Innate-like control of human iNKT cell autoreactivity via the hypervariable CDR3beta loop

Abstract

Invariant Natural Killer T cells (iNKT) are a versatile lymphocyte subset with important roles in both host defense and immunological tolerance. They express a highly conserved TCR which mediates recognition of the non-polymorphic, lipid-binding molecule CD1d. The structure of human iNKT TCRs is unique in that only one of the six complementarity determining region (CDR) loops, CDR3beta, is hypervariable. The role of this loop for iNKT biology has been controversial, and it is unresolved whether it contributes to iNKT TCR:CD1d binding or antigen selectivity. On the one hand, the CDR3beta loop is dispensable for iNKT TCR binding to CD1d molecules presenting the xenobiotic alpha-galactosylceramide ligand KRN7000, which elicits a strong functional response from mouse and human iNKT cells. However, a role for CDR3beta in the recognition of CD1d molecules presenting less potent ligands, such as self-lipids, is suggested by the clonal distribution of iNKT autoreactivity. We demonstrate that the human iNKT repertoire comprises subsets of greatly differing TCR affinity to CD1d, and that these differences relate to their autoreactive functions. These functionally different iNKT subsets segregate in their ability to bind CD1d-tetramers loaded with the partial agonist alpha-linked glycolipid antigen OCH and structurally different endogenous beta-glycosylceramides. Using surface plasmon resonance with recombinant iNKT TCRs and different ligand-CD1d complexes, we demonstrate that the CDR3beta sequence strongly impacts on the iNKT TCR affinity to CD1d, independent of the loaded CD1d ligand. Collectively our data reveal a crucial role for CDR3beta for the function of human iNKT cells by tuning the overall affinity of the iNKT TCR to CD1d. This mechanism is relatively independent of the bound CD1d ligand and thus forms the basis of an inherent, CDR3beta dependent functional hierarchy of human iNKT cells.

Figure 1. Distinct iNKT cell subpopulations revealed by OCH-CD1d tetramer staining.

OCH- and K7-CD1d tetramer stainings of (A) a representative K7-stimulated human iNKT line after 14 d in vitro culture and (B) a healthy human volunteer's PBMC ex vivo are shown. While K7-CD1d tetramer staining identifies a single homogeneous population of iNKT cells (upper row), OCH-CD1d tetramer staining reveals the presence of different distinct iNKT populations within these samples (lower row).

Figure 2. Clonal variation in OCH-CD1d tetramer binding by human iNKT cells is not related to TCR expression levels.

Flow cytometric analysis of one representative CD4+ human Vα24+/Vβ11+ iNKT line (A) and three representative CD4+ human Vα24+/Vβ11+ iNKT clones from different donors (B) demonstrates clonal variation in binding to OCH-CD1d (upper row), but not K7-CD1d (lower row) tetramers. (C) K7- and OCH-CD1d tetramer staining in pure human iNKT lines (n = 68) and clones (n = 256) was related to expression levels of iNKT TCR Vα24 and Vβ11. The intensity (MFI) of K7- but not OCH-CD1d tetramer staining was strongly associated with Vα24 and Vβ11 expression, as determined by Pearson correlation analysis, but not with CD4+ (blue markers) or CD4−CD8− double negative (red markers) phenotype.

Figure 3. Differential binding of OCH^HIGH and OCH^LOW iNKT cells to βGC-CD1d tetramers.

Ex vivo sorted human Vα24+/Vβ11+ iNKT clones were stained with different, α- or β-glycosylceramide loaded CD1d-tetramers. (A) A representative pair of CD4+ OCH^HIGH and OCH^LOW iNKT clones from one donor is shown. βGC-CD1d tetramers only stained OCH^HIGH but not OCH^LOW iNKT clones above background (as determined by PE-streptavidin binding). TCR Vα24 and Vβ11 surface expression levels for the two clones shown were very similar (for PE-conjugated anti-Vα24, MFI 2673 (OCH^HIGH) and 2710 (OCH^LOW); for FITC-conjugated anti-Vβ11, MFI 106 (OCH^HIGH) and 97 (OCH^LOW)). (B) βGC-CD1d tetramer staining intensity (MFI) of a panel of OCH-LOW (red markers), OCH-INT (green markers), and OCH-HIGH (blue markers) iNKT clones showed almost linear correlation with OCH-CD1d tetramer staining, but no correlation with either Vα24 or Vβ11 surface expression.

