Journal: PLoS Pathogens
Article Title: Plasmacytoid dendritic cells have divergent effects on HIV infection of initial target cells and induce a pro-retention phenotype
Figure Lengend Snippet: Addition of pDCs increased intracellular HIV expression but not extracellular virus production in memory CD4 T cells. (A) Workflow for generating memory T cell-pDC cocultures: memory CD4 T cells were sorted into resting TCM, TTM and TEM subsets. Sorted cells were infected with either HIV BaL or HIV TF (MOI of 0.75) overnight. They were cultured for further 2 days before the addition of pDCs. (B) Representative dot plots showing p24 expression at 5 dpi in resting TCM, TTM and TEM that were infected with HIV BaL in the absence (HIV) or presence of pDCs (HIV+pDC). (C) Mean percentage of p24 + cells at 5 dpi with HIV BaL (n = 11). (D) CCR5 expression across T cell subsets in the presence and absence of pDCs (n = 11). (E) Mean percentage of p24 + cells at 5 dpi with the HIV TF in the presence or absence of pDCs (n = 7). (F) Assessment of extracellular viral production by the reverse transcriptase assay using supernatants derived from infected resting CD4 T cell subsets in the absence (HIV) or presence of pDCs (HIV+pDC) (n = 2–5). (G) Same as in F but using p24 ELISA on supernatants derived from infected resting TEM cells (n = 2). For all data, *p
Article Snippet: These reagents included: an inhibitor of the intercellular adhesion molecule and component of the immunologic synapse (anti-ICAM-1, 10μg/mL, R & D systems, clone BBIG-I1); the CCR5 inhibitor maraviroc (10μM, AIDS Reagent Program); the reverse transcription inhibitor Zidovudine (50μM, AIDS Reagent Program); neutralising antibodies to receptors of IFN (IFNAR, 20μg/mL, PBL Assay Science, clone MMHAR-2) and TNF (TNFR1, 10μg/mL, R & D systems, clone 16803) in addition to neutralising antibodies for secreted IFNα8 (20000U/mL, PBL Assay Science), IFNβ (5000U/mL, PBL Assay Science) and TNFα (1μg/mL, R & D systems) to block signalling pathways of INF and TNF; recombinant (r) human TNFα (10ng/mL, R & D systems) and rIFNα8 (PBL Assay Science) was used at 350ng/mL for MDDCs and MDMs and at a graded concentration of 300, 600 and 900ng/mL for TCM, TTM and TEM based on the amount of IFNα detected by ELISA in infected MDDCs ( ) and T cell subsets ( ) upon their co-culture with pDCs.
Techniques: Expressing, Transmission Electron Microscopy, Infection, Cell Culture, Reverse Transcriptase Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay