Ag processing in the endoplasmic reticulum (ER) by the ER aminopeptidase associated with Ag processing (ERAAP) is central to presentation of a normal peptide–MHC class I (MHC I) repertoire. Alternations in ERAAP function cause dramatic changes in the MHC I–presented peptides, which elicit potent immune responses. An unusual subset of CD8+ T cells monitor normal Ag processing by responding to a highly conserved FL9 peptide that is presented by Qa-1b, a nonclassical MHC Ib molecule (QFL) in ERAAP-deficient cells. To understand the structural basis for recognition of the conserved ligand, we analyzed the αβ TCRs of QFL-specific T cells. Individual cells in normal wild-type and TCRβ-transgenic mice were assessed for QFL-specific TCR α- and β-chains. The QFL-specific cells expressed a predominant semi-invariant TCR generated by DNA rearrangement of TRAV9d-3–TRAJ21 α-chain and TRBV5–TRBD1–TRBJ2-7 β-chain gene segments. Furthermore, the CDR3 regions of the α- as well as β-chains were required for QFL ligand recognition. Thus, the αβ TCRs used to recognize the peptide–Qa-1 ligand presented by ERAAP-deficient cells are semi-invariant and likely reflect a conserved mechanism for monitoring the fidelity of Ag processing in the ER.

Molecules of MHC class I (MHC I) present a large repertoire of peptides on cell surface. These peptides are derived from virtually all intracellular proteins as well as those derived from infecting microbes or mutations in cancer cells. Because the peptide–MHC I (pMHC I) complexes serve as ligands for the TCRs expressed on CTLs, the abnormal cells displaying foreign peptides are efficiently detected and eliminated (13).

Generation of pMHC I is a function of the Ag processing pathway, which involves a series of concerted steps (4, 5). The protein precursors are first fragmented by the proteasome in the cytoplasm, and the fragments are transported into the endoplasmic reticulum (ER) by the TAP transporter (6). In the ER, the peptide intermediates are further trimmed to generate the final peptides that are loaded onto MHC I molecules, and the pMHC I complex is transported to the cell surface (7, 8). Failure of any of these steps leads to profound immunological consequences (6, 913). Thus, it is critical to monitor the Ag processing pathway for abnormal functioning.

ER aminopeptidase associated with Ag processing (ERAAP) is the protease that plays a major role in customizing peptides in the ER, where it trims peptide precursors with amino terminal extensions to their final length (7, 8, 14). The critical function of ERAAP in the Ag processing pathway was established by studies that showed that impaired ERAAP function is associated with profound changes in the pMHC I repertoire that, in turn, elicit potent immune responses (11, 1517). Furthermore, polymorphic versions of ERAP1, the human ortholog of mouse ERAAP, are associated with autoimmune diseases as well as poor prognosis of tumors (11, 1820). Notably, a unique CD8+ T cell population was discovered to be responsible for monitoring the normal function of ERAAP (21). These T cells recognize the peptide FYAEATPML (FL9) derived from the Fam49b gene that is conserved from humans to zebrafish. The conserved FL9 peptide is presented by Qa-1b, a nonclassical MHC Ib molecule (Qa-1–FL9 or QFL) exclusively by ERAAP-deficient cells. In addition to their highly conserved ligand specificity, the QFL-specific T cell population is characterized by its relative abundance and Ag-experienced phenotype in naive mice (21). How the αβ TCRs of QFL-specific T cells recognize their conserved ligand is not known.

T cell responses usually involve recognition of particular pMHC I ligands by diverse TCRs, although examples of biased TCR usage have been described (22). Notably, mouse invariant NKT (iNKT) cells specific for the glycolipid α-galactosylceramide presented by CD1d, another nonclassical MHC Ib molecule, express an invariant TCR α-chain paired with a limited number of β-chains (23). Similarly, T cells that recognize vitamin B metabolites presented by MR1, the MHC I–like molecule (called mucosal-associated invariant T [MAIT] cells) also bear invariant TCR α-chains paired with a limited array of β-chains (24, 25).

In this study, we analyzed the α and β subunits of TCRs used by the QFL-specific T cells. We show that similar to the TCRs of iNKT and MAIT cells specific for nonpeptidic materials presented by the MHC Ib molecule, the FL9 peptide Qa-1 MHC Ib–specific TCRs are also semi-invariant. However, the QFL-specific T cells share some but not all characteristics with their iNKT and MAIT cell counterparts.

The cDNA encoding the V and J regions of the TCR α- and β-chains of BEko8Z hybridoma was amplified with the following primers: BEko.α, forward, 5′-GCTGGATCCAGCCTTCTCAAGGCTCAGTCATGCTCC-3′, reverse, 5′-ATGCGGCCGCAGTCTTCTCCAGGCTTTCATGCC-3′; BEko.β, forward, 5′-GCGGCCGCATGTCTAACACTGCC-3′, reverse, 5′-ATGCGGCCGCGCATAAAAGTTTGTCTCAGG-3′. MSCV-IRES GFP(pMIG) vector was a gift from W. Sha (University of California, Berkeley, Berkeley, CA). The BEko.α and BEko.β DNA fragments were cloned into the BamHI-NotI and NotI-NotI sites of the pMIG vector. Generation of retrovirus and transduction of suspension cells have been described (26).

