by Éric Aubin, Réal Lemieux, and Renée Bazin Indirect inhibition of in vivo and in vitro T-cell responses by intravenous immunoglobulins due to impaired antigen presentation by Éric Aubin, Réal Lemieux, and Renée Bazin Blood Volume 115(9):1727-1734 March 4, 2010 ©2010 by American Society of Hematology

Effect of IVIg on the generation and activation of OVA-specific T cells. Effect of IVIg on the generation and activation of OVA-specific T cells. (A) Groups of 4 mice were immunized with OVA and treated or not with IVIg, as described in “OVA immunization and antigenic recall.” Spleens were recovered after 28 days, and the splenocytes (10 × 106 cells/mL for the secretion of IFN-γ and IL-4 and 5 × 106 cells/mL for IL-10) were stimulated in vitro with OVA-IC. After 3 days, the presence of OVA-specific T cells was evaluated by measuring the secretion of IFN-γ, IL-4, and IL-10 using ELISA. (B) Groups of 4 mice were immunized with OVA in the absence of IVIg. Spleens were recovered after 28 days, and the splenocytes were stimulated in vitro for 3 days with OVA-ICs, in the presence or not of 10 mg/mL IVIg. OVA-specific T-cell activation was evaluated by measuring cytokine secretion by ELISA. The results are representative of 2 independent experiments. *P < .001. Éric Aubin et al. Blood 2010;115:1727-1734 ©2010 by American Society of Hematology

Effect of IVIg on the in vivo OVA-specific T-cell proliferation. Effect of IVIg on the in vivo OVA-specific T-cell proliferation. A total of 5 × 106 CFSE-stained spleen cells from DO11.10 transgenic mice were transferred to naive BALB/c mice. Mice (n = 4) were treated or not with IVIg before stimulation with OVA-ICs. OVA-specific T-cell expansion in the spleen of recipient BALB/c mice was determined 3 days later (A) by measuring the CFSE fluorescence intensity of splenic CD4+ cells by flow cytometry and (B) by evaluating of the number of OVA-specific T cells using the KJ1-26 mAb specific for the TCR expressed by DO11.10 mice. Results are representative of 3 independent experiments. *P < .001. Éric Aubin et al. Blood 2010;115:1727-1734 ©2010 by American Society of Hematology

Effect of IVIg on the in vivo antigen-specific B-cell response. Effect of IVIg on the in vivo antigen-specific B-cell response. Anti-OVA IgG titers were measured in the serum of OVA-immunized mice treated (n = 7) or not (n = 7) with IVIg, 28 days after the first immunization. The results were compared using the Wilcoxon-Mann-Whitney 2-sample rank-sum test. The median values in the IVIg-treated and control groups were 62 424 and 119 258, respectively. The analysis confirmed that IVIg treatment resulted in significantly lower OVA-specific antibody titers compared with the control group (Mann-Whitney U = 14, n1 = n2 = 7, α = 0.05). The anti–human IgG response was also determined in a similar ELISA, using IVIg instead of OVA as capture antigen and revealed the absence of a significant anti–human IgG response in all mice treated with IVIg at the dilutions tested (titers < 1000). Éric Aubin et al. Blood 2010;115:1727-1734 ©2010 by American Society of Hematology

Effect of IVIg on the in vitro antigen-specific T-cell activation. Effect of IVIg on the in vitro antigen-specific T-cell activation. (A) OVA-specific DO-11.10 hybridoma cells were cultured for 24 hours with IFN-γ–stimulated P388D1 cells or BMDCs in the presence of absence of IVIg and OVA-IC. DO-11.10 cell activation was evaluated by measuring the IL-2 secretion using ELISA. (B) CD4+ T cells isolated from DO11.10 transgenic mice were stained with CFSE and cultured with IFN-γ–stimulated P388D1 cells or BMDCs in the presence or absence of IVIg (10 mg/mL) and OVA-ICs. Proliferation of OVA-specific CD4+ T cells was evaluated after 3 days, by flow cytometric analysis of CFSE dilution. The percentage of proliferating CD4+ T cells is shown in the upper left quadrant. Results are representative of 3 independent experiments. *P < .001; **P < .01; ***P < .05. Éric Aubin et al. Blood 2010;115:1727-1734 ©2010 by American Society of Hematology

