Key Residues at Third CDR3β Position Impact Structure and Antigen Recognition of Human Invariant NK TCRs

Kenji Chamoto, Tingxi Guo, Stephen W. Scally, Yuki Kagoya, Mark Ancruzowski, Chung-Hsi Wang, Muhammed A. Rahman, Kayoko Saso, Marcus O. Butler, Priscilla P. L. Chiu, Jean-Philippe Julien and Naoto Hirano
J Immunol February 1, 2017, 198 (3) 1056-1065; DOI: https://doi.org/10.4049/jimmunol.1601556

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Abstract

The human invariant NK (iNK) TCR is largely composed of the invariant TCR Vα24-Jα18 chain and semivariant TCR Vβ11 chains with variable CDR3β sequences. The direct role of CDR3β in Ag recognition has been studied extensively. Although it was noted that CDR3β can interact with CDR3α, how this interaction might indirectly influence Ag recognition is not fully elucidated. We observed that the third position of Vβ11 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-Vα24 Abs depends on whether Ser or Arg is encoded. When stained with an anti–Vα24-Jα18 Ab, human primary invariant NKT cells could be divided into Vα24 low- and high-intensity subsets, and Arg-encoding TCR Vβ11 chains were more frequently isolated from the Vα24 low-intensity subpopulation compared with the Vα24 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 CDR3β loop, as well as the CDR3α loop. Collectively, these results indicate that the Arg/Ser encoded at the third CDR3β residue can effectively modulate the overall structure of, and Ag recognition by, human iNK TCRs.

Introduction

Invariant NKT (iNKT) cells are a subset of evolutionarily conserved αβ T cells that recognize lipids presented by the MHC class I homolog CD1d. These cells are rapid responders that play immunological roles in various settings, such as autoimmunity, cancer, and infection. Recognition of the canonical glycolipid α-galactosylceramide (α-GalCer) or its analog, PBS-57, presented by CD1d is a defining feature of iNKT cells. In addition to α-GalCer and PBS-57, iNKT cells are activated by various microbial lipids, self-lipids, and synthetic glycolipids, such as OCH. Another feature of iNKT cells is their biased TCR repertoire. Human invariant NK (iNK) TCR V region genes are largely limited to the invariant TCR Vα24-Jα18 (Vα24i) and semivariant TCR Vβ11 chains. Murine iNKT cells preferentially express an invariant TCR Vα14-Jα18 and TCR Vβ8, 7, and 2 chains. Despite the limited V gene usage, the CDR3β sequences are highly heterogeneous in both species (1–7).

Two methods are widely used to detect human iNKT cells: staining with α-GalCer/PBS-57–loaded CD1d tetramer and costaining with anti-Vα24 and anti-Vβ11 mAbs. The CD1d tetramer was first described by Benlagha et al. (8) and has since been widely adopted in the iNKT biology field. Two anti-Vα24 mAbs are commercially available for detecting human iNKT cells. Clone C15 is a pan-Vα24 mAb that was first described by Padovan et al. (9) and binds to Vα24, regardless of the Jα gene usage. Clone 6B11 was developed by Exley et al. (10) to specifically detect human iNKT cells, and it recognizes the Vα24-Jα18 CDR3 loop. The 6B11 mAb was generated by immunizing CD1d−/− mice with a cyclic form of the Vα24i CDR3 peptide. Point mutation at the Vα24-Jα18 junction of an iNK TCR decreased 6B11 reactivity, thus confirming its cognate epitope (10). Furthermore, the frequency of 6B11 positivity was comparable to that of α-GalCer–loaded CD1d tetramer-positive cells when tested with PBMCs (10, 11).

Recognition of self-lipids by iNKT cells is important in their thymic development and peripheral activation. The endogenous antigenic lipids for human iNKT cells include glycolipids, phospholipids, and plasmalogens (7, 12–18). The molecular basis of self-recognition is similar to that of α-GalCer/CD1d recognition, where CDR1α, CDR3α, and CDR2β mediate critical interactions. The difference lies in the role of CDR3β, whose direct interaction with CD1d is critical when recognizing self-antigens (19–21). Because CDR3β does not directly contact the ligand, previous studies highlighted a ligand-nonselective role in its control of the overall affinity of iNK TCRs for CD1d–lipid complexes (20, 22, 23). We previously identified three CDR3β amino acid sequence motifs that are associated with greater human iNK TCR autoreactivity strength, regardless of the lipid presented by CD1d (24). Interestingly, studies of human iNK TCR crystal structures suggested an additional role for CDR3β in influencing Ag recognition indirectly via interactions with the CDR3α loop (21, 25).

