Characterization of human invariant natural killer T subsets in health and disease using a novel invariant natural killer T cell‐clonotypic monoclonal antibody, 6B11

Abstract

Summary Identification of human CD1d‐restricted T‐cell receptor (TCR)‐invariant natural killer T (iNKT) cells has been dependent on utilizing combinations of monoclonal antibodies or CD1d tetramers, which do not allow for the most specific analysis of this T‐cell subpopulation. A novel monoclonal antibody (clone 6B11), specific for the invariant CDR3 loop of human canonical Vα24Jα18 TCR α chain, was developed and used to specifically characterize iNKT cells. In healthy individuals studied for up to 1 year, a wide but stable frequency of circulating iNKT cells (range: 0·01–0·92%) was observed, with no differences in frequency by gender. Four stable iNKT cell subsets were characterized in peripheral blood based on the expression of CD4 and CD8, with CD8+ iNKT cells being a phenotypic and functionally different subset from CD4+ and double negative iNKT cells; in particular, LAG‐3 was preferentially expressed on CD8+ iNKT cells. In addition, a strong negative linear correlation between the frequency of total iNKT cells and percentage of the CD4+ subset was observed. In terms of their potential association with disease, patients at risk for type 1 diabetes had significantly expanded frequencies of double negative iNKT cells when compared to matched controls and first‐degree relatives. Moreover, peripheral blood CD4+ iNKT cells were the highest producers of interleukin‐4, while the production of interferon‐γ and tumour necrosis factor‐α was similar amongst all iNKT cell subsets. These differences in iNKT cell subsets suggest that in humans the relative ratio of iNKT cell subsets may influence susceptibility vs. resistance to immune‐mediated diseases.

Application of user-guided automated cytometric data analysis to large-scale immunoprofiling of invariant natural killer T cells

“…However, the proportion of CD4 + iNKT cells was not correlated with sex (P = 0.12) or age (P = 0.75). Some of these trends had been observed in previous data sets (24). With a larger sample size, we confirmed the correlations with statistical significance.…”

“…Functional characterization of iNKT cells requires comprehensive assessment of surface expression of homing receptors, lectins, and cell adhesion molecules, as well as cytokine production. Immunoprofiling studies have yet to assay such a comprehensive set of markers in primary iNKT cells in a sufficiently large cohort (18,(20)(21)(22)(23)(24). Here, we profiled iNKT cells in 110 subjects with 19 surface and intracellular markers.…”

Selective activation, expansion, and monitoring of human iNKT cells with a monoclonal antibody specific for the TCR α‐chain CDR3 loop

“…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 cells for experimental, diagnostic, and therapeutic studies of human and non-human primate iNKT cells. The availability of multiple reagents active in various assays to identify and for manipulating iNKT cells will enable understanding their role in different patient population subsets (e.g.…”

Testing the NKT cell hypothesis in lenalidomide-treated myelodysplastic syndrome patients

“…It is now widely accepted that the optimal technique for identifying the primary (that is, type 1, invariant) NKT cells is by positive flow cytometric examination with either aGC-loaded CD1d tetramer or the 6B11 mAb, which provide similar specificity. 1,33,46 The three earlier studies linking MDS to NKT cell deficiency used less stringent approaches, including a definition of NKT cells as Va24 þ Vb11 þ CD4ÀCD8À 11 (excluding legitimate CD4 þ NKT cells, which comprised B40% of NKT cells in our study), or Va24 þ CD161 þ cells 12 (excluding legitimate CD161À NKT cells, while including Va24 þ Vb11ÀCD161 þ non-NKT cells). The third study identified NKT cells as Va24 þ Vb11 þ , 10 which usually provides a reasonably accurate approximation of NKT cell frequency, 46,47 despite not necessarily identifying all NKT cells.…”

“…1,33,46 The three earlier studies linking MDS to NKT cell deficiency used less stringent approaches, including a definition of NKT cells as Va24 þ Vb11 þ CD4ÀCD8À 11 (excluding legitimate CD4 þ NKT cells, which comprised B40% of NKT cells in our study), or Va24 þ CD161 þ cells 12 (excluding legitimate CD161À NKT cells, while including Va24 þ Vb11ÀCD161 þ non-NKT cells). The third study identified NKT cells as Va24 þ Vb11 þ , 10 which usually provides a reasonably accurate approximation of NKT cell frequency, 46,47 despite not necessarily identifying all NKT cells. 48 Therefore, we suggest a more likely explanation for the discrepancy is that the earlier study pooled results from treated (not with lenalidomide) and untreated patients, whereas we separately assessed the NKT cell frequency of patients before and after treatment with lenalidomide.…”