A variety of methods have been used to identify iNKT. Historically, this population was defined by Vα24/CD4-CD8-, Vα24//CD161 or perhaps most precisely by Vα24/Vβ11 staining. These approaches are less than optimal because they include a proportion of non-iNKT, or as is the case with Vα24/CD161 staining, also exclude bona fide iNKT. [5
] These earlier methods have been supplanted by staining with CD1d tetramers loaded with α-GalCer or other relevant iNKT antigens. [4
] Even when B cell non-specific binding is excluded, CD1d tetramer staining alone invariably includes non-Vα24 T cells and in some subjects the fraction of variant CD1d-restricted T cells can be upwards of 30% of cells. (, ) [28
] The biological function of such variant CD1d-restricted T cells is poorly understood. There is good evidence that variant populations are selected by, and recognizes self-antigens processed from different cellular compartments. [29
] Even co-expression of Vα24 and Vβ11 does not completely overlap with CD1d-tetramer reactivity in humans, and can be complicated by competition between CD1d tetramers and anti-TCR mAbs or their often bulky chromophores (e.g. anti-Vα24-PE). [28
] Concurrent incubation with anti-Vα24 has been used to block tetramer binding to iNKT, and thus identify non-invariant CD1d-restricted populations. [28
] Previous estimates of the relative contribution of iNKT subset to the total population of CD1d-restricted T cells has been limited by the lack of a comprehensive set of self antigens, and the use of various strains of mice with iNKT-related gene deletions. [35
] However, the introduction of MHC deletions are known to induce major perturbations in lymphocyte homeostasis, and in particular, MHC class II-null mice have an expanded repertoire of variant CD1d-restricted T cells. [36
] Hence, the ability to specifically identify, manipulate, and expand iNKT will likely be important. Toward this end we developed antibodies specific for human iNKT by modeling the invariant TCR CDR3 region using a cyclic peptide as an immunogen, one of which, 6B11, fulfilled these criteria.
As the accurate detection of iNKT is the first step in their analysis, we suggest that a more precise phenotypic definition of iNKT than use of Vα24 and / or Vβ11 should be based on the invariant TCR α chain rearrangement (Vα24Jα18), as detected by 6B11. Therefore, alone or in combination with anti-Vα24, anti-Vβ11, or anti-CD3, 6B11 is an excellent criterion for such analysis of human iNKT. Functional high avidity binding of α-GalCer loaded_CD1d tetramers provides a similar but not identical identification: all clones so obtained were CD1d-restricted but a minority were Vα24-negative, unlike with 6B11, all of which were CD1d-restricted and Vα24+
. The 6B11 antibody was cloned from a large number of peptide-reactive hybridomas generated using a novel approach of immunization with cyclic peptides predicted to define particular loop structures within the target protein of interest. The specificity of 6B11 was defined by binding to the cyclic peptide used as antigen, reactivity with Jurkat cells transfected to express the iNKT TCR, reactivity to T cells derived from a mouse that expressed a human invariant AV24AJ18 TCR α-chain, and selection for and activation of iNKT clones and lines in vitro
. Although raised in CD1d KO mice, 6B11 did not react with murine iNKT (or other murine) cells (data not shown), but does react with iNKT of Rhesus macaques, non-human primates [37
]. To our knowledge, this antibody is the only anti-CDR3 idiotype-specific mAb available for a TCR of known specificity. Thus, this novel antibody can be used to specifically identify, enumerate [38
], and as we show here to localize in tissues, isolate, and expand authentic iNKT for experimental, diagnostic, and therapeutic studies of human and nonhuman primate iNKT. The availability of multiple reagents active in various assays to identify and for manipulating iNKT will enable understanding their role in different patient population subsets (e.g. patients with various cancers (47), adult and pediatric acute versus chronic asthmatics, untreated or on various treatments; 48–53) and their various tissues, as well as and intervening therapeutically. Given that several approaches to exploit iNKT have entered clinical trials [39
], development of such a selective reagent seems timely.