In the current study, we provide novel evidence for this indirect mechanism of CDR3β in regulating iNK TCR Ag recognition. We found that staining with anti-Vα24 mAbs was altered depending on whether the Vβ11 CDR3 sequence encoded Ser or Arg at the third position, which occurs as a result of somatic rearrangement. In this article, the amino acid following the conserved cysteine is defined as the first residue of the CDR3β sequence, according to The International Immunogenetics Information System annotation. Furthermore, recognition of self-lipids and OCH differed between the Arg- and Ser-encoding iNK TCRs. Finally, molecular modeling indicated that a mutation at this particular CDR3β residue is able to influence the conformation of the CDR3α and CDR3β loops, which could account for the altered anti-Vα24 mAb staining and Ag reactivity. Together, these data highlight a role for CDR3β in regulating the structure of the invariant TCRα-chain of human iNKT cells.

Materials and Methods

Cells and reagents

PBMC and thymus samples were obtained with institutional review board approval from the University Health Network and appropriate informed consent. SupT1, Jurkat 76, K562, C1R cells, and their derivatives were cultured in RPMI 1640 supplemented with 10% FCS and gentamicin. α-GalCer was purchased from Axxora (San Diego, CA). Recombinant human IL-2 was purchased from Novartis (New York, NY).

High-throughput sequencing of the CDR3β region of primary human iNKT cells

Primary human iNKT cells were initially purified from PBMCs or thymi using anti-iNKT MicroBeads (clone 6B11; Miltenyi Biotec, Auburn, CA). Subsequently, the cells were stained with anti-Vα24 (clone C15) and anti-Vβ11 mAbs. The double-positive population was sorted with a FACSAria (BD Biosciences, Mississauga, ON, Canada). The purity of the sorted cells was consistently >95%. TCRβ sequencing was performed at Adaptive Biotech using the ImmunoSEQ platform (Seattle, WA). This method was used to capture the frequencies of individual TCRs in biologic samples with accurate reproducibility and a sensitivity of 1/100,000 T cells (26).

Flow cytometry analysis

The following mAbs recognizing the indicated Ags were used: human TCR Vα24 (clone C15) and Vβ11 (both from Beckman Coulter, Mississauga, ON, Canada); human pan TCR (clone BMA031; Thermo Fisher Scientific, Burlingame, CA); and human TCR Vα24-Jα18 (clone 6B11), human CD3, human CD1d, human CD69, human pan TCR (clone IP26), and isotype controls (BioLegend, San Diego, CA). 7-Aminoactinomycin D (BioLegend) was used for live/dead staining. Human CD1d monomers, unloaded, OCH loaded, and PBS-57 loaded, were kindly provided by the National Institutes of Health Tetramer Core Facility. Unloaded monomers were produced in HEK293 cells and, therefore, presented HEK293-derived endogenous ligands. CD1d monomers were multimerized with streptavidin-PE (Life Technologies, Grand Island, NY), according to a protocol provided by the Tetramer Core Facility. All data shown were gated on singlets and live cells. Data involving Cl6 transfectants were further gated on CD3+ cells unless otherwise specified.

cDNAs

Full-length cDNAs encoding the Vα24i, Vβ11, and CD1d genes were molecularly cloned via RT-PCR using gene-specific primers into the pMX vector (24, 27–29). Nucleotide sequencing was performed at the Centre for Applied Genomics, The Hospital for Sick Children. CDR3β sequences were defined according to ImMunoGeneTics (http://www.imgt.org/).

