IVIg induces the formation of numerous germinal centers.

<p>(A) Spleen sections of Ova-immunized mice were co-stained with PNA (Peanut agglutinin from <i>Arachis hypogaea</i>, in red) and the rat monoclonal GL7 antibody (green) to verify specificity of the staining. Magnification: x 20. (B-C) Mice were immunized with Ova as described previously and 50 mg of IVIg or bevacizumab were co-injected as indicated (B) or IVIg was injected alone or in combination to adjuvant AddaVax^® (C). Spleen and draining lymph nodes were stained for germinal centers with GL7 antibody (green). Graphs show the number of germinal centers in each group of treatment. Each picture is generated from one representative animal. n = 6 animals from 2 independent experiments for (B) and n = 3 animals for (C). Magnification: x 0.25.</p

IVIg increases IgG-Immunoglobulin secreting cells in lymph nodes and raises a specific antibody response in mice.

<p>(A-C) Mice were immunized with Ova and 50 mg of IVIg were co-injected. Cells were processed from lymphoid organs and plated for ELISPOT assay to assess (A) Proportion of Ova-specific IgG antibody secreting cells (ASC) relative to total IgG immunoglobulin-secreting cells (ISC) (B) Total IgG ISC or (C) Total IgM ISC. Data are derived from one experiment (n = 3). Statistical significance was tested using two-way ANOVA (Sidak’s test for (A) and Dunnett’s test for (B-C) where each group was compared to the control group ‘Adj + Ova’). (D) Mice were immunized with Ova and increasing doses of IVIg were co-injected as indicated. IVIg-specific mouse IgG or total mouse IgG were measured by ELISA. Values as expressed as relative units to standard. Data were pooled from 2 independent experiments (n = 6). Statistical significance was tested using one-way ANOVA (Dunnett’s test) where each group was compared to the control group ‘Adj + Ova’. (E) Addition of IVIg in vitro in mouse serum inhibits the detection of IVIg-specific mouse IgG by ELISA but not the detection of total mouse IgG. Blocking buffer, IVIg or bevacizumab were added at 10, 20 or 50 mg/mL in serum from mice treated 3 times weekly with 1mg of IVIg (n = 3). Statistical significance was tested using two-way ANOVA (Dunnett’s test) where each group was compared to the control group ‘Buffer’. (F) ELISA measuring mouse IgG anti-IVIg-Fc and (Fab)’₂ fragments. Data pool 2 independent experiments (n = 6). Bars represent mean ± SD. Star maker significant difference *: p < 0.05; **p<0.01; ***: p<0.001; ****: p<0.0001. n.d: not detected.</p

Bevacizumab, a humanized monoclonal IgG₁κ antibody, does not reproduce IVIg effects.

<p>Mice were immunized with Ova as described previously using either adjuvant AddaVax^® (MF59) or adjuvant LT(R192G/L211A). IVIg or bevacizumab were co-injected as indicated. (A) Anti-Ova IgGs in mouse serum were measured by ELISA. Values are expressed as relative units to standard. For experiment using adjuvant AddaVax^® (MF59), results pool data from 4 independent experiments (n = 12) and for adjuvant LT(R192G/L211A) results are from one experiment (n = 3). Bars represent mean ± SD. Statistical significance was tested using one-way ANOVA (Dunnett’s test) where each group was compared to the control group ‘Adj + Ova’. (B) Draining lymph nodes and spleen were harvested, weighed and flow cytometry was performed on isolated cells (adjuvant AddaVax^® was used). Results pool data from 2 independent experiments (n = 6), except for CD69 measured on B cells for which data are derived from one experiment (n = 3). Bars represent mean ± SD. Statistical significance was tested using one-way ANOVA (Dunnett’s test) where each group was compared to the control group ‘Adj + Ova’. (C) Flow cytometry histograms are shown for one representative animal. Star maker significant difference *: p< 0.05; **p<0.01; ***: p<0.001; ****: p<0.0001. ns: not significant. n.d: not detected. MFI: Median of fluorescence intensity. LN: lymph nodes.</p

IVIg does not decrease the antibody response against a thymus-independent antigen but recruits mature B cells to draining lymph nodes.

