The differentiation of naïve conventional T cells to distinct effector populations is central to the development of effective immune response.  Current vaccine strategies include targeting unconventional T cells, such as invariant NKT cells, to influence this antigen specific conventional T cell targeting The aim of our study was to evaluate whether fluorine modification of glycolipid ligands represented a novel approach to increase the binding affinity to human CD1d with functional consequences upon both direct iNKT effector responses and indirect adjuvant effects upon the priming of naive T cells.

We developed an in vitro assay to study CD4 naïve T cell commitment to effector cell differentiation with the purpose of investigating the influence of different adjuvants. We analysed compounds known to have distinctive Th1/Th2 biasing influences and 3 novel compounds that demonstrated increased binding affinity to human CD1d and iNKT activation.

Competitive binding studies demonstrated that two of our modified compounds demonstrated a four-fold improved affinity for CD1d compared to α-GalCer. Our results suggested that fluorine modification of iNKT ligands enhanced CD1d affinity and improved direct and indirect iNKT effector responses. In particular, iNKT cytokine diversity was greater for all modified compounds with IL-13 and IL-17 being significantly increased compared to α-GalCer. One of our ligands with a CF₂ modification at the C4’ position of the sphingosine chain displayed a distinctive multi-functional CD4 response with significant Th1 (IFN-γ), Th2 (IL-13), Th17 (IL-17) and iTreg (IL-10) responses.  The novel development of CF₂ modified CD1d ligands identifies iNKT as important targets for the design of new vaccine adjuvants that target distinctive effector T cell responses. In vitro correlates of novel adjuvants may provide a useful adjunct to vaccine design strategies for distinct pathogen targets requiring bespoke effector responses.