Stimulatory Effect of CMV Immunoglobulin on Innate Immunity and on the Immunogenicity of CMV Antigens

Figures

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  FIGURE 1.. Stimulatory effect of CMVIG and IVIG on PBMCs from CMV-seronegative and -seropositive donors. Freshly isolated PBMCs from healthy individuals were incubated for 19 h with the indicated stimulants, and the number of IFN-γ–producing cells was measured by IFN-γ ELISpot. Differences between groups were assessed using the 2-sided nonparametric Mann-Whitney U test. Statistically significant P values are shown in bold. A, The response of PBMCs from 15 donors (5 CMV-seronegative and 10 CMV-seropositive [donors 1–10]) was significantly increased in the presence of CMV immunoglobulin (CMVIG; 80 μL/mL or 8 U/mL) compared with the unstimulated control (Medium). B, ELISpot results from (A), depicted according to the donors’ CMV serostatus, showed a comparable stimulatory effect of CMVIG (80 μL/mL) in CMV-seronegative and -seropositive subjects. C, PBMCs from the 5 CMV-seronegative donors shown in (A) and (B) were stimulated with CMVIG (80 μL/mL), CMV antigens (1 μg/mL pp65 or CMV lysate), or opsonized CMV antigens (ops. pp65 or ops. CMV lysate, that is, 1 μg/mL antigens preincubated with 80 μL/mL CMVIG). The stimulatory effect of CMVIG was slightly reduced in the presence of CMV antigens. D,…

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  FIGURE 2.. Stimulatory activity of CMV antigens opsonized with CMVIG on PBMCs from CMV-seropositive donors. Freshly isolated PBMCs from 3 healthy CMV-seropositive individuals (donors 1–3 shown as circle, triangle, and square, respectively) were incubated for 19 h with increasing amounts of CMV antigens (A, 0.02–1 μg/mL pp65; B, 0.04–1 μg/mL CMV lysate) or of opsonized CMV antigens (ie, antigens preincubated with CMVIG; [A] ops. pp65; [B] ops. CMV lysate). Medium or CMVIG served as controls. CMVIG was adjusted to a final concentration of 80 μL/mL (in CMVIG alone and opsonized antigens conditions). The number of IFN-γ–producing cells was measured by IFN-γ ELISpot. A and B, The dose-dependent response of PBMCs to CMV antigens was slightly increased in the presence of CMVIG (ops. pp65 and ops. CMV lysate). Differences between groups (medium vs CMVIG and nonopsonized vs opsonized antigens for each antigen concentration) using the Mann-Whitney U test were not statistically significant, most likely because of the low number of measurements (n = 3). CMV, cytomegalovirus; CMVIG, CMV immunoglobulin; ELISpot, enzyme-linked immunospot; IFN, interferon; ops., opsonized; PBMC, peripheral blood…

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  FIGURE 3.. Immunomodulatory effect of CMVIG on NK cells. Freshly isolated PBMCs from 5 healthy CMV-seropositive individuals (donors 1–5) were stimulated, stained, and analyzed by flow cytometry as described in Materials and Methods. CMV pp65 antigen was used at 1 μg/mL, CMV lysate at 0.33 μg/mL, and CMVIG at 80 μL/mL. PMA/iono served as positive control. The number of CD56+CD3– NK cells expressing the cytokines TNF (A), IFN-γ (B), IL-2 (C), or IL-4 (D) was quantified and expressed as percentage of gated NK cells. Two-group comparisons (medium vs CMVIG, nonopsonized vs opsonized antigens, and CMVIG vs opsonized antigens) were assessed using the Mann-Whitney U test. Statistically significant P values are shown in bold. The frequency of TNF- and IFN-γ–producing NK cells was significantly increased in the presence of CMVIG. CD, cluster of differentiation; CMV, cytomegalovirus; CMVIG, CMV immunoglobulin; IFN, interferon; IL, interleukin; ops., opsonized; NK, natural killer; PMA/iono, phorbol 12-myristate 13-acetate/ionomycin; PBMC, peripheral blood mononuclear cells; pp65, phosphoprotein 65; TNF, tumor necrosis factor.