<p>Mice were immunized with NP-OVA or NP-Ficoll together with adjuvant AddaVax^®, with or without IVIg as indicated. (A) ELISA for NP-specific mouse IgG (serum diluted 1:500) and for NP-specific mouse IgM (serum diluted 1:50). Data are derived from one experiment (n = 6). Statistical significance was tested using one-way ANOVA (Tukey’s test) where each group was compared with every other. (B) IVIg does not affect the B-cell compartment in the spleen but reduces the proportion of mature re-circulating B cells in the bone-marrow and promotes pre-B cell formation. Gating strategy is shown for the different B-cell compartments in the bone-marrow. Statistical significance was tested using two-way ANOVA (Tukey’s test) where each group was compared with every other. Data are from one experiment (n = 6). Bars represent mean ± SD. Star maker significant difference *: p < 0.05; **p<0.01; ***: p<0.001; ****: p<0.0001. ns: not significant.</p

Massive immune response against IVIg interferes with response against other antigens in mice: A new mode of action?

<div><p>Administration of high dose intravenous immunoglobulin (IVIg) is widely used in the clinic to treat autoimmune and severe inflammatory diseases. However, its mechanisms of action remain poorly understood. We assessed the impact of IVIg on immune cell populations using an <i>in vivo</i> ovalbumin (Ova)-immunization mouse model. High dose IVIg significantly reduced the Ova-specific antibody response. Intriguingly, the results obtained indicate an immediate and massive immune reaction against IVIg, as shown by the activation and expansion of B cells and CD4+ T cells in the spleen and draining lymph nodes and the production of IVIg-specific antibodies. We propose that IVIg competes at the T-cell level with the response against Ova to explain the immunomodulatory properties of IVIg. Two monoclonal antibodies did not succeeded in reproducing the effects of IVIg. This suggests that in addition to the mouse response against human constant domains, the enormous sequence diversity of IVIg may significantly contribute to this massive immune response against IVIg. While correlation of these findings to IVIg-treated patients remains to be explored, our data demonstrate for the first time that IVIg re-directs the immune response towards IVIg and away from a specific antigen response.</p></div

A proposed mechanism of action of IVIg based on T-cell competition effects occurring at several stages of the adaptive immune response.

<p>(A) Classical immune response and germinal center formation after Ova immunization. T cells primed by Ova-derived epitopes presented on APCs differentiate into effector T helper cells (T_H) and migrate at the T cell–B cell border of the lymph node. Ova-activated B cells take up and present antigenic peptides to T_H. After receiving co-stimulatory signals they initiate the formation of a germinal center (GC). After a cycle of proliferation and somatic hypermutations in the dark zone, B cells moves to the light zone to meet with their cognate antigen exposed on follicular dendritic cells (FDCs). If the affinity of the mutated B cell receptor (BCR) is very low, the B cell will not receive survival signals and will undergo apoptosis. The surviving B cells need to compete for help from T follicular helper cells (T_FH), thus favoring B cells with high affinity BCRs. B cells can then either re-enter the dark zone to further mature the BCR affinity, or exit the GC as plasma cells or memory B cells. Blue lines depict IVIg potential competition mechanisms. (B) T-cell competition for DCs access. In addition to Ova-peptides, DCs present a multitude of diverse IVIg-derived epitopes. This favors the competition between the many different IVIg-specific T cell clones with the Ova-T cell clones to receive priming from DCs. (C) Competition for accessing T cell-B cell zone border. The few Ova-T cell clones that may have been primed by DCs will be outnumbered by the numerous IVIg-T_H clones accessing the T cell-B cell zone border. This reduces the likelihood of OVA-T cell clones engaging with their cognate Ova-B cells. Likewise IVIg-B cells will spatially compete with Ova-specific B cells for accessing their cognate T_H cells. This results in apoptosis of Ova-activated B cells. (D) T_FH competition for centrocytes priming in GC. Only few Ova-T cell clones will be able to be primed by DCs, deliver helper signals to their cognate Ova-activated B cells and induce the formation of a GC. However, the help delivered by T_FH to centrocytes in GC is very limited. As the numerous IVIg- T_FH will compete with the few Ova- T_FH for access to centrocytes, the latter will be more likely to die by apoptosis.</p

IVIg inhibits anti-OVA IgG response <i>in vivo</i> in a dose-dependent manner but increases the weight of spleen and draining lymph nodes.

