Anti-CD1d Monoclonal antibody, clone 62.2 [APC]

The human invariant NK (iNK) TCR is largely composed of the invariant TCR V alpha 24-J alpha 18 chain and semivariant TCR V beta 11 chains with variable CDR3 beta sequences. The direct role of CDR3 beta in Ag recognition has been studied extensively. Although it was noted that CDR3 beta can interact with CDR3 alpha, how this interaction might indirectly influence Ag recognition is not fully elucidated. We observed that the third position of V beta 11 CDR3 can encode an Arg or Ser residue as a result of somatic rearrangement. Clonotypic analysis of the two iNK TCR types with a single amino acid substitution revealed that the staining intensity by anti-V alpha 24 Abs depends on whether Ser or Arg is encoded. When stained with an anti-V alpha 24-J alpha 18 Ab, human primary invariant NKT cells could be divided into V alpha 24 low-and high-intensity subsets, and Arg-encoding TCR V beta 11 chains were more frequently isolated from the V alpha 24 low-intensity subpopulation compared with the V alpha 24 high-intensity subpopulation. The Arg/Ser substitution also influenced Ag recognition as determined by CD1d multimer staining and CD1d-restricted functional responses. Importantly, in silico modeling validated that this Ser-to-Arg mutation could alter the structure of the CDR3 beta loop, as well as the CDR3 alpha loop. Collectively, these results indicate that the Arg/Ser encoded at the third CDR3 beta residue can effectively modulate the overall structure of, and Ag recognition by, human iNK TCRs.

Glycosphingolipids (GSLs) bearing the agalactosyl headgroup and the acyl chain terminated with a phenyl derivative were found to be more potent than agalactosyl ceramide (alpha GalCer) to stimulate both murine and human invariant natural killer T (iNKT) cells and to induce an antibody isotope switch to IgG. In this study, we replaced the galactosyl group with glucose (alpha Glc) and its fluoro-analogs and found that phenyl GSLs with alpha Glc (C34-Glc) and its fluoro-analog 6F-C34-Glc were stronger than those with alpha Gal in stimulating human iNKT cells but weaker in mice. Their activities have a strong correlation with the binding avidities of the ternary interaction between the iNKT-cell receptor (iNKTCR) and CD1d-GSL complex. It was the iNKTCR rather than CD1d that dictated the species-specific responses. C34-Glc was further used as an adjuvant for a SSEA4-crm-197 vaccine, and after immunization in mice, the vaccine was highly effective against Lewis lung carcinoma.