The genomic DNA fragments encoding TRBV5–TRBJ2-7 were amplified from BEko8Z with the following primers: 5′ primer: XholI-TRBV5 forward, 5′-TCTCTCGAGATGAGCTGCAGG-3′; 3′ primer: SacII–TRBJ2-7 reverse, 5′-CATGCCGCGGCACCACCCACC-3′. The DNA fragment was cloned into the cassette vector for TCR expression (27). The DNA construct was linearized and injected into fertilized (C57BL/6 × SJL) F2 embryos. The transgenic founders were screened by amplification of the transgene with oligonucleotide primers described above. Transgene-positive founders are backcrossed to C57BL/6 mice. ERAAP-KO and ERAAP-TAP-DKO mice have been described (21). Wild-type C57BL/6 mice were purchased from The Jackson Laboratory. All experiments involving mice were done with the approval of Institutional Animal Care and Use Committee of the University of California, Berkeley.

Abs for flow cytometry were from BD Biosciences (anti-B220 [RA3-6B2], anti-CD3ε [145-2C11], anti-CD8α [53-6.7], anti-CD4 [RM4-5], anti-CD44 [IM7], anti-CD25 [PC61], anti-Vα3.2 [RR3-16], anti-Vα8.3 [B21.14], anti-Vα2 [B20.1], anti-Vβ5.1/5.2 [MR9-4], anti-Vβ8.1/8.2 [MR5-2], anti-Vβ3 [KJ25], anti-Vβ2 [B20.6], anti-Vβ8.3 [1B3.3], anti-Vβ6 [RR4-7], anti-Vβ7 [TR310], anti–retinoic acid–related orphan receptor [ROR]γt [Q31-378], and anti-promyelocytic leukemia zinc finger [PLZF; R17-809]), eBioscience (anti-TCRβ [H57-597] and anti–T-bet [eBio4B10]), and BioLegend (anti-CD122 [TM-β1] and anti-CD127 [A7R34]). The BEko.8Z hybridoma and C6VL.22α-, C6VL51.β-, and 58α-β–mutant cell lines have been described earlier (21, 28).

The Qa-1b–FL9 monomers were obtained from the Tetramer Core Facility of the National Institutes of Health and tetramerized with PE- or allophycocyanin-labeled streptavidin from ProZyme. Homogenized mice spleen cells were resuspended in 200 μl of sorter’s buffer (PBS with 2% FCS and 0.1% sodium azide) and stained with PE- or allophycocyanin-labeled QFL tetramers at a final dilution of 1:200 and 1:100 at 23°C for 50 min. QFL tetramer+ T cells were enriched, gated, and counted as described (21). The enriched fraction of cells was stained with anti-Vα3.2, anti-Vα8, anti-Vα2, anti-Vβ5.1/5.2, anti-Vβ8.1/8.2, and anti-Vβ2 for TCR analysis and anti-CD44, anti-CD122, anti-CD25, and anti-CD127 for surface marker characterization. For transcription factor staining, the enriched fraction of cells was first stained with anti-CD4, anti-B220, anti-CD8, anti-TCRβ, and anti-CD44, fixed and permeabilized for 45 min, and then stained with anti–T-bet, anti-RORγt, and anti-PLZF for 45 min using a transcription factor set (BD Biosciences). Cells were analyzed on an LSRFortessa X-20, and data were analyzed with FlowJo (Tree Star) software.

The enriched QFL tetramer+ T cells from three mice were pooled and suspended in PBS containing 2% BSA and 200 U RNasin/ml (Promega) and sorted as single cells in to 96-well PCR plates using a BD Influx cell sorter (BD Biosciences). The multiplex nested RT-PCR protocol and the oligonucleotide primers used for CDR3 region amplification have been previously described (29). The sequences presented in this article have been submitted to GenBank (https://www.ncbi.nlm.nih.gov/genbank/) under accession numbers KY271957 and KY271958.

BEko8Z, a β-galactosidase (LacZ) inducible T cell hybridoma, was derived from C57BL/6 mice immunized with ERAAP-deficient cells (21). This hybridoma responds specifically to splenocytes from ERAAP-deficient mice but not to self-C57BL/6 splenocytes (Fig. 1A). Previous studies have also revealed that the ligand recognized by BEko8Z T cells is the FYAEATPML (FL9) peptide presented by Qa-1b, the nonclassical MHC Ib molecule, and the Qa-1b–FL9 complex is referred to as the QFL ligand (21). We first analyzed the TCR subunits used by the BEko8Z using a nested PCR–based strategy (29). The transcripts of the TCR α- and β-chain (BEko.α- and BEko.β-chain) were amplified simultaneously. We found that TRAV9d-3–TRAJ21 and TRBV5–TRBD1–TRBJ2-7 rearrangements were most likely used to encode the BEko.α- and BEko.β-chains. The full-length nucleotide sequence of BEko.α and BEko.β cDNA, which was determined using oligonucleotide primers specific for TRAV9d-3 and TRBV5, revealed that the gene segments were productively rearranged (Fig. 1B). We concluded that TRAV9d-3–TRAJ21 (Vα3.2–Jα21) and TRBV5–TRBD1–TRBJ2-7 (Vβ1–Dβ1–Jβ2-7) were used to generate the full-length TCR α- and β-chains in BEko8Z T cells.