Figure 4. The CDR3β loop strongly impacts on human iNKT TCR affinity to CD1d, independent of the CD1d-bound ligand.

(A) Binding of two recombinant human iNKT TCRs, one OCH^HIGH (4C1369) and one OCH^LOW (4C12), to K7-, OCH-, βGC-, and LacCer-CD1d at equilibrium is shown (see also panel C and Table 2). (B) The affinity of the seven recombinant iNKT TCRs to OCH-CD1d, as determined by SPR, was linearly related to the staining intensity (MFI) of the original iNKT clone with OCH-CD1d tetramers. (C) The seven recombinant human iNKT TCRs followed a strict hierarchy of binding to ligand-CD1d complex, which was not affected by the specific CD1d-bound ligand. These iNKT TCRs differed only with regard to their CDR3beta sequence (Table 1).

Figure 5. Differential autoreactive functional responses by human OCH^HIGH and OCH^LOW iNKT clones.

Matched pairs of human OCH^HIGH (red columns and markers) and OCH^LOW (blue columns and markers) iNKT clones were compared for their ability to proliferate, secrete cytokines, and exhibit cytotoxicity in response to lipid-pulsed or endogenous lipid presenting CD1d-positive antigen presenting cells. (A) Proliferation of three representative pairs of OCH^HIGH and OCH^LOW iNKT clones from different healthy donors in response to K7-, OCH-, or vehicle-pulsed human CD1d-expressing T2 cells (T2-CD1d) or to K7-pulsed CD1d negative T2 cells (T2-) is shown. OCH^HIGH clones consistently displayed greater proliferation than OCH^LOW clones in response to OCH or vehicle pulsed T2-CD1d. cpm, counts per minute. Mean values ± s.e.m. are shown. (B) Cytokine secretion profiles of a representative pair of matched OCH^HIGH and OCH^LOW iNKT clones in response to the strong agonist ligand K7 and the partial agonist ligand OCH, presented by T2-CD1d, are shown. OCH^HIGH iNKT clones exhibited much stronger cytokine secretion than OCH^LOW iNKT cells in response to OCH-pulsed T2-CD1d, while cytokine secretion was similar for both in response to K7-pulsed T2-CD1d. (C) Autoreactive cytokine release in response to T2-CD1d in the absence of added exogenous ligands is shown for four matched pairs of OCH^HIGH and OCH^LOW iNKT clones. OCH^HIGH but not OCH^LOW iNKT clones consistently exhibited substantial autoreactive cytokine secretion. (D) Specific lysis of K7- (filled markers) and OCH- (unfilled markers) pulsed T2-CD1d targets is shown for three matched pairs of OCH^HIGH and OCH^LOW iNKT clones from different donors.

Figure 6. Differential binding of OCH^HIGH and OCH^LOW iNKT clone derived TCR tetramers to endogenous lipid presenting CD1d molecules.

PE-conjugated recombinant iNKT TCR tetramers derived from OCH^HIGH (4C1369; red lines) and OCH^LOW (4C12; blue lines) iNKT clones, at increasing concentrations, were used to stain T2-CD1d lymphoblasts. Clear staining of vehicle-pulsed T2-CD1d (unfilled markers) was only seen with the OCH^HIGH TCR tetramer, whereas both iNKT TCR tetramers strongly bound to K7-pulsed T2-CD1d (filled markers). The black bar shows background staining of T2- cells with iNKT TCR tetramers.