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

Abstract

A significant fraction of CD1d-restricted T cells express an invariant T cell receptor (TCR) a-chain. These highly conserved invariant NKT (iNKT) populations are important regulators of a wide spectrum of immune responses. The ability to directly identify and manipulate iNKT cells is essential to understanding their function and to exploit their therapeutic potential. To this end, we sought monoclonal and polyclonal antibodies specific for iNKT cells by immunizing CD1d KO mice, which lack iNKT cells, with a cyclic peptide modeled after the TCRa CDR3 loop. One mAb (6B11) was specific for cloned and primary human but not rodent iNKT cells and the human invariant TCRa, as shown by transfection and reactivity with human invariant TCRa transgenic T cells ex vivo and in situ. 6B11 was utilized to identify, purify, and expand iNKT cells from an otherwise minor component of human peripheral blood lymphocytes and to specifically identify human iNKT cells in tissue. Thus, we report a novel and general strategy for the generation of mAb specific for the CDR3 loop encoded by the TCR of interest. Specifically, an anti-Va24Ja18 CDR3 loop clonotypic TCR mAb is available for the enumeration and therapeutic manipulation of human and non-human primate iNKT populations.Key words: Anti-TCR Á Clonotypic Á Cyclic peptide Á IL-4 Á Invariant NKT Introduction Natural killer T (NKT) cells expressing surface markers found on NK cells (e.g. CD161, CD56, CD94 and/or KIR) comprise up to 20% of human peripheral blood lymphocytes (PBL) [1], whilst only *0.05% of PBL are CD1d restricted [2][3][4]. A major proportion of such PBL CD1d-reactive T cells are invariant NKT (iNKT) cells in that they express an invariant TCR a-chain, Va24Ja18 in humans and Va24Ja18 in mice [3,5,6]. Both human and murine iNKT cells can be activated in a CD1d-dependent fashion by the synthetic glycolipid a-galactosylceramide (a-GalCer) and self-or pathogen-derived glycolipid molecules [7][8][9]. Activation of iNKT cells leads to rapid production of large amounts of numerous cytokines and consequent stimulation of Mark A. Exley et al. Eur. J. Immunol. 2008. 38: 1756-1766 [3,[10][11][12][13][14], perhaps most significantly by regulating dendritic cell (DC) function [10,15]. The frequency and functional activity of iNKT cells have been shown to be important determinants for the progression of autoimmune disorders, tumors, and viral infections [10,[16][17][18]. In humans, a reduction in the number of circulating iNKT cells is accompanied by a striking Th1 polarization in certain autoimmune diseases such as type 1 diabetes, systemic sclerosis and rheumatoid arthritis [18][19][20][21]. Cancer patients have decreased numbers of iNKT cells in their blood and impaired in vitro proliferative potential, reversible with IL-2 [22][23][24]. As well as the reduced numbers of these cells found in many cancer patients, there is a striking reversal of cytokine polarization relative to that found in diabetes, which may reflect a reduction in iNKT anti-tumor activity during progression [...

Natural variations at position 93 of the invariant Vα24‐Jα18 α chain of human iNKT‐cell TCRs strongly impact on CD1d binding

“…Ex vivo identification of natural iNKTa variants in CD1d-restricted ''invariant'' Va24-Ja18/Vb11 TCRs Exley et al have recently published that a significant fraction, up to 30%, of human iNKT-cell clones expressed p93 variants of the ''invariant'' TCR Va24-Ja18 chain [4]. Consistent with this, we identified four different iNKTa variants with single-nucleotide variations at the middle position of codon 93 within the Va24-Ja18 junction from a small panel of 12 ex vivo FACS-sorted human TCR Va241/Vb111 T-cell clones.…”

“…In addition, we have recently shown that the only variable region of human iNKT-cell TCRs, i.e. the CDR3b loop, strongly modulates the affinity to CD1d [3].Human iNKT-cell clones with single-amino acid variations in the ''invariant'' Va24-Ja18 iNKTa chain have been previously identified [4,5], and a recent study found that 4-30% of human iNKT-cell clones carried such variations [4]. However, the effect of these variations on iNKT-cell TCR binding to CD1d has not been examined.…”

Effective functional maturation of invariant natural killer T cells is constrained by negative selection and T-cell antigen receptor affinity

“…Interestingly, a rather substantial proportion of human iNKT cell clones (4-30%), which also express a conserved Vα24-Jα18 rearrangement, has been found with variations to the canonical sequence (37,38). Moreover, these TCR-α variations strongly influenced the affinity of human iNKT TCRs for various antigenCD1d complexes, irrespective of the TCR-β chain with which they were paired (39).…”

Severe loss of invariant NKT cells exhibiting anti–HTLV-1 activity in patients with HTLV-1–associated disorders

“…14,15 A small population of T cells within this NKT cell subset expresses a highly conserved V␣24J␣18 TCR chain that associates preferentially with V␤11. [16][17][18] These T cells are referred to as invariant NKT (iNKT) cells. Very recently, a novel clonotypic monoclonal antibody 6B11 specific for the V␣24J␣18 TCR chain has been shown to selectively stain human iNKT cells.…”

“…Very recently, a novel clonotypic monoclonal antibody 6B11 specific for the V␣24J␣18 TCR chain has been shown to selectively stain human iNKT cells. 18,19 Activation of human iNKT cells requires the presentation of glycolipids, such as ␣-galactosylceramide (␣-GalCer) on the major histocompatibility complex class I-like molecule CD1d. 20 ␣-GalCer stimulation induces rapid cytokine production and potent antitumor and antipathogen responses by iNKT cells.…”