FIGURE 1.

The sequence of αβ TCR expressed by BEko8Z hybridoma. (A) Specificity of the BEko8Z hybridoma. The LacZ response of BEko8Z hybridoma cells to a varying number of wild-type (WT) or ERAAP-knockout (ERAAP-KO) spleen cells was measured by absorbance (A595) of the cleaved lacZ substrate chlorophenol red β-d-galactopyrandoside. Data are representative of three independent experiments. (B) Schematic structure, including the nucleotide and the translated amino acid sequences of the TCR α-chain (BEko.α) and β-chain (BEko.β). Annotations above and below the sequence show the various rearranged V, D, and J segments for the α- and β-chains.

FIGURE 1.

The sequence of αβ TCR expressed by BEko8Z hybridoma. (A) Specificity of the BEko8Z hybridoma. The LacZ response of BEko8Z hybridoma cells to a varying number of wild-type (WT) or ERAAP-knockout (ERAAP-KO) spleen cells was measured by absorbance (A595) of the cleaved lacZ substrate chlorophenol red β-d-galactopyrandoside. Data are representative of three independent experiments. (B) Schematic structure, including the nucleotide and the translated amino acid sequences of the TCR α-chain (BEko.α) and β-chain (BEko.β). Annotations above and below the sequence show the various rearranged V, D, and J segments for the α- and β-chains.

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We next assessed whether the TCR α- and β-chains identified above recognized the Qa-1–FL9 complex (QFL). The cDNAs were used to prepare constructs with the BEko.α and BEko.β cloned into a retroviral vector that also encoded the IRES-GFP cassette to allow identification of transfected cells. We transduced mutant T cell lines C6VL.22, C6VL.51, and 58αβ that lacked expression of endogenous TCR α- or TCR β-chain alone or both TCR α- and TCR β-chains. As a consequence, none of these recipient cells expressed αβ TCR or the CD3 complex on the cell surface (28, 30). Among the GFP+ transduced cells, the TCR/CD3-expressing cells were obtained by sorting for the CD3+GFP+ population indicating that each TCR α- or β-chain successfully paired with its respective β or α counterpart as well as with CD3 polypeptides (Fig. 2A). These CD3+ cell lines were then stained with QFL tetramer reagent prepared using the QFL monomer obtained from the Tetramer Core Facility of the National Institutes of Health (21). The specificity of the QFL tetramer was confirmed by staining BEko8Z T cells (Fig. 2B). However, despite high levels of TCR/CD3 expression by all TCR transduced cells, only the 58αβ cells expressing both the BEko.α- and β-chains bound the QFL tetramer as compared with the vector only control (Fig. 2B). The cells expressing either of the BEko.α- or β-chain paired with the endogenous β- or α-chain of the recipient cells did not stain with the fluorescent tetramer reagent. Thus, the QFL ligand recognition by the BEko.TCR required both the BEko.α- and BEko.β-chains.

FIGURE 2.

The BEko.α- and β-chains and their CDR3 regions are required for binding the QFL ligand. (A) CD3 and GFP expression by C6VL.22(α) recipient cells transduced with BEko.α, C6VL.51(β) recipient cells transduced with BEko.β and 58 (αβ) recipient cells transduced with both BEko.α- and BEko.β-encoding retroviruses. (B) Fluorescence intensity of transduced cell lines expressing the BEko.α- and/or β-chains stained with the QFL tetramers labeled with PE. (C) The amino acid sequences of the BEko.α- and β-chain CDR3 regions and the 4A and 3A alanine substitution mutants. (D) CD3 and GFP expression by the 58 (αβ) cells transduced with both intact BEko.α- and β-chain, BEko.α pairing with BEko.β3A(3A), or BEko.β pairing with BEko.α4A(4A). (E) Fluorescence intensity of PE-labeled QFL tetramer staining of the cell lines that express BEko.α- and β-chain (α+β), 4A paired with BEko.β-chain (4A+β), or 3A paired with BEko.α-chain (α+3A). Plots shown are representative of three independent experiments.

FIGURE 2.

The BEko.α- and β-chains and their CDR3 regions are required for binding the QFL ligand. (A) CD3 and GFP expression by C6VL.22(α) recipient cells transduced with BEko.α, C6VL.51(β) recipient cells transduced with BEko.β and 58 (αβ) recipient cells transduced with both BEko.α- and BEko.β-encoding retroviruses. (B) Fluorescence intensity of transduced cell lines expressing the BEko.α- and/or β-chains stained with the QFL tetramers labeled with PE. (C) The amino acid sequences of the BEko.α- and β-chain CDR3 regions and the 4A and 3A alanine substitution mutants. (D) CD3 and GFP expression by the 58 (αβ) cells transduced with both intact BEko.α- and β-chain, BEko.α pairing with BEko.β3A(3A), or BEko.β pairing with BEko.α4A(4A). (E) Fluorescence intensity of PE-labeled QFL tetramer staining of the cell lines that express BEko.α- and β-chain (α+β), 4A paired with BEko.β-chain (4A+β), or 3A paired with BEko.α-chain (α+3A). Plots shown are representative of three independent experiments.

