Antigen/immune complexes internalized through FcγRI are presented more efficiently to CD4⁺ T cells. (A) A hemagglutination inhibition assay was used on serum samples from a panel of influenza A–vaccinated human donors to identify samples contained high levels of anti–influenza A (strain A/PR/8/34) hIgG. (B) Flow cytometric analysis of live influenza A virus labeled with the lipophilic fluorescent dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine binding to U937 cells (top histogram); 1 μM of hIgG isolated from donor 1 inhibits the binding of the live virus (bottom histogram). (C) Purified immune complexes (IC) generated by incubating live influenza A virus with hIgG from donor 1 (Flu/Ab) exhibit protein bands from both the virus (Flu) and hIgG (Ab). (Di) Cytosolic calcium signaling in primary human monocytes stimulated with IC. IC-mediated FcγRI Ca²⁺ signaling was analyzed in the presence of a blocking antibody to FcγRII, IV.3. FcγRIIa signaling was analyzed in presence of excess hIgG to ensure receptor occupancy and blockade of FcγRI. (Dii) IC-mediated FcγRIIa induced cytosolic Ca²⁺ in primary monocytes treated with the PLC-γ inhibitor U73122 versus untreated controls. (Diii) IC-mediated FcγRI induced cytosolic Ca²⁺ in primary monocytes treated with the PLD inhibitor butan-1-ol (0.3%) versus butan-2-ol (0.3%) and untreated controls. (E) Top panel: FcγRIIa is blocked with antibody IV.3 to allow for FcγRI-mediated internalization of IC into primary human monocytes into enodocytic compartments that stain positive for HLA-DM, the putative MIIC marker, and the influenza A matrix protein (M1 matrix). Middle panel: FcγRI is blocked with excess hIgG to allow for IC internalization via FcγRIIa. Bottom panel: IC uptake via both FcγRI and FcγRIIa in the absence of receptor blockade. (F) Human monocytes that internalize influenza A/hIgG IC via FcγRI versus FcγRIIa stimulate greater proliferation in influenza-A-specific CD4⁺ human T lymphocyte cell lines; 5 cell lines derived from 3 unrelated human donors were tested with syngeneic monocytes pulsed with IC for 10 minutes in the presence or absence of FcγRI or FcγRIIa-blocking antibodies. 3H-Thymidine incorporation was used to measure cell proliferation and the resulting data expressed as a proliferation index. (G) ELISPOT analyses of influenza-A-specific CD4 T-cell responses in primary human cells. Monocytes and CD4⁺ T cells were enriched by CD8 depletion before internalization of IC via FcγRI and/or FcγRIIa. More than 9 independent experiments were carried out on unrelated human donors, the numbers of resulting ELISPOTs were assayed from 14 to 18 hours poststimulation using a CTL ImmunoSpot S4 analyzer. The fold difference between the number of spots per treatment condition over controls was calculated. An ANOVA analysis, followed by a protected t test, was conducted between the different conditions used for the proliferation assay and the IFN-γ ELISPOT, and the significance levels are as reported. All intracellular calcium measurements were carried out in the presence of 1.5 M extracellular Calcium and results shown are typical of 3 independent experiments.