, 2010a and Bere et al., 2010b). Here, cervical cells from 7 HIV-infected women were thawed to investigate whether cryopreserved cytobrush-derived T cells could be expanded in vitro with anti-CD3 and rhIL-2 after thawing ( Fig. 2). From these 7 cytobrushes, a median of 80 000 CD3+ T cells (IQR 35 040–110 880) was isolated prior to cryopreservation. After thawing, 30% of these CD3+ T cells was recovered (median of 23 680 CD3+ T cells; IQR 13 968–47 168; p = 0.0278). Four of the 7 thawed cervical samples expanded successfully during

14 days of polyclonal culture with anti-CD3 and rhIL-2 ( Fig. 2). A median yield of 23 845 CD3+ T cells (IQR 12 100–91 220) was obtained from these 4 samples after thawing and was expanded to a median of 252 291 CD3+ T cells (IQR 190 308–701 000; 10-fold; p = 0.0286) after buy Venetoclax 14 days of culture. We investigated the impact of cytobrush handling

and processing on the ability of cervical T cells to produce IFN-γ following stimulation with PMA/Ionomycin (positive control). The rate of PMA/Ionomycin failure (no production of IFN-γ following PMA stimulation) was determined in cervical CD8 and CD4 T cells processed immediately ex vivo (n = 98) or subjected to delayed processing [24 h at 37 °C (n = 24), 4 °C (n = 5) or room temperature (n = 22)]. We found that ex vivo CD3 cell counts in cervical cytobrush samples correlated significantly with the frequency of T cells producing IFN-γ following stimulation with PMA/Ionomycin (Rho = 0.5, P < 0.0001). Furthermore, cervical samples which failed to respond to PMA/Ionomycin had significantly lower CD3+ events PF-562271 [median 18 (IQR 4–143)] than cytobrush samples that yielded positive IFN-γ responses to PMA [median 98 (IQR 6–154); Fig. 3; p = 0.0007]. From this finding, samples with CD3+ event counts < 100 or were unresponsive to PMA/Ionomycin were excluded from further analysis. No significant differences were observed between the rate of PMA/Ionomycin failure by CD8 or CD4 T cells in cervical samples

subjected to delayed processing Baf-A1 manufacturer after 24 h at 37 °C, 4 °C or room temperature compared to those processed immediately (Table 3). Furthermore, the odds of obtaining a positive PMA response after 24 h at any of the mock transport conditions were similar to that ex vivo ( Table 3). In addition, delayed processing (using any of the conditions tested) did not significantly alter the magnitude of PMA/Ionomycin-stimulated IFN-γ responses by CD8+ or CD4+ T cells compared to ex vivo ( Fig. 4 left panels). Similarly, we found that delayed processing did not result in significantly reduced rates or magnitudes of T cell responses following mitogenic stimulation with PHA (data not shown). In addition to IFN-γ responses to mitogens PMA/Ionomycin and PHA, we evaluated the ability of cervical cytobrush-derived T cells to produce IFN-γ in response to CEF peptides, a pool of common viral epitopes from Cytomegalovirus, Epstein–Barr virus and Influenza virus (Currier et al.