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The CDR3 regions, which comprise the junctions of the V(D)J on the TCR α- and β-chains, are key elements that define the ligand specificity of the TCR (31, 32). To assess whether this canonical binding pattern applied to the BEko.TCR as well, we identified the CDR3 regions of BEko.α and BEko.β TCR chains by analyzing their DNA sequence using the International ImMunoGeneTics Information System database (http://www.imgt.org) (33). The 4 and 3 aa, respectively, within the prospective CDR3 junctional region of the BEko.α- and β-chains were replaced with alanine residues (Fig. 2C). The cell lines expressing BEko.α 4A or BEko.β 3A mutants paired with the wild-type BEko.β- or α-chain were generated by transducing the 58αβ cells described above. Again, each α- and β-chain of TCRs paired with each other and the CD3 subunits (Fig. 2D). However, staining the GFP+CD3+ cells with the fluorescent QFL tetramer showed that in contrast to cells expressing the wild-type BEko.TCR (α and β), tetramer binding was completely abolished in cells expressing either the 4A α-chain or the 3A β-chain mutants paired with their wild-type β or α counterpart (Fig. 2E). We conclude that the CDR3 regions on both the α- and β-chain of the BEko.TCR are essential for QFL ligand recognition.

To assess the diversity of TCR α-chains that could be used to generate QFL-specific T cells in vivo, we generated transgenic mice expressing the BEko.β-chain (βTg). The rearranged genomic fragment encoding the VDJ segments of the BEko.β-chain was cloned into the TCRβ cassette vector and microinjected into fertilized eggs (Fig. 3A) (27). We identified four TCR transgenic mice by PCR amplification of the injected construct in tail DNA samples (Supplemental Fig. 1A). The founder line no. 3 referred to as βTg was chosen for subsequent analysis because the transgene was well expressed and efficiently transmitted to the progeny. The fraction of the CD4+ and CD8+ population as well as the total number of T cells were normal in βTg mice as compared with wild-type mice and the transgene-negative littermates (Supplemental Fig. 1B). Analysis of CD4+ and CD8+ T cell subsets in thymocytes and splenocytes from the βTg mice with a set of TCR Vβ Abs showed the expected staining in wild-type and transgene-negative littermates (Supplemental Fig. 1C). In contrast, these TCR Vβ+ cells were not detected in βTg mice even though these mice contained cells expressing TCR β-chains detected by the Ab specific for the Cβ C region. We conclude that transgene expression suppressed expression of diverse endogenous TCR β-chains, leaving the T cells to pair the TCR β transgene with endogenously rearranged TCR α-chains to allow development of normal numbers of CD4+ and CD8+ T cells.

FIGURE 3.

Relative frequency of QFL-specific T cells in wild-type and βTg mice. (A) Schematic representation of the rearranged TRBV5–TRBJ2-7 DNA cloned in the pTβcass vector for transgene expression. (B) Flow cytometry analysis of spleen cells from naive ERAAP-TAP-DKO mice as negative control, wild-type mice, and βTg mice stained with QFL tetramer labeled with PE (QFL-PE) or allophycocyanin (QFL-APC) before (left) and after (right) enrichment of the tetramer+ cells. Enrichment was carried out by positive selection of the magnetically labeled QFL-PE+ cells. Numbers in plots indicate the average number of QFL+ T cells per spleen. (C) Numbers of QFL+ T cells enriched per spleen in wild-type and βTg mice. Each symbol represents an individual animal. (D) Flow cytometry analysis of the enriched QFL+ T cells from wild-type and βTg mice for CD44 expression. Numbers in plots indicate average percentage of CD44hiTCRβ+ cells. (E) Frequency of CD44hi cells among QFL+ T cells and total CD8+ T cells in wild-type and βTg mice. Results are shown as the mean ± SD of at least three mice (B and C) or three mice (D and E) per genotype and are representative of three independent experiments. *p < 0.01, **p < 0.001, Student t test.

FIGURE 3.

Relative frequency of QFL-specific T cells in wild-type and βTg mice. (A) Schematic representation of the rearranged TRBV5–TRBJ2-7 DNA cloned in the pTβcass vector for transgene expression. (B) Flow cytometry analysis of spleen cells from naive ERAAP-TAP-DKO mice as negative control, wild-type mice, and βTg mice stained with QFL tetramer labeled with PE (QFL-PE) or allophycocyanin (QFL-APC) before (left) and after (right) enrichment of the tetramer+ cells. Enrichment was carried out by positive selection of the magnetically labeled QFL-PE+ cells. Numbers in plots indicate the average number of QFL+ T cells per spleen. (C) Numbers of QFL+ T cells enriched per spleen in wild-type and βTg mice. Each symbol represents an individual animal. (D) Flow cytometry analysis of the enriched QFL+ T cells from wild-type and βTg mice for CD44 expression. Numbers in plots indicate average percentage of CD44hiTCRβ+ cells. (E) Frequency of CD44hi cells among QFL+ T cells and total CD8+ T cells in wild-type and βTg mice. Results are shown as the mean ± SD of at least three mice (B and C) or three mice (D and E) per genotype and are representative of three independent experiments. *p < 0.01, **p < 0.001, Student t test.

