CD1d-dependent expansion of NKT follicular helper cells in vivo and in vitro is a product of cellular proliferation and differentiation

Abstract

NKT follicular helper cells (NKTfh cells) are a recently discovered functional subset of CD1d-restricted NKT cells. Given the potential for NKTfh cells to promote specific antibody responses and germinal center reactions, there is much interest in determining the conditions under which NKTfh cells proliferate and/or differentiate in vivo and in vitro. We confirm that NKTfh cells expressing the canonical semi-invariant Vα14 TCR were CXCR5(+)/ICOS(+)/PD-1(+)/Bcl6(+) and increased in number following administration of the CD1d-binding glycolipid α-galactosylceramide (α-GC) to C57Bl/6 mice. We show that the α-GC-stimulated increase in NKTfh cells was CD1d-dependent since the effect was diminished by reduced CD1d expression. In vivo and in vitro treatment with α-GC, singly or in combination with IL-2, showed that NKTfh cells increased in number to a greater extent than total NKT cells, but proliferation was near-identical in both populations. Acquisition of the NKTfh phenotype from an adoptively transferred PD-1-depleted cell population was also evident, showing that peripheral NKT cells differentiated into NKTfh cells. Therefore, the α-GC-stimulated, CD1d-dependent increase in peripheral NKTfh cells is a result of cellular proliferation and differentiation. These findings advance our understanding of the immune response following immunization with CD1d-binding glycolipids.

Coordination between T helper cells, iNKT cells, and their follicular helper subsets in the humoral immune response against Clostridium difficile toxin B

“…For intracellular staining, cells were fixed and permeabilized using the FoxP3 Staining Kit, according to the manufacturer's instructions (eBioscience). Isotype controls and fluorescence-minus-one controls were used to set backgrounds, with both approaches yielding similar results (detailed in Rampuria and Lang [21]). Unloaded CD1d tetramer lacking ligand was also used to confirm specificity of iNKT cell detection.…”

“…Recently a subset of iNKT cells, known as iNKTfh cells, was discovered [19]. Like Tfh cells, iNKTfh cells express Bcl-6, CD44, CXCR5, PD-1, ICOS, and IL-21 [19][20][21]. However, it is not well understood whether iNKTfh cells directly contribute to the B cell response to protein antigens.…”

B Cell Help by CD1d-Rectricted NKT Cells

“…Interestingly, however, iNKTFH cells expansion is neither enhanced or suppressed by exogenous IL-2 administration [65] that, by contrast, impairs TFH differentiation, suggesting some difference in the molecular cues regulating the follicular helper differentiation between the two cell subsets. Unlike the mouse iNKT cell subsets that display bona-fide TH1, TH2 and TH17 effector phenotypes, which are developmentally acquired in the thymus, iNKTFH cells are not a pre-formed thymic subset and result from the combined proliferation and differentiation in the periphery of apparently uncommitted precursors [65].…”

Diacylglycerol Kinases in T Cell Tolerance and Effector Function

“…Recently, iNKT cells have also been defined into multiple terminally differentiated effector lineages, such as IFN-γ-producing iNKT1, IL-4-producing iNKT2, and IL-17-producing iNKT17 lineage (Matsuda et al, 2006; Michel et al, 2007, 2008). In addition, IL-10-producing iNKT10, T follicular helper (Tfh)-like iNKT cells (iNKT FH ), and regulatory T cell (Treg)-like iNKT cells have also recently been described (Chang et al, 2012; Tonti et al, 2012; Sag et al, 2014; Lynch et al, 2015; Rampuria and Lang, 2015). iNKT1 and iNKT17 cells mostly reside in the CD44 + NK1.1 + and the CD44 + NK1.1 − ICOS + populations, respectively (Watarai et al, 2012; Constantinides and Bendelac, 2013; Lee et al, 2013; Wu et al, 2014b).…”