients (Fig.?2A). Figure 2 . Open in a separate window Detection of antitumor effects RNA was knocked down in both cell lines using an shRNA targeted.