Generation of TCR transfectants

Using the 293GPG-based retrovirus system (30), TCRα− β− Jurkat 76 cells were transduced with a β2-microglobulin short hairpin RNA (Origene, Rockville, MD) and the Vα24i gene. The Jurkat 76.clone 6 (Cl6) expressing Vα24i and low levels of CD1d was established by the limiting dilution method. Cl.6 cells were further transduced with various clonotypic TCR Vβ11 genes encoded by the 293GPG virus (24, 31). Jurkat 76 cells transduced with the HLA-A2/TAX–restricted TCR clone A6 were used as a control (32). The CD3 expression levels for each transfectant were >90%.

Expansion of CD1d-restricted iNKT cells

Human CD3+ T cells purified from healthy donors were plated in 24-well plates at a density of 2 × 106 cells per well in RPMI 1640 with 10% human AB serum. Then, CD1d-expressing K562-based artificial APCs (aAPCs) pulsed with 500 ng/ml of α-GalCer were irradiated (200 Gy) and added to the responder cells at a responder/stimulator ratio of 20:1 (day 0), as previously described (24). The T cells were restimulated every 7 d and supplemented with 100 IU/ml of IL-2 every 3 d.

Cytokine ELISPOT analysis

IL-2 ELISPOT assays were conducted as described elsewhere (33–36). Briefly, polyvinylidene difluoride plates (Millipore, Etobicoke, ON, Canada) were coated with capture mAb (R&D Systems, Minneapolis, MN). One hundred thousand Cl6 transfectants were incubated with the indicated numbers of target cells per well for 22–24 h at 37°C. Plates were washed and incubated with biotin-conjugated detection mAb (R&D Systems), followed by washing and incubation with streptavidin-conjugated alkaline phosphatase (Jackson ImmunoResearch, West Grove, PA). Maximal response was determined by stimulating iNK TCR transfectants with a K562-based aAPC expressing a membranous form of the anti-human CD3 mAb clone OKT3 (34).

Molecular dynamics simulation

Molecular dynamics (MD) simulations were performed with the MOE program (v2015.10). The atomic coordinates were derived from the crystal structures of the Vα24–Jα18–Vβ11 iNK TCRs in complex with CD1d–α-GalCer (PDB ID 2PO6), CD1d–β-galactosylceramide (PDB ID 3SDX), or CD1d-lysophosphatidylcholine (PDB ID 3TZV) (19–21). These iNK TCRs are identical in their CDR except for varying CDR3β sequences. CD1d–lipid molecules were deleted from each model. Missing side chains and hydrogen atoms were generated using QuickPrep and Protonate3D. The iNK TCR were then energy minimized using the AMBER10 force field. Sodium ions and water molecules were added as a droplet around the molecule and energy minimized once more. iNK TCR CDR loops and water molecules were experimentally probed for their dynamics, whereas framework residues were fixed in position. MD simulations were performed using the NPA algorithm on the wild-type Vα24–Jα18–Vβ11 iNK TCRs and an Arg95β mutant. During the MD simulations, the sample was heated to 26.85°C over 100 ps, followed by an equilibration of 100 ps. A production run of 500 ps was performed, followed by a cooling step to −273.15°C over 100 ps. A time step of 1 fs and no bond constraints were used.

Statistics

Statistical analyses were performed using GraphPad Prism version 6.0. Two-way ANOVA tests with Bonferroni post hoc tests were used. Analyses were paired between the Ser and Arg version of each clone or paired between each E:T ratio for the ELISPOT experiments. All tests were two-tailed, and p < 0.05 was considered statistically significant.

Results

A natural polymorphism at the third position of CDR3β sequences alters the antigenicity of human iNK TCRs

We previously isolated 54 unique human TCR Vβ11 genes and individually reconstituted them along with the Vα24i chain on the human Vα24−Vβ11− T cell line, SupT1. Among the 54 SupT1 iNK TCR transfectants (24), two clones, Cl.3007 and Cl.2133, exhibited noticeably lower staining by the pan anti-Vα24 mAb (clone C15), despite expressing comparable levels of CD3 (Supplemental Fig. 1). These two clones encoded CASR at the beginning of CDR3β (with Arg at the third residue), whereas all of the others encoded CASS or CAST. Two clones encoded CAST instead of CASS; however, they did not demonstrate lower staining by the C15 mAb. To confirm that peripheral human iNKT cells naturally encode CASR at the CDR3β loop, we purified Vα24-Jα18+Vβ11+ cells from PBMCs and thymi of 12 donors (n = 6 for each) and analyzed their TCRβ sequences by high-throughput sequencing (Fig. 1A). The majority of the TCR Vβ11 genes encoded Ser at the third position. However, CASR sequences were indeed detected in both tissues from all donors (Fig. 1B). These results demonstrate that Arg at the third CDR3β position is a natural polymorphism of human Vβ11 TCRs.

