Background/Purpose:  Complement opsonized immune complexes (ICs) are key players of disease pathology. We showed in systemic lupus erythematosus (SLE), ICs and complement (C’) drive activation and differentiation of naïve CD4⁺ cells into the T effector population. We also established activation-induced expression of FcγRIIIA in naïve CD4⁺ population that is partly responsible for this activation. To further understand the role IC and C’ mediated activation in the development of autoantibody secreting plasma cells, we examined the development of Bcl6⁺PD1⁺CXCR5⁺ follicular helper cells (T_FH). We asked whether ICs play a role in cognate contact between T_FH cells and naïve B cells in pre germinal centers (GC) and/or in secondary GC that could lead to the formation B memory and/or plasma cells.

Methods:  Peripheral human naïve CD4⁺ T cells were stimulated in vitro using ICs and the late complement complex C5b-9. They were grown in the presence of IL-6 and IL-21. Post 72 h, cells were analyzed by flow for the expression of Bcl6, PD1, and CXCR5.  RNA expression was quantified using QT-PCR. To analyze whether ICs facilitate the contact among CD4⁺T cells, we used an autoimmune gastritis mouse model (TxA23 TCR-Tg). In this model, the T and B cells association was analyzed by flow staining for a cell specific marker in the presence of Alexa Fluor 647 labeled ICs and GC reaction by staining with anti-GL-7.

Results: Antigen-specific B cell memory progressively develops under the cognate guidance of T_FH cells, following initial priming and secondary antigenic challenge. Cognate contact of naïve B cells with T_FH initiates immunoglobulin class switching and naïve B cell differentiation into plasma cells outside the GC. Such cell contacts also induce GC reaction. Our previous work established the presence of FcγRIIIA on CD4⁺ T cells, which upon ICs engagement produced IFN-g.  It also, showed T cell activation. The naïve CD4⁺ T cell activation by ICs and C5b-9 showed three-fold increase in expression CXCR5⁺Bcl6⁺ T cell population. This was comparable to anti-CD3 and anti-CD28 stimulation. However, CD4⁺IC⁺ gated population showed 52% cells that were CXCR5⁺PD1⁺Bc6⁺, in response to ICs and C5b-9 activation compared to 20% using anti-CD3 and anti-CD28.  These results suggest that naïve CD4⁺ cells upon activation with ICs and C5b-9 express T_FH markers and receptors that bound to ICs. To further establish the role of these cells in starting the formation of GC reaction, we used TxA23 TCR-Tg mice that demonstrate formation of ectopic GCs. Cells obtained from spleen/lymph nodes of these mice with disease activity upon staining with anti-CD4, anti-CD19, and labeled ICs showed 39% of CD4⁺; 34.8 % of CD19⁺ and 6.2 % of cells that were CD4⁺CD19⁺. The CD4⁺CD19⁺ showed a very high percentage of cells (83.9%) that bound to ICs, compared to 4% CD4⁺ cells and 43% CD19⁺ B cells. The side scatter of CD4⁺CD19⁺was two fold higher suggesting a large size. This suggests the formation of T and B cell conjugates by ICs.

Conclusion: These results thus suggest an important role for ICs and complement in generation of T_FH cells and formation of secondary GCs.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/generation-of-cd4-follicular-helper-t-cells-by-complement-and-immune-complexes/