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We analyzed the QFL-specific T cells in βTg mice by staining splenocytes with QFL tetramers labeled with two different fluorophores, followed by magnetic bead–based enrichment of the tetramer+ cells (34, 35). As observed earlier, ∼900 QFL-specific T cells were found per spleen in naive wild-type mice (Fig. 3A) (21). In contrast, the number of QFL-specific T cells was enhanced ∼7-fold to an average of 6400 QFL-specific T cells in a naive βTg spleen. As a negative control, QFL-specific T cells were undetectable in mice lacking the TAP transporter (Fig. 3B, 3C). We further analyzed QFL-specific T cells for CD44 expression, which was earlier shown to be expressed in a surprisingly high fraction of QFL-specific T cells in naive wild-type mice (21). We found that ∼86% of the QFL-specific T cells from naive βTg mice were CD44hi, which is substantially higher than the 48% frequency detected in naive wild-type mice (Fig. 3D, 3E). In contrast, there was no significant difference between wild-type and the βTg mice in the frequency of CD44hi cells among all CD8+ cells (Fig. 3E). We infer that QFL-specific T cells developed robustly in βTg mice with a similar CD44hi Ag-experienced phenotype as in wild-type mice.

To identify the corresponding TCR α-chains expressed by the QFL-specific T cells, we isolated single T cells by sorting the QFL tetramer+ T cells enriched from splenocytes of naive βTg mice. The rearranged TCR α- and β-chain–encoding mRNAs were then amplified from the sorted cells using the same multiplex nested PCR protocol as above (29). Strikingly, in ∼98% (82 of 84) of the cells analyzed, the TCR α-chain contained the TRAV9d-3 segment (Fig. 4A). Among these TRAV9d-3 Vα rearrangements, ∼80% (66 of 82) were joined to the Jα segment TRAJ21. The remaining 16 TRAV9d-3 Vα segments as well as the 2 other Vα segments detected were rearranged to 9 distinct Jα segments (Fig. 4A). Strikingly, this dominant TCR α-chain formed by the Vα–Jα rearrangement was virtually identical to the BEko.α-chain, which was also formed by TRAV9d-3 rearranged with TRAJ21 and paired with the same BEko.β-chain encoded by the transgene (Figs. 1B, 4B). We further confirmed the high frequency of cells expressing TRAV9 by staining the QFL-specific T cells with Abs specific for different TRAV regions (36, 37). Consistent with the result from the single-cell TCR analysis, ∼98% of the QFL-specific T cells were stained with anti-TRAV9 Ab compared with <2% of TRAV9+ cells among all CD8+ T cells (Fig. 4C, 4D). In contrast, TRAV12- or TRAV14-expressing cells were barely detectable among the QFL tetramer+ cells. We conclude that the QFL-specific T cells in βTg mice expressing a single TCR β-chain developed using a predominant and unique Vα–Jα rearrangement of TCR α-chains.

FIGURE 4.

The structure and function of the highly invariant TCR α-chain expressed by QFL-specific T cells in naive βTg spleen cells. (A) Frequency of Vα and Jα segments used by QFL-specific T cells was determined by single-cell multiplex RT-PCR followed by sequencing 84 cells from three mice. (B) Numbers and frequency of the QFL-specific T cells that expressed BEko.α-chain, BEko.β-chain, or both BEko.α- and β-chains in βTg mice. (C) Staining of the QFL-specific T cells with TRAV9-, TRAV12-, and TRAV14-specific Abs. Numbers in the plots indicate percentage of TRAV+ cells detected. (D) Frequency of the TRAV9+, TRAV12+, and TRAV14+ cells among QFL+ or CD8+ T cells from βTg mice. (E) The QFL-specific T cells bearing the wild-type or four natural variants within the CDR3 region of BEko.α-chain found in βTg mice. (F) Schematic representation of the wild-type (1), natural variants (V>A or T) (2, 3), or artificial mutants (S/N/Y>A) (4–6) in the CDR3 region of the BEko.α-chain. (G) CD3 and GFP expression of the 58 (αβ) cells transduced with both intact BEko.α- and β-chain (1) or BEko.β pairing with the BEko.α CDR3 region variants/mutants (2–6). Plot is representative of the six αβ TCR transfectants. (H) QFL tetramer fluorescence intensity of cell lines described in (F). Data are shown as mean ± SD and are representative of three experiments with three mice each.

FIGURE 4.

