Background: Murine studies showed immune cell derived C3a/C5a are costimulatory intermediaries required for T cell activation. We investigated how these concepts apply to human alloreactive T cells.

Methods: Human monocyte derived DCs and naÏve CD45RA+CD45RO-CD4+ T cells were isolated by magnetic beads/sorting. Experimental strategies included RT-PCR, flow cytometry/CFSE dilution, ELISA, and siRNA transfection. Human PBMC were transferred into NOD/scid/IL-2RΓ−/− mice to induce graft vs. host disease (GVHD).

Results: C3aR/C5aR were detected on naÏve human CD4+ T cells and were upregulated upon stimulation with anti-CD3/CD28, but not either alone. When we stimulated naÏve CD4+ T cells with anti-CD3±C3a or C5a we observed a 3-8-fold increase in proliferation (p<0.05) along with increased intracellular pAKT, the latter biochemically linking C3aR/C5aR to T cell signaling pathways. During MLRs with allogeneic DCs, C3a/C5a were released in culture supernatants, indicating alloreactions cause complement production/activation. DCs express C3aR/C5aR and produce mRNA for C3, factor B (fB), and C5. LPS increased C3/fB mRNA ∼150-fold (p<0.05) and augmented C3a/C5a in culture supernatants. No C3, fB or C5 mRNA was detected in resting or anti-CD3/CD28 stimulated T cells. C3aR- and/or C5aR-blockade reduced alloreactive T cell proliferation in MLRs (C3aR block: -28%, C5aR block: -60%; p<0.05). Increasing local C3a/C5a by downregulating (siRNA) DC expression of decay accelerating factor (DAF), a surface protein that restrains complement activation, increased C3a/C5a (∼1.5-fold, p<0.05) and enhanced T cell proliferation (26%, p<0.05). The augmented responses were abrogated by C3aR- and/or C5aR blockade (p<0.05). siRNA-induced C3 silencing in DC reduced CD4+ T cell proliferation (-38% p<0.05). Recombinant C3a/C5a bypassed the need for costimulation, augmenting CD4+ T cell proliferation in MLRs despite CTLA4Ig. To study in vivo effects we administered a C5aR antagonist (C5aRA) as a preventative therapy for human anti-mouse GVHD in NOD/scid/IL-2RΓ−/− mice. We observed prolonged survival (>10d) in C5aRA-treated animals vs. vehicle controls (n=12 per group, p<0.05).

Conclusions: Our findings indicate for the first time that immune cell derived C3a/C5a mediate human alloreactive T cell responses, providing new mechanistic insight, and supporting the need for testing whether C3aR/C5aR can be exploited as targets for treating human GVHD or allograft rejection.

« Back to 2013 American Transplant Congress