Contribution of NKT Follicular Helper Cells to T-dependent and T-independent Humoral Immunity Against Bacterial Toxins and Polysaccharides

Abstract Activation of CD1d-restricted Natural Killer T (NKT) cells with the CD1d-binding glycolipid α-galactosylceramide (α-GC), promotes humoral immunity against co-administered T-dependent and T-independent antigens. The discovery of a Bcl6hi/CXCR5+/PD-1hi, IL-21-secreting ‘follicular helper’ NKT subset (NKTfh) led to the hypothesis that NKTfh cells promote humoral immune responses to vaccine candidates including bacterial toxins and surface polysaccharides. We used Cre-Lox technology to ablate Bcl6 expression by CD4-expressing cells in mice and examine the impact on NKT-driven humoral immunity to Clostridium difficile-derived Toxin B (TcdB) and surface carbohydrates (PSI, II, and III) as well as model polysaccharides (Ficoll). In the CD4Cre;Bcl6fl/floffspring, we observed ablation of Bcl6hi/CXCR5+/PD-1hi NKT cells without depleting the parent NKT population. Adoptive transfer of NKT cells from B6 donors capable of NKTfh differentiation demonstrated a contribution to the T-dependent TcdB-specific antibody response. As expected, NP-conjugated Ficoll produced a strong NP-specific IgM and IgG3 response when administered alone, and selective class switch to IgG1 was observed when α-GC was co-administered. In NKTfh cell-ablated mice, the IgG1 response was abolished while the IgM and IgG3 responses remained intact. Experiments are underway to determine if this result will be replicated using C. difficile-derived polysaccharides. In conclusion, NKTfh differentiation driven by glycolipid adjuvants can enhance and/or broaden humoral immune responses against pathogen-derived T-dependent and T-independent antigens.