The structure and function of the highly invariant TCR α-chain expressed by QFL-specific T cells in naive βTg spleen cells. (A) Frequency of Vα and Jα segments used by QFL-specific T cells was determined by single-cell multiplex RT-PCR followed by sequencing 84 cells from three mice. (B) Numbers and frequency of the QFL-specific T cells that expressed BEko.α-chain, BEko.β-chain, or both BEko.α- and β-chains in βTg mice. (C) Staining of the QFL-specific T cells with TRAV9-, TRAV12-, and TRAV14-specific Abs. Numbers in the plots indicate percentage of TRAV+ cells detected. (D) Frequency of the TRAV9+, TRAV12+, and TRAV14+ cells among QFL+ or CD8+ T cells from βTg mice. (E) The QFL-specific T cells bearing the wild-type or four natural variants within the CDR3 region of BEko.α-chain found in βTg mice. (F) Schematic representation of the wild-type (1), natural variants (V>A or T) (2, 3), or artificial mutants (S/N/Y>A) (4–6) in the CDR3 region of the BEko.α-chain. (G) CD3 and GFP expression of the 58 (αβ) cells transduced with both intact BEko.α- and β-chain (1) or BEko.β pairing with the BEko.α CDR3 region variants/mutants (2–6). Plot is representative of the six αβ TCR transfectants. (H) QFL tetramer fluorescence intensity of cell lines described in (F). Data are shown as mean ± SD and are representative of three experiments with three mice each.

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Interestingly, among the 66 cells that had the TRAV9d-3–TRAJ21 rearrangement, 12 cells were identified with variations within the CDR3 region, whereas the remaining 54 cells expressed the TCR α-chain identical to BEko.α. We noted that the first valine in the CDR3 region was occasionally substituted with alanine (7 of 66), threonine (2 of 66), or serine (1 of 66). Likewise, the second serine residue was substituted by asparagine residue in 2 of 66 sequences (Fig. 4E). To test the functional significance of these substitutions, we generated GFP fusion constructs encoding TCRα with these two amino acid substitutions as well as alanine substitutions for each of the other three amino acids within the junctional CDR3 region (Fig. 4F). The TCRα mutants together with the wild-type BEko.β-chain were introduced into 58αβ cells that lacked endogenous TCR α- and TCR β-chains as above. The TCR+ cells were sorted by flow cytometry as a CD3+GFP+ population and tested for their ability to bind the QFL tetramer (Fig. 4G). The cells that expressed the two natural variants (V>A or V>T) of BEko.α-chain bound the QFL tetramer as well as the ones that expressed the BEko.α-chain. In contrast, substitution of the other amino acids within the junctional region either partially or completely disrupted ligand recognition (Fig. 4H). We conclude that in βTg mice, the QFL-specific T cells not only express predominantly invariant Vα and Jα segments, but also a near invariant junctional CDR3 region for recognizing the QFL ligand.

To determine the natural diversity among the TCR α- and β-chains of QFL-specific T cells, we analyzed splenocytes of naive wild-type mice. The single cells were obtained and analyzed using methods similar to those used for the βTg mice described above. We found that the TRAV9d-3 and TRAJ21 used by the BEko.α-chain were again the most frequent Vα–Jα rearrangement. About 84% (42 of 50) of the cells analyzed used the TRAV9d-3 Vα segment, and 36% (18 of 50) of these used the TRAJ21 Jα segment (Fig. 5A). Virtually all the Vα–Jα rearrangements had the same CDR3 junction identical to that of BEko.α-chain with the exception of a single CDR3 that contained a valine to alanine substitution (Fig 5B). The remaining TRAV9d-3 Vα segments as well as the 6 other Vα segments detected were joined to 22 different Jα segments (Fig. 5A). Thus, the TCR repertoire of QFL-specific T cells is somewhat more diverse in wild-type relative to βTg mice. Staining of the enriched QFL-specific T cells with Abs specific for different Vα regions showed that ∼80% of the QFL-specific T cells expressed TRAV9, relative to a few (∼3%) of TRAV12+ cells that were also detected. However, as a negative control, TRAV14+ cells remained undetectable (Fig. 5C). In contrast, the frequency of cells that used each of the three TRAVs in total CD8+ T cell population was ∼4% (Fig. 5D). We conclude that similar to the BEko8Z hybridoma and T cells in βTg mice, the QFL-specific T cells in wild-type mice also used the TRAV9d-3 Vα segment that was most frequently rearranged to the TRAJ21 Jα segment.

FIGURE 5.

The BEko.α-chain is expressed predominantly by QFL-specific T cells enriched from naive wild-type spleen cells. (A) Frequency of Vα and Jα segments used by 50 QFL-specific T cells in three wild-type mice. (B) Numbers of the QFL-specific T cells that used the wild-type or variant BEko.α-chain CDR3 region in wild-type mice. (C) Staining of the QFL T cells with TRAV9, TRAV12, and TRAV14 Abs. Numbers in the plot indicate percentage of TRAV+ cells. (D) Frequency of the TRAV9+, TRAV12+,and TRAV14+ cells among QFL+ or CD8+ T cells. Data are representative of three experiments and are shown as mean ± SD of nine mice.

FIGURE 5.

The BEko.α-chain is expressed predominantly by QFL-specific T cells enriched from naive wild-type spleen cells. (A) Frequency of Vα and Jα segments used by 50 QFL-specific T cells in three wild-type mice. (B) Numbers of the QFL-specific T cells that used the wild-type or variant BEko.α-chain CDR3 region in wild-type mice. (C) Staining of the QFL T cells with TRAV9, TRAV12, and TRAV14 Abs. Numbers in the plot indicate percentage of TRAV+ cells. (D) Frequency of the TRAV9+, TRAV12+,and TRAV14+ cells among QFL+ or CD8+ T cells. Data are representative of three experiments and are shown as mean ± SD of nine mice.