<p>(A) Mouse immunization protocol. C57Bl/6NCrl mice were injected 3 times weekly subcutaneously in the neck with 50 μg Ovalbumin (Ova) and adjuvant AddaVax^® (MF59), and terminated 24h after last injection. IVIg doses ranging from 1 mg to 50 mg were co-injected as indicated. (B) IgG mouse response against Ova measured by ELISA performed on mouse serum. Values are expressed as relative units to standard. Results pool data from 2 independent experiments (n = 6). Bars represent mean ± SD. n.d: not detected. Statistical significance was tested using one-way ANOVA (Dunnett’s test) where each group was compared to the control group ‘Adj + Ova’. (C) Pictures of spleen and draining lymph nodes (LN) from one representative animal in each group of treatment. (D) Weight of spleen and draining LN with number of B cells and CD4+ T cells as measured by flow cytometry. For weight, values are expressed as ratio to the ‘Adj + Ova’ treated group. For cell numbers, values are expressed as absolute cell numbers. Dead cells were excluded using a viability fluorescent dye. B cells were gated on CD19+ cells and T cells on CD4+ CD8- cells. Bars represent mean ± SD. Data pool 4 independent experiments (n = 12). Statistical significance was tested using one-way ANOVA (Dunnett’s test) where each group was compared to the control group ‘Adj + Ova’. Star maker significant difference *: p<0.05; **p<0 0.01; ***: p<0.001; ****: p<0.0001. n.d: not detected.</p

IVIg activates B and T cells in a dose-dependent manner and only when co-injected with adjuvant.

<p>(A) Mice were immunized with Ova as described previously and IVIg doses ranging from 1 mg to 50 mg were co-injected. Expression of activation markers and co-stimulatory molecules on B and T cells was measured by flow cytometry. Results pool data from 2 independent experiments (n = 6). Values are expressed as ratio to the ‘Adj + Ova’ treated group. Bars represent mean ± SD. Statistical significance was tested using one-way ANOVA (Dunnett’s test) where each group was compared to the control group ‘Adj + Ova’. (B) Presence of adjuvant is required together with IVIg to mediate increase of weight in lymphoid organs and B cells activation. Mice were immunized with Ova and 50 mg of IVIg was injected alone or in combination to Ova and/or adjuvant AddaVax^® (MF59), as indicated. Results are pooled data from 2 independent experiments (n = 6). Values are expressed as ratio to the ‘Adj + Ova’ treated group. Bars represent mean ± SD. Statistical significance was tested using one-way ANOVA (Tukey’s test) where each group was compared with every other. Star maker significance *: p< 0.05; **p<0.01; ***: p<0.001; ****: p<0.0001. MFI: Median of fluorescence intensity. LN: lymph nodes.</p

Correlation of HLA-DR associated peptides and peptide clusters measured by MAPPs to the amount of protein present in subvisible particles.

<p>Linear regression analyses of the increase of the HLA-DR associated peptides and clusters as functions of the calculated amount of protein present in the subvisible particles. Left up: HLA-DR associated peptides of mAb1 vs protein amount in subvisible particles (r² = 0.994), left down: HLA-DR associated peptide clusters of mAb1 vs protein amount in subvisible particles (r² = 0.993), right up: HLA-DR associated peptides of mAb2 vs protein amount in subvisible particles (r² = 0.86), right down: HLA-DR associated peptide clusters of mAb2 vs protein amount in subvisible particles (r² = 0.943). HS: aggregates generated by heat and shake stress; FT: aggregates generated by freeze and thaw stress, S: aggregates generated by shear stress mAb1: monoclonal antibody 1, mAb2: monoclonal antibody 2, 1: stress level 1, 2: stress level 2. For further details, please, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086322#s4" target="_blank">Materials and Methods</a>.</p

Illustration of MAPPs assay procedure and data output example.

<p>A: Illustration of MAPPs assay procedure. Monocytes are isolated from human buffy coats and differentiated to immature DCs in the presence of cytokines. Immature DCs are loaded with the model mAb and induced to maturation with lipopolysaccharide. After 24 hours, mature DCs are frozen. After lysis of mature DCs, HLA-DR:peptide complexes are isolated via immunoprecipitation using anti-HLA-DR coated beads. After several wash steps, peptides are eluted from HLA-DR complexes by pH shift and analysed by nano LC-MS with subsequent sequence identification via SEQUEST database search. B: MAPPs assay data output example. HLA-DR associated peptides can originate from different sequence regions of a protein and can occur in multiple length variants. Peptides in a sample with unique sequence are termed “different peptides”, highlighted with blue box. Nested sets of peptide length variants occurring within a sequence region sharing the same HLA-DR binding core are termed “clusters”, highlighted with green boxes. The number of different peptides per amino acid position can be summarized in a heatmap in which the cell colors are reflecting the number of different peptides, highlighted with red box. Different donors can differ in the pattern of presented peptides depending on binding propensities of their 2 HLA-DR alleles.</p