Figure 4. Viral replication, TGFβ signaling, and regulatory dendritic cells promote T_FR expansion.

Tonsil cells were mock-spinoculated or spinoculated with X4 or R5 virus and cultured for 2 days under a variety of conditions. (a) Tonsil cells were treated with the integrase inhibitor raltegravir (RAL, 10 μM) during culture to allow viral entry but prevent replication and percentages of T_FR determined (n=5). (b) Tonsil cells were cultured under the conditions shown and TGFβ-1 levels were measured in culture supernatant by ELISA (n=8). (c) Tonsil cells were cultured in the presence of TGFβ blocking antibodies (2 μg/mL) and the percentages of T_FR were measured (n=4). Addition of anti- TGFβ antibodies did not influence cell viability. (d) Tonsil cells were cultured under the conditions shown and then analyzed for the presence of immature myeloid DCs (CD11c+CD1c+HLA-DRlo) and mature myeloid DCs (CD11c+CD1c+HLA-DR+CD83+) (n=6). (e) Samples in (d) were also analyzed for activated plasmacytoid DCs (CD123+HLA-DR+, n=6). (f) Cell culture supernatants and lysates from (d, e) were analyzed by ELISA to quantitate IDO production (n=6). The horizontal bars of each graph indicate the median value and are listed where appropriate for clarity. Statistical analyses were performed Wilcoxon matched-pairs tests (a, c) or Whitney ranked sums tests (b, d-f) and significance is denoted by asterisks where * = p < 0.05, ** = p < 0.01 and *** = p < 0.001.