Acute B-Cell Inhibition by Soluble Antigen Arrays Is Valency-Dependent and Predicts Immunomodulation in Splenocytes

Abstract

Antigen valency plays a fundamental role in directing the nature of an immune response to be stimulatory or tolerogenic. Soluble antigen arrays (SAgAs) are an antigen-specific immunotherapy that combats autoimmunity through the multivalent display of autoantigen. Although mechanistic studies have shown SAgAs to induce T- and B-cell anergy, the effect of SAgA valency has never been experimentally tested. Here, SAgAs of discrete antigen valencies were synthesized by click chemistry and evaluated for acute B-cell signaling inhibition as well as downstream immunomodulatory effects in splenocytes. Initial studies using the Raji B-cell line demonstrated SAgA valency dictated the extent of calcium flux. Lower valency constructs elicited the largest reductions in B-cell activation. In splenocytes from mice with experimental autoimmune encephalomyelitis, the same valency-dependent effects were evident in the downregulation of the costimulatory marker CD86. The reduction of calcium flux observed in Raji B-cells correlated strongly with downregulation in splenocyte CD86 expression after 72 h. Here, a thorough analysis of SAgA antigenic valency illustrates that low, but not monovalent, presentation of autoantigen was ideal for eliciting the most potent immunomodulatory effects.

Fig. 1.

Synthesis of varied valency conjugates. Antigen-only (SAgA_PLP) and antigen plus inhibitor (SAgA_PLP:LABL) conjugates were synthesized by click chemistry according to varied target conjugation efficiencies for each 16 kD HA backbone. RP-HPLC was used for characterization, and calculated peptide conjugations are reported.

Fig. 2.

Calcium flux was used as a measure of acute B-cell response by varied valency conjugates. A) Raji B-cells were loaded with Fluo-4 as a fluorescent calcium indicator and stimulated with IgM. Fluo-4 signal was increased from baseline (left) after stimulation (right). B) Acute B-cell inhibition was measured by stimulating Raji B-cells for 60 s, followed by treatment with varied valency conjugates. After treatment, mean fluorescence intensity was monitored for 180s and compared to the 60 second stimulation period. C) Reduced calcium signaling by one-signal varied valency conjugates (SAgA_PLP, blue), two-signal varied valency conjugates (SAgA_PLP:LABL, red), monovalent PLP and azide-modified HA alone (white). (n > 3/group, *p < 0.05, **p < 0.01).

Fig. 3.

Mixed splenocytes were harvested from EAE mice at peak of disease, treated with varied valency conjugates, and rechallenged with 25 μM PLP for 72h. Following the incubation, cells were fluorescently labeled and analyzed by flow cytometry, where changes in A) CD86, B) CD80, and C) CD3 were compared to healthy control splenocytes (HC). All values are expressed in terms of fold-change as compared to vehicle treated EAE splenocytes. D) CD86 changes are shown by dot plot for PBS-treated EAE spenocytes, as well as those treated with low-valency SAgA_PLP:LABL or SAgA_PLP:LABL of typical valency from previous reports. (n = 3/group, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 4.

A) EAE splenocytes treated with varied valency conjugates and 25 μM PLP rechallenge. Groups were incubated with resazurin after 72 hr to assess differences in cell metabolism. B) Likewise, healthy splenocytes were treated with varied valency conjugates and 25 μM PLP and subsequently incubated with resazurin after 72 hr. C) Supernatants were collected from conjugate-treated EAE splenocytes and analyzed for GM-CSF, IFN- γ, Il-10, Il-12, Il-17, Il-2, Il-23, Il-6, and TNF-α. (n = 3/group, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 5.

Inhibitory outcomes from treating EAE splenocytes with varied valency conjugates were assessed according to PLP valency, including A) acute inhibition of B-cells and B) CD86 expression. Pearson correlation coefficients were calculated for each comparison. C) Correlation between acute B-cell inhibition and downstream CD86+ expression was also investigated. D) The relationship between PLP valency, calcium flux reduction, and CD86 expression changes was collectively analyzed to form a correlation matrix where Pearson correlation coefficients were expressed. In Figures 5A and 5C, data points for 90% SAgA_PLP:LABL are omitted for clarity, but readouts from this group are applied for the analysis in Figures 5D.