FIGURE 1.
FIGURE 1.

Frequency of peripheral and thymic human iNK TCRs encoding Ser or Arg at the third CDR3β position. (A) The sorting strategy is shown for one PBMC donor. Thymic iNKT cells were similarly sorted. (B) Vα24+Vβ11+ cells were purified from PBMC and thymi samples (six donors each), and the TCRβ repertoire of sorted cells from each sample was analyzed by high-throughput sequencing. Frequencies of CASS- or CASR-encoding clones among total or unique sequences are shown. Means ± SD are shown in the graphs.

Next, we generated CASR- or CASS-encoding point mutants of Cl.3014, Cl.2037, Cl.3010, and Cl.2119 (CASS to CASR), as well as Cl.3007 and Cl.2133 (CASR to CASS). The parental and mutated sequences are shown in Table I. Note that the third CDR3β position encoding Ser or Arg was the only difference between each pair of TCR Vβ11 genes. The CASS and CASR versions of each clone were individually reconstituted in the Cl6 cell line, which was engineered to stably express the Vα24i chain and low surface CD1d levels (Supplemental Fig. 2). Jurkat 76 cells lack endogenous TCR expression (37). Cl6 with low CD1d expression was used to avoid fraternal activation mediated by the transduced iNK TCRs. Each transfectant was stained with the two distinct anti-Vα24 mAbs: C15 and 6B11. The C15 mAb stained all CASR-encoding transfectants with lower mean fluorescence intensity (MFI) compared with their respective CASS counterparts, although the differences were smaller with Cl.3014, Cl.2037, and Cl.3010 (Fig. 2A). The 6B11 MFIs were substantially lower for all of the CASR-encoding transfectants with the exception of Cl.2037, for which the CASR version stained better than the CASS version (Fig. 2B). These data demonstrate that the antigenicity of the Vα24i chain can be modulated by the third amino acid of the CDR3β sequence, which suggests that this residue may alter the structure of the Vα24i chain. Furthermore, our initial high-throughput sequencing analysis may have underestimated the frequency of the CASR-encoding TCR Vβ11 genes, because the cells were purified with anti-Vα24 mAbs.

View this table:
Table I. CDR3β sequences of human TCR Vβ11 clones
FIGURE 2.
FIGURE 2.

Ser or Arg at the third CDR3β residue impacts antigenicity of the Vα24i chain. (A) CASS- or CASR-encoding Cl.3014, Cl.2037, Cl.3010, Cl.2119, Cl.3007, or Cl.2133 TCR Vβ11 chain was reconstituted on Cl6 cells. Transfectants were stained with anti-Vα24 clone C15 and anti-CD3 mAbs. (B) Transfectants were similarly analyzed with clone 6B11. MFI of staining with anti-Vα24 mAbs was normalized by respective CD3 MFI. Data are representative of three repeated experiments. Means ± SD are shown in the bar graphs. ****p < 0.0001.

CASR-encoding iNK TCRs are preferentially expressed by human iNKT cells with low 6B11 reactivity