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The β-chains pairing with these α-chains were then similarly assessed for the Vβ and Jβ usage in the same cells. We found that TRBV5 (the Vβ segment used by BEko.β) was used by 40% (20 of 50) of the QFL-specific cells analyzed (Figs. 1B, 6A). Likewise, the TRBJ2-7 (also the Jβ used by BEko.β) was used by 50% (25 of 50) of wild-type cells. Thus, in 40% (20 of 50) of TCR β-chains, TRBV5 was joined to the TRBJ2-7 segment as the predominant rearrangement similar to the BEko.β TCR chain. All of the TCR β-chains that were identical to BEko.β-chains were paired exclusively to the TCR α-chain identical to BEko.α (Fig. 6B). Two variations of amino acid sequence within CDR3 were detected in three of the cells that used the TRBV5–TRBJ2-7 rearrangement. Unlike the single amino acid substitution found in the α-chains, the junctional region of CDR3 was substantially altered among TCR β-chain variants. However, none of the three variants was paired with the BEko.α-chain (Fig. 6C). A relatively high frequency of TCR β-chains also used TRBV12-1/12-2 rearranged to a diversity of TRBJs. Thirty-two percent (16 of 50) of the QFL-specific T cells expressed TRBV12-2/12-1 and 4% (2 of 50) used TRBV26 (Fig. 6A). This was directly confirmed by staining the QFL-specific T cells with Abs specific for different TRBVs. The QFL-specific T cell population contained ∼30% cells that stained with the anti–TRBV12-2/12-1 Ab and 1.8% that stained with the anti-TRBV26 Ab, but TRBV13-3/13-2+ cells were undetectable (Fig. 6D, 6E). Although Ab specific for TRBV5 is not available, we deduced that ∼70% TRBV12 cells include the TRBV5+ cells. We conclude that in wild-type mice, the QFL-specific T cells comprise a major subset bearing the invariant TCR α- and β-chains and a minor subset bearing the same TRAV9 Vα segment but rearranged to a variety of other TRAJ segments. These TCR α-chains are paired with a limited number of TCR β-chains such as TRBV5 and TRBV12-2/12-1 joined with several distinct TRBJs.

FIGURE 6.

The TCR β-chains used by QFL-specific T cells enriched from naive wild-type spleen cells. (A) Frequency of the Vβ and Jβ segments used by the TCR β-chains pairing with the TCR α-chains analyzed above. (B) Numbers and frequency of QFL-specific T cells that expressed the BEko.α-chain, BEko.β-chain, or both the BEko.α- and β-chains in wild-type mice. (C) Number of QFL-specific T cells that express wild-type or variant BEko.β-chain CDR3 region in wild-type mice. (D) Staining of the QFL-specific T cells with TRBV12-2/12-1, TRBV13-3/13-2, and TRBV26 Abs. Numbers in plot indicate percentage of TRBV+ cells. (E) Frequency of the TRBV12-2/12-1+, TRBV13-3/13-2+, or TRBV26+ cells among QFL+ or CD8+ T cells. Data are representative of three experiments and are shown as mean ± SD of nine mice.

FIGURE 6.

The TCR β-chains used by QFL-specific T cells enriched from naive wild-type spleen cells. (A) Frequency of the Vβ and Jβ segments used by the TCR β-chains pairing with the TCR α-chains analyzed above. (B) Numbers and frequency of QFL-specific T cells that expressed the BEko.α-chain, BEko.β-chain, or both the BEko.α- and β-chains in wild-type mice. (C) Number of QFL-specific T cells that express wild-type or variant BEko.β-chain CDR3 region in wild-type mice. (D) Staining of the QFL-specific T cells with TRBV12-2/12-1, TRBV13-3/13-2, and TRBV26 Abs. Numbers in plot indicate percentage of TRBV+ cells. (E) Frequency of the TRBV12-2/12-1+, TRBV13-3/13-2+, or TRBV26+ cells among QFL+ or CD8+ T cells. Data are representative of three experiments and are shown as mean ± SD of nine mice.

Close modal

The predominant use of a particular TCR α-chain and limited TCR β-chains by QFL-specific T cells suggested a striking similarity of QFL TCRs with TCRs used by the iNKT and MAIT cell subsets. Notably, the iNKT and MAIT cells also predominantly use highly invariant TCR α-chains and a limited number of TCR β-chains (38). The iNKT cells are also characterized by their memory-like and poised effector phenotype (39, 40). We therefore further investigated the expression of iNKT surface markers and transcription factors by the QFL-specific T cells. The enriched QFL-specific T cell population was assessed for CD44, CD122, CD25, and CD127 expression by flow cytometry. We found that the CD44hiCD122hi Ag–experienced T cells were present at an ∼15% higher frequency in the CD8+QFL+ population as compared with the CD8+QFL population (Fig. 7A, 7B). This observation was further supported by the somewhat higher level of CD25 and CD127 expressed on the CD8+QFL+ T cells compared with the CD8+QFL T cells, suggesting that this population of T cells had the phenotype of readily activated, long-living memory T cells (41) (Fig. 7C, 7D). It has been proposed that the iNKT cell population can be subdivided into Th1-like, Th2-like, and Th17-like cells based on the expression of the master regulators T-bet, PLZF, and RORγt (39). We thus assessed expression of these transcriptional factors in QFL-specific T cells. Remarkably, we found that the CD44hiQFL+ T cells were distinguished by the T-bet+ but PLZFRORγt expression pattern, as compared with the CD44hiNK1.1+ cell subset that contained cells expressing each of the three transcription factors (Fig. 7E, 7F). This observation agrees with the CD122hi phenotype of the QFL-specific T cells and our previously reported observation of a substantial fraction of the QFL+ T cells producing IFN-γ in response to the ERAAP-knockout APCs (21). We conclude that most QFL-specific T cells share some characteristics with Ag-experienced and effector memory–like T cells.