IVIg did not inhibit T-cell responses by direct interaction with T cells. IVIg did not inhibit T-cell responses by direct interaction with T cells. (A) IFN-γ–stimulated P388D1 cells were incubated in the presence of IVIg (10 mg/mL) or the corresponding volume of RPMI 1640 medium (control), for 24 hours. IVIg (or RPMI) was then removed and DO-11.10 hybridoma cells were added with OVA-IC for an additional 24 hours. DO-11.10 cell activation was measured by IL-2 secretion. (B) IFN-γ–stimulated P388D1 cells were pulsed with OVA-ICs for 24 hours and fixed with paraformaldehyde. DO-11.10 cells were then added in the presence or not of IVIg (10 mg/mL), and IL-2 secretion was measured 24 hours later. (C) OVA-specific CD4+ T cells isolated from spleens of DO11.10 mice were cultured with OVA-IC—pulsed, P388D1-fixed cells in the presence or not of IVIG (10 mg/mL). IL-2 secretion was measured 24 hours later. (D-E) CFSE-stained spleen cells from naive BALB/c mice were incubated with or without Dynabeads Mouse CD3/CD28 T-cell expander in the presence or absence of IVIg (10 mg/mL): (D) polyclonal T-cell activation was evaluated by measuring IL-2 secretion 24 hours later, and (E) T-cell proliferation was evaluated by analysis of CFSE dilution by flow cytometry after 72 hours of culture. All results are representative of 2 independent experiments. *P < .001. Éric Aubin et al. Blood 2010;115:1727-1734 ©2010 by American Society of Hematology

Costimulatory molecules and inhibitory FcγRIIb are not involved in the inhibition of OVA-IC presentation. Costimulatory molecules and inhibitory FcγRIIb are not involved in the inhibition of OVA-IC presentation. (A) The cell-surface expression of MHC II, CD80, and CD86 was measured on BMDCs cultured for 48 hours in the presence or absence of IVIg (10 mg/mL). The MFIs are indicated in the upper right corner of the histograms. (B) BMDCs from wild-type or fcgr2b−/− BALB/c mice were used in the OVA-IC antigen presentation assay done with DO-11.10 cells in the presence (■) or absence (□) of IVIg (10 mg/mL). The results are expressed as the percentage of the amount of IL-2 secreted in the presence of IVIg compared with the control condition in the absence of IVIg. Results shown are representative of 2 independent experiments. *P < .001. Éric Aubin et al. Blood 2010;115:1727-1734 ©2010 by American Society of Hematology

Interaction between IVIg and low-affinity FcγR is required for the inhibition of OVA-IC presentation. Interaction between IVIg and low-affinity FcγR is required for the inhibition of OVA-IC presentation. (A) Biotin-OVA-IC (1.66 μg/mL) was incubated with IFN-γ-activated P388D1 cells (1 × 105 cells) for 1 hour at 4°C in the presence or not of IVIg (10 mg/mL) or 2.4G2 mAb (30 μg/mL). Binding of biotin–OVA-ICs was monitored by flow cytometry using allophycocyanin-conjugated streptavidin. The MFIs are indicated in the histograms. (B) IVIg and F(ab′)2 fragments of IVIg were used at 10 mg/mL in the OVA-IC presentation assay. IL-2 secretion by DO-11.10 cells was measured as before. Results are representative of 3 independent experiments. *P < .001. Éric Aubin et al. Blood 2010;115:1727-1734 ©2010 by American Society of Hematology