To address whether these in vitro findings apply to human primary iNKT cells, we stimulated peripheral T cells with α-GalCer–loaded aAPCs to polyclonally expand peripheral iNKT cells (24). Without ex vivo expansion, this experiment would not be feasible given the rarity of peripheral iNKT cells, especially 6B11-low iNKT cells, in the majority of donors. After expansion, we observed Vα24-Jα18–high, -low, and -negative populations among the PBS-57 tetramer-positive cells when stained with the 6B11 mAb. All three populations expressed comparable levels of TCR Vβ11 (Fig. 3A). Vα24− α-GalCer–reactive human iNKT cells were described previously (38). Vα24-Jα18–high and -low populations were purified by flow cytometry–guided sorting, and Vβ11 genes were cloned and analyzed from respective populations. Two unique TCRβ sequences encoding CASR were identified in each cohort. Interestingly, the two CASR sequences identified in the 6B11-low population were two of the most frequent within that group, together encompassing >40% of all Vβ11 genes cloned from that cohort. In contrast, only one copy of each of the CASR sequences in the 6B11-high population was identified from a total of 120 Vβ11 genes (Fig. 3B, 3C). These results demonstrated that human peripheral Vβ11+ iNKT cells with lower 6B11 reactivity preferentially encode CASR within the CDR3β.

FIGURE 3.
FIGURE 3.

Peripheral iNKT cells weakly stained by 6B11 express CASR-encoding Vβ11+ TCRs more frequently than those stained strongly by 6B11. (A) T cells were isolated from PBMCs and stimulated by aAPCs pulsed with α-GalCer (500 ng/ml). After three stimulations, the T cells were stained with PBS-75 CD1d tetramer, anti-Vα24 (clone 6B11), and anti-Vβ11 mAbs. Data for staining with anti-Vα24 and Vβ11 mAbs are shown after gating on tetramer-positive cells. Data are representative of three donors. (B) Vα24-high and Vα24-low cells were isolated from three donors by flow cytometry (purity > 95%). After synthesizing cDNA, we molecularly cloned 120 and 187 Vβ11+ TCRs from the Vα24-high and Vα24-low populations, respectively, and determined their CDR3β sequences. The frequency of each unique Vβ11+ TCR is shown as a section of the respective pie graph. Different colors indicate clones encoding different CASR sequences. (C) CDR3β sequences of CASR-encoding Vβ11 genes are shown. The same color is used to denote identical sequences in (B) and (C).

Encoding Ser or Arg alters recognition of self-lipids and OCH but not PBS-57

Given the dominant role of Vα24i in Ag recognition for human iNK TCRs, we explored whether the potentially altered Vα24i conformation induced by the CDR3β single amino acid substitution impacted Ag recognition. We first stained each pair of iNK TCR transfectants with unloaded or PBS-57–loaded CD1d tetramers. PBS-57 tetramer staining was comparable between CASR- and CASS-encoding clones (Fig. 4A). When stained with the unloaded tetramer, which presents HEK293-derived endogenous lipids, the transfectants expressing CASR-encoding TCRs possessed low tetramer positivity (Fig. 4B). We also compared the autoreactivity of the CASS- or CASR-encoding transfectants in functional assays. Each Cl6 transfectant was stimulated with K562 or C1R transduced with CD1d, and CD69 upregulation was measured by flow cytometry. Parental K562 and C1R cells, which do not endogenously express surface CD1d, were used as negative controls. With the exception of Cl.2133, a significant decrease in CD69 expression was observed for the Arg-encoding transfectants compared with those encoding Ser, regardless of the target cells used (Fig. 5). Functional autoreactivity between CASS- and CASR-encoding clones was also compared by IL-2 ELISPOT assays. The number of spot-forming units was normalized to the maximal response obtained upon stimulation with an aAPC expressing a membranous form of anti-CD3 mAb (34). At the highest E:T ratio tested, all CASS-encoding transfectants secreted IL-2 in greater proportions in response to C1R expressing CD1d compared with CASR clones. Significantly higher cytokine secretion was also detected at limiting target cell numbers by all CASS-encoding iNK TCRs with the exception of Cl.2133 (Fig. 6), which inherently possesses low autoreactivity (Figs. 4, 5). These data indicate that CASS-encoding iNK TCRs are more sensitive at recognizing self-antigens than the CASR type.

FIGURE 4.
FIGURE 4.

CASR-encoding iNK TCR transfectants possess diminished self-lipid tetramer staining. The transfectants in Fig. 2 were stained with 1 μg/ml of PBS-57–loaded CD1d tetramer (A) or 10 μg/ml of unloaded CD1d tetramer (B) along with anti-CD3 mAb. MFI of tetramer staining was normalized by respective CD3 MFI. Data are representative of three repeated experiments. Means ± SD are shown in the bar graphs. ***p < 0.001, ****p < 0.0001.