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  FIGURE 4.. Immunomodulatory effect of CMVIG on monocytes. Freshly isolated PBMCs from 5 healthy CMV-seropositive individuals (donors 2–6) were stimulated, stained, and analyzed by flow cytometry as described in Materials and Methods. CMV pp65 antigen was used at 1 μg/mL, CMV lysate at 0.33 μg/mL, and CMVIG at 80 μL/mL. LPS and PMA/iono served as positive controls. The number of CD14+ cells (monocytes) expressing the cytokines IL-12 (A) and IL-10 (B) was quantified and expressed as percentage of gated monocytes. Two-group comparisons (medium vs CMVIG, nonopsonized vs opsonized antigens, and CMVIG vs opsonized antigens) were assessed using the Mann-Whitney U test. Statistically significant P values are shown in bold. The proportion of IL-12–producing monocytes was slightly but significantly increased in the presence of CMVIG. In contrast, the proportion of IL-10–producing monocytes did not increase in the presence of CMVIG or CMV antigens (nonopsonized or opsonized). CD, cluster of differentiation; CMV, cytomegalovirus; CMVIG, CMV immunoglobulin; IL, interleukin; LPS, lipopolysaccharide; ops., opsonized; PBMC, peripheral blood mononuclear cells; PMA/iono, phorbol 12-myristate…

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  FIGURE 5.. Immunomodulatory effect of CMVIG on Th cells. Freshly isolated PBMCs from 5 healthy CMV-seropositive individuals ([A–D] donors 1–5: T-cell protocol; [E] donors 2–6: APC protocol) were stimulated, stained, and analyzed by flow cytometry as described in Materials and Methods. CMV pp65 antigen was used at 1 μg/mL, CMV lysate at 0.33 μg/mL, and CMVIG at 80 μL/mL. PMA/iono served as positive control. The number of CD4+CD3+ T helper (Th) cells expressing the cytokines TNF (A), IFN-γ (B), IL-2 (C), IL-4 (D), or IL-10 (E) was quantified and expressed as percentage of gated Th cells. Two-group comparisons (medium vs CMVIG, nonopsonized vs opsonized antigens, and CMVIG vs opsonized antigens) were assessed using the Mann-Whitney U test. Statistically significant P values are shown in bold. The frequency of TNF- and IFN-γ–producing Th cells was slightly but not significantly increased in the presence of CMVIG. APC, antigen-presenting cells; CD, cluster of differentiation; CMV, cytomegalovirus; CMVIG, CMV immunoglobulin; IFN, interferon; IL, interleukin; ops., opsonized; PBMC, peripheral blood mononuclear cell; PMA/iono, phorbol 12-myristate 13-acetate/ionomycin; pp65, phosphoprotein 65; Th,…

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  FIGURE 6.. Immunomodulatory effect of CMVIG on CTL. Freshly isolated PBMCs from 5 healthy CMV-seropositive individuals (donors 1–5) were stimulated, stained, and analyzed by flow cytometry as described in Materials and Methods. CMV pp65 antigen was used at 1 μg/mL, CMV lysate at 0.33 μg/mL, and CMVIG at 80 μL/mL. PMA/iono served as positive control. The number of CD8+CD3+ CTL expressing the cytokines TNF (A), IFN-γ (B), IL-2 (C), or IL-4 (D) was quantified and expressed as percentage of gated CTL. Two-group comparisons (medium vs CMVIG, nonopsonized vs opsonized antigens, and CMVIG vs opsonized antigens) were assessed using the Mann-Whitney U test. Statistically significant P values are shown in bold. The frequency of TNF- and IFN-γ–producing CTL was significantly increased in the presence of CMVIG. CD, cluster of differentiation; CMV, cytomegalovirus; CMVIG, CMV immunoglobulin; CTL, cytotoxic T lymphocyte; IFN, interferon; IL, interleukin; ops., opsonized; PBMC, peripheral blood mononuclear cell; PMA/iono, phorbol 12-myristate 13-acetate/ionomycin; pp65, phosphoprotein 65; TNF, tumor necrosis factor.

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  FIGURE 7.. Sensitivity of CMVIG-treated PBMCs to the immunosuppressive agent cyclosporine A. PBMCs freshly isolated from 3 healthy CMV-seropositive individuals ([A and D] donor 2; [B and E] donor 3; [C and F] donor 7) were incubated for 19 h with the indicated stimulants (8.8 μL/mL CMVIG, 0.11 μg/mL CMV lysate, opsonized CMV lysate, or medium as control) in the presence of increasing amounts of cyclosporine A. The number of IFN-γ–producing cells was measured by IFN-γ ELISpot. Geometric means of SFC/2 × 105 PBMCs (±SD) are shown. To better visualize the stimulating effect of CMVIG alone, the results of medium and CMVIG conditions are shown separately at a different scale (A–C), in addition to all conditions (D–F). The beneficial anti-CMV effects mediated by CMVIG were also observed in PBMCs treated with clinically relevant concentrations of cyclosporine A. CMV, cytomegalovirus; CMVIG, CMV immunoglobulin; ELISpot, enzyme-linked immunospot; IFN, interferon; ops., opsonized; PBMC, peripheral blood mononuclear cell; SFC, spot-forming cell.