CD14⁺ cells are needed for the inflammatory cytokine response against RSV in PBMC cultures.

<p>(<b>A</b>) Confirmation of specific depletion of CD14⁺ and BDCA-4⁺ cells by FACS. M: monocyte gate, L: lymphocyte gate. Depletion of CD14⁺ cells leaves B cells (in M+L, CD19⁺ third row) and CD16⁺ monocyte/DC (in M, CD14^neg., CD16⁺, 2^nd row), cDC (in M+L, lineage^neg., MHCII⁺, CD11c⁺, 4^rd row) and pDC (in M+L, lineage^neg., MHCII⁺, BDCA-4⁺, 4^rd row) compartments intact. Depletion of BDCA-4⁺ cells removes pDC but does not affect CD14⁺CD16^neg., CD14⁺CD16⁺, CD14^neg.CD16⁺ cells in the M gate (2^nd row), nor B cells (3^rd row) and cDC (4^rd row). The contribution of single cell types to anti-RSV cytokine responses in PBMC cultures was evaluated by depletion of specific cell populations. Cytokines in supernatant from the remaining cell populations were measured after 20 hrs. exposure to RSV or UV-RSV in the presence or absence of autologous serum. (<b>B</b>) Depletion of CD14⁺ cells and BDCA-4⁺ cells was confirmed by stimulation of depleted cell populations by TLR ligands, ultra-pure LPS to confirm the absence of TLR4⁺ (monocytes) and ODN 2216 to confirm the absence of TLR9⁺ (pDC). (<b>C</b>) PBMC, CD14 monocyte-depleted PBMC, or pDC-depleted PBMC were cultured with live and UV inactivated RSV (A2, MOI 5) in the presence or absence of 10% autologous serum. Data represent the mean ± SEM of 3 measurements in 1 donor and were analyzed using one way ANOVA followed by a Bonferroni post-test, *P< 0.05, **P <0.01. Experiments were performed in 3 different donors with similar results.