FIGURE 5.
FIGURE 5.

CASR-encoding transfectants possess diminished functional autoreactivity. (A) The transfectants in Fig. 2 were stimulated with an E:T ratio of 5:1 K562 or K562 expressing CD1d. (B) Transfectants were also stimulated with an E:T ratio of 10:1 C1R or C1R expressing CD1d. After a 4-h stimulation, cells were stained with anti-CD69 and CD3 mAbs and analyzed by flow cytometry. Raw data for stimulation with CD1d-expressing target cells are shown. Data are representative of three repeated experiments. Means ± SD are shown in the bar graphs. **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 6.
FIGURE 6.

CASR-encoding transfectants possess diminished cytokine secretion at various E:T ratios. A total of 1 × 105 cells of each transfectant was stimulated with 5 × 104 parental C1R or indicated numbers of CD1d-expressing C1R target cells. Responses were measured by IL-2 ELISPOT assays. All spots counted were normalized to a maximal response, which was measured by stimulating each transfectant with 5 × 104 aAPCs expressing a membranous form of the anti-human CD3 mAb clone OKT3. Data are representative of two independent experiments performed in triplicates. Means ± SD are shown in the graphs. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

OCH is a synthetic α-linked glycolipid that is also recognized by iNKT cells (39). Although human iNK TCR–OCH–CD1d trimolecular complexes have yet to be structurally analyzed, structural analysis of a murine iNK TCR recognizing OCH–CD1d demonstrated that the CDR3β loop did not mediate direct contact with the Ag complex, similar to how α-GalCer is recognized (40). For four of the six clones, the CASR-encoding transfectants were stained with weaker intensities by the OCH-loaded CD1d tetramer compared with the CASS transfectants. Encoding Arg or Ser had no observable effect on OCH–CD1d recognition by Cl.3007 and Cl.2133 (Supplemental Fig. 3). We cannot rule out the possibility that the diminished reactivities are due, at least in part, to a CDR3β-intrinsic effect of CASR sequence rather than modulation of Vα24i, because CDR3β can mediate direct contact with the CD1d molecule (20, 21, 23). However, these results indicate that the third residue of CDR3β also influences the Ag recognition of human iNK TCRs, possibly by modulating the conformation of other CDR loops within the TCR.

The third amino acid of Vβ11 CDR3β modulates the structure of CDR3α

To determine the potential for an Arg residue in the third position of the CDR3β sequence to alter the structure of the iNK TCR CDRα loops, we performed MD simulations on the wild-type CASS and its respective Arg95β CASR mutant of three Vα24-Jα18-Vβ11 iNK TCRs (PDB ID 2PO6, 3SDX and 3TZV. Amino acid numbering based on 2PO6) (19–21). MD simulations of the iNK TCRs revealed only minor differences between the wild-type and Arg95β mutant models in the CDR1/2 of TCRα and TCRβ-chains, with an average main-chain root-mean-square deviation (RMSD) of 0.38 and 0.45 Å in the CDR1/2α loops and 0.68 and 0.60 Å in the CDR1/2β loops, respectively. In contrast, the CDR3 loops displayed a higher degree of variation, with the wild-type and Arg95β mutant CDR3α and CDR3β loops differing by an average RMSD of 1.13 and 2.17 Å, respectively (Fig. 7A, 7B).

FIGURE 7.
FIGURE 7.

MD simulations of the wild-type (Ser95β) and Arg95β-mutated Vα24–Jα18–Vβ11 iNK TCRs from PDB 2PO6, 3SDX, and 3TZV. Average RMSD difference in CDRα (A) and CDRβ (B) between the wild-type and Arg95β-mutated iNK TCR models. Means ± SD are shown in the bar graphs. Superposition of the CDR loops of the wild-type and Arg95β iNK TCR for 2PO6 (C), 3SDX (D), and 3TZV (E). The CDR3β sequence of the iNK TCRs is shown. The wild-type iNK TCRs are colored in gray, whereas the TCRα- and TCRβ-chains of the Arg95β mutant iNK TCRs are colored in green and wheat, respectively (upper panels). Interactions between the CDR3α/β loops of the wild-type and Arg95β mutant iNK TCRs (lower panels). Hydrogen bonds are shown as black dashes.

