Background Autoimmunity in connective tissue diseases, such as systemic lupus erythematosus (SLE) is associated with elevated levels of immune complexes (ICs), complement activation and hyperactive T cell responses. ICs activate complement, however role for ICs in modulating T-cell responses has never been explored previously.
Objectives To understand the role for ICs and complement in deriving CD4+ T-cell responses i.e. generation of Th1, Th17 and Tfh, CD4+effector populations.
Methods Peripheral human naïve CD4+ T-cells were treated in vitro with ICs and sublytic C5b-9 in the presence of anti-CD3. The activated cells were cultured under various cytokine milieu required for development of CD4+ effector T-cells. Cells were phenotyped for the expression of effector T-cell markers and associated transcription factors. Flow analysis for IFN-γ, IL17A, IL-21, CXCR5, PD1, ICOS, Bcl6, and T-bet were carried out. Culture soups were analyzed for cytokines by multiplex assays. Up-regulation of FcγRIIIA and T-bet transcripts were analyzed by QT-PCR.
Results Naïve CD4 cells activated with ICs and C5b-9 costimulation demonstrated the capability to differentiate into a T effector population. When cultured post stimulation in the presence of IL-2, IL-1b and IL-23, they produced IL-17A, IL-17F and IL-22. Post 48 hrs after costimulation with IC and C5b-9, we observed a three fold increase in the expression of IL-21, CXCR5 and PD1, markers of Tfh. We also observed a CD4+FcγRIIIA+ population. This population did not express T-bet, a Th1 transcription factor. The ligation of FcγRIIIA by ICs resulted in receptor internalization in conjunction with IFN-γ production suggesting a new signaling pathway.
Conclusions For activation of T-cell primary pep-MHC signal and costimulation from CD28 is required. All regulators of CD4+ T-cells, CD28, ICOS, PD1, CTLA-4 are membrane proteins. We show a first non-membrane bound costimulator that is present at the site of tissue inflammation and tissue damage. ICs and C5b-9 successfully replaced costimulatory requirement for the development of effector Th1, Th17 and Tfh populations. We also show expression of low affinity FcγRIIIA on CD4 cells and these cells upon ligation with ICs produced IFN-γ. Naïve CD4+ cells in perivascular spaces of tissues with immune deposits will thus allow localized development of effector CD4+ T-cells such as Th1 and Th17 populations. Both of these cell types are known to particiapte in the development of autoimminty.
Disclosure of Interest A. Chauhan Shareholder of: ProGen Biologics LLC, T. Moore: None Declared