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. Author manuscript; available in PMC: 2011 Mar 27.
Published in final edited form as: Transplantation. 2010 Mar 27;89(6):667–676. doi: 10.1097/TP.0b013e3181ca9048

Figure 2. Cross stimulation by HEL-specific MD-4 B cells and HEL-modified CTL.

Figure 2

(A) 5×104 resting HEL- or vector-modified CTL transduced and flow cytometrically purified for the expression of CD8 and GFP were re-stimulated with the indicated number of irradiated MD-4 BCR Tg or control non-Tg C57BL/6 splenocytes 9 days after initial transduction. After 48 h, cultures were pulsed with 3H-thymidine and 3H incorporation determined 16 h later by scintillation counting. No proliferative response was observed. Viability of the modified CTL was confirmed by stimulation with control splenocytes in the presence of the mitogen conA. (B) 5×104 purified vector- of HEL-modified CTL, 9 days after transduction, were cultured alone, or co-cultured with 2.5×105 irradiated MD-4 Tg or non-Tg C57BL/6 splenocytes. 48 h later cell free supernatant was isolated and IFN-γ production measured by ELISA. Cells were additionally stimulated with non-Tg splenocytes in the presence of conA as a positive control. ND, none detected. (C) To determine if MD-4 B cell engagement of HEL on HEL-modified CTL induced B cell lysis, MD-4 splenocytes (∼50% B lymphocytes) were co-cultured in triplicate with HEL-CTL or vector-modified CTL for 6 h at a 1:1 ratio. B lymphocytes were then enumerated by staining with B220 and MD-4 allotype specific IgMa reactive antibodies and quantitative flow cytometry. A significant increase in survival (2-sided t-test, p<0.05) is apparent among B cells co-cultured with HEL-CTL in the experiment shown, but was inconsistently seen in additional analyses. (D, E) To determine if MD-4 B lymphocytes are stimulated to proliferate and expand in response to the HEL-CTL, CFSE-labeled MD-4 splenocytes were co-cultured with HEL- or vector-transduced CTL, or cultured untreated or in the presence of LPS for up to 3 days. Cells were analyzed at daily intervals by quantitative flow cytometry. Total number of viable cells detected are plotted (D). (E) Plots showing CFSE dilution with proliferation. Representative data is shown for each subfigure from 2 or more experimental repeats.