A comparison of the wild-type and mutant iNK TCR–simulated models revealed key differences in the interactions between the CDR3α loop and the Ser95β- or Arg95β-encoding CDR3β loop. In the wild-type models, Ser95β formed a lone van der Waals contact with Thr98α of the CDR3α loop (Fig. 7C) or did not contact the CDR3α loop at all (Fig. 7D, 7E). In contrast, in two simulated Arg95β models, the Arg95β residue of the mutant models hydrogen bonded extensively with main-chain atoms or residues of the CDR3α loop, which resulted in a shift in the CDR3α loops position compared with the wild-type model (Fig. 7C, 7D). Conversely, as observed in a third MD simulation, the Arg95β residue could also be prevented from contacting the CDR3α loop by a neighboring Glu97β residue (Fig. 7E), indicating that neighboring residues in the CDR3β loop could potentially prevent Arg95β from contacting CDR3α in some settings.

In an effort to support our MD simulations, we also compared the crystal structure of a Vα10-Vβ8.1 murine iNK TCR with our MD-simulated Arg95β iNK TCR models (41). The Vα10-Vβ8.1 murine iNK TCR naturally contains the CASR sequence in its CDR3β loop. Similar to two of our Arg95β iNK TCR MD-simulated models, the third Arg of the CDR3β loop also contacts a main-chain carbonyl group (Supplemental Fig. 4). Based on these in silico observations, we propose that, by contacting the CDR3α of the iNK TCR and slightly reorienting the CDR3α loop, the Arg at the third position of the CDR3β loop has the potential to affect the ability of human iNK TCRs to recognize Ags.

Discussion

In this study, we demonstrated that the third residue of Vβ11 CDR3 influences the antigenicity, Ag recognition, and TCRα-chain structure of human iNK TCRs. Ser or Arg at this position significantly altered the binding by two anti-Vα24 mAbs: clones C15 and 6B11. It is unclear where clone C15 binds on the TCR Vα24 V region, but clone 6B11 is specific for the Vα24-Jα18 CDR3 loop. Therefore, this result strongly suggests that Arg-encoding CDR3β alters the conformation of the CDR3α region. Furthermore, the altered 6B11 staining for iNK TCRs encoding Arg did not appear to be entirely due to masking of the epitope, because Cl.2037 had increased 6B11 staining intensity upon mutating from Ser to Arg. Nevertheless, staining with these two anti-Vα24 mAbs was generally decreased in the CASR clones. It is important to note that, although C15 staining among CASS clones was fairly consistent, 6B11 staining was variable, even among the clones encoding CASS. Thus, although encoding Ser or Arg at the third position can influence 6B11 staining, other CDR3β residues likely play a role in dictating the reactivity of this mAb to Vα24 and possibly the conformation of CDR3α.

Based on the modeling from previous human iNK TCR crystal structures, we observed that the CDR3α and CDR3β loop conformations were affected by whether Ser or Arg was encoded at the third position of CDR3β. Specifically, the Arg residue at this position in the CDR3β was able to make direct interactions with CDR3α in two of the three MD-simulated models that we performed. Previous structural analyses of human iNK TCRs also found interactions between the CDR3α and CDR3β residues, although they were not mediated by Arg at the same position as described in this article. Kjer-Nielsen et al. (25) described a van der Waals interaction between Gly96 of CDR3α and Tyr101 of CDR3β. In a human iNK TCR–CD1d–lysophosphatidylcholine trimolecular complex, López-Sagaseta et al. (21) observed several contacts between similar regions of CDR3α and CDR3β that were coordinated by water molecules. Therefore, CDR3β and CDR3α of human iNK TCRs form interloop interactions that influence their respective structures.

Our finding has interesting implications for the indirect role of CDR3β in regulating Ag recognition. Numerous structural and mutational analyses identified residues within CDR1α, CDR3α, and CDR2β that are important for the recognition of the α-GalcCer–CD1d complex (20, 23, 42–45). Because CDR3β is the only V region among iNK TCRs with the same Vβ usage and does not mediate direct contact with the ligand, it is thought that CDR3β variability is able to influence the overall affinity toward CD1d–lipid complexes but not lipid specificity (20, 22, 23). Our data suggest that CDR3β could indirectly influence Ag recognition by altering the CDR3α loop structure in a sequence-dependent manner. This may be the mechanism by which certain clonotypic murine Vβ8.2 iNK TCRs with unique CDR3β sequences can possess lipid selectivity for human CD1d–self-lipid complexes (31). However, this effect might only be apparent for low-affinity interactions between iNK TCRs and CD1d presenting weak lipid ligands but not potent agonists, such as α-GalCer and PBS-57.

Detection of iNKT cells using 6B11 and anti-CD3 mAbs tends to yield higher frequencies compared with detection using C15 and anti-Vβ11 mAbs (46, 47), possibly because not all Vα24i pairs with Vβ11. However, the analyses in those studies were not conducted in a pairwise manner. Montoya et al. (11) demonstrated that these two methods produced similar results when they were compared within each donor, albeit with a smaller sample size. Additionally, it is obvious that 6B11 cannot detect Vα24-Jα18–independent human iNKT cells; they can only be detected by the α-GalCer/PBS-57–loaded CD1d tetramer. In addition to these data, this study suggests that the variability in CDR3β sequences could be another element that affects the accuracy of detecting human iNKT cells using the 6B11 mAb.

Florence et al. (42) and Scott-Browne et al. (44) demonstrated that a Gly-to-Ala mutation at the third position of the murine Vβ8.2 iNK TCR CDR3 sequence did not influence α-GalcCer–mouse CD1d or PBS-57–mouse CD1d recognition, as measured by functional response and/or tetramer staining. Murine Vβ8.2 is homologous to human Vβ11, and Gly is typically encoded at this position by mouse Vβ8.2 compared with the Ser encoded by human Vβ11. In the same studies, the Gly-to-Ala mutant moderately decreased reactivity against mouse CD1d-presenting iGb3, which is a self-lipid for murine iNKT cells. These data are similar to what we observed for human iNK TCRs when Ser was mutated to Arg. However, it is possible that the Gly-to-Arg mutation in murine iNK TCRs may behave differently than Gly-to-Ala or Ser-to-Arg mutations in humans.

We demonstrated previously that three CDR3β sequence motifs are associated with strong autoreactivity of human iNK TCRs (24). However, in this study we found that a feature of the CDR3β amino acid sequence, CASR, was associated with weak autoreactivity of iNK TCRs. Additionally, the CASR sequence altered binding of Vα24-specific mAbs, especially clone 6B11, which binds the CDR3α region. In silico analysis also highlighted a role for the third CDR3β residue in regulating the CDR3β and CDR3α loop conformations. Taken together, our study provides evidence for the role of CDR3β in modulating the structure of the iNK TCRα-chain, which could influence the function of the overall TCR.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank the National Institutes of Health Tetramer Core Facility for the provision of human CD1d monomers. Jurkat 76 cells were a generous gift from Dr. M.H. Heemskerk, Leiden University Medical Center.

Footnotes

  • This work was supported by National Institutes of Health Grant R01 CA148673 (to N.H.), Ontario Institute for Cancer Research Clinical Investigator Award IA-039 (to N.H.), a BioCanRx catalyst grant (to N.H.), the Princess Margaret Cancer Foundation (to M.O.B. and N.H.), a Knudson postdoctoral fellowship (to K.C.), a Canadian Institutes of Health Research Canada graduate scholarship (to T.G.), the Province of Ontario (to T.G. and M.A.); and a Natural Sciences and Engineering Research Council of Canada postgraduate scholarship (to T.G.).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    aAPC
    artificial APC
    Cl6
    Jurkat 76.clone 6
    α-GalCer
    α-galactosylceramide; iNK, invariant NK
    iNKT
    invariant NKT
    MD
    molecular dynamics
    MFI
    mean fluorescence intensity
    RMSD
    root-mean-square deviation
    Vα24i
    invariant TCR Vα24-Jα18.

  • Received September 6, 2016.
  • Accepted November 21, 2016.

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