FIGURE 7.

Characterization of QFL-specific T cells in naive wild-type C57BL/6 mice. (A) Flow cytometry of spleen cells, stained with QFL-PE tetramer and assessed after magnetic enrichment of the tetramer+ cells. Numbers adjacent to the outlined area indicate percentage of CD44+CD122+ cells among CD8+QFL+ or the CD8+QFL cells. (B) Frequency of CD44+CD122+ cells among CD8+QFL+ cells or CD8+QFL cells after enrichment. (C) Fluorescence intensity of the QFL tetramer+ (QFL-Tet+) or QFL tetramer (QFL-Tet) cells stained with anti-CD25 or anti-CD127. (D) Mean fluorescence intensity (MFI) of CD25 or CD127 expression by QFL-Tet+ or QFL-Tet cells. (E) Flow cytometry of transcription factors T-bet, PLZF, or RORγt expression in the CD44hiNK1.1+ or CD8+QFL+ cells. Numbers in the plots indicate average percentage of T-bet+, PLZF+, or RORγt+ cells. (F) Frequency of T-bet+, PLZF+, or RORγt+ cells among the CD44hiNK1.1+ or CD8+QFL+ cells. Data are representative of three experiments and shown as mean ± SEM of three mice.

FIGURE 7.

Characterization of QFL-specific T cells in naive wild-type C57BL/6 mice. (A) Flow cytometry of spleen cells, stained with QFL-PE tetramer and assessed after magnetic enrichment of the tetramer+ cells. Numbers adjacent to the outlined area indicate percentage of CD44+CD122+ cells among CD8+QFL+ or the CD8+QFL cells. (B) Frequency of CD44+CD122+ cells among CD8+QFL+ cells or CD8+QFL cells after enrichment. (C) Fluorescence intensity of the QFL tetramer+ (QFL-Tet+) or QFL tetramer (QFL-Tet) cells stained with anti-CD25 or anti-CD127. (D) Mean fluorescence intensity (MFI) of CD25 or CD127 expression by QFL-Tet+ or QFL-Tet cells. (E) Flow cytometry of transcription factors T-bet, PLZF, or RORγt expression in the CD44hiNK1.1+ or CD8+QFL+ cells. Numbers in the plots indicate average percentage of T-bet+, PLZF+, or RORγt+ cells. (F) Frequency of T-bet+, PLZF+, or RORγt+ cells among the CD44hiNK1.1+ or CD8+QFL+ cells. Data are representative of three experiments and shown as mean ± SEM of three mice.

Close modal

Conventional CD8+ T cell responses are initiated by specific recognition of the pMHC Ia complex on the surface of APCs. The diversity of αβ TCRs that recognize a particular pMHC Ia complex is the defining property of most conventional CD8+ T cell responses. In the present study, we described a unique population of CD8+ T cells that express semi-invariant TCRs to monitor ERAAP deficiency by recognizing a conserved peptide presented by Qa-1b, a nonclassical MHC Ib molecule. These TCR and ligand characteristics together with their high frequency and Ag-experienced phenotype in naive mice make the QFL-specific CD8+ T cells uniquely different from conventional pMHC Ia–restricted CD8+ T cells but akin to innate-like T cells.

We analyzed the αβ TCRs expressed by the BEko8Z hybridoma that was originally generated by fusing anti-ERAAP–specific CD8+ T cells with a fusion partner lacking an endogenous αβ TCR (21, 42). This lacZ inducible hybridoma was used earlier to identify the QFL ligand that was induced by ERAAP deficiency. The QFL was the immunodominant ligand that elicited CD8+ T cells in wild-type mice by ERAAP-deficient cells. Remarkably, the TCR α- and β-chains from this hybridoma were also commonly expressed by other QFL-specific CD8+ T cells in naive wild-type mice and by virtually all QFL-specific CD8+ T cells in TCR β-chain transgenic mice. The Vα segment TRAV9d-3 rearranged to the TRAJ21 Jα segment was therefore the most prevalent among all TCR α-chains expressed by QFL-specific T cells. Likewise, the TRBV5-encoded Vβ segment dominated the TCR β-chains, but other TRBV12-1/12-2 rearrangements were also used by a substantial fraction of T cells. Collectively, the results show that the QFL-specific T cells express a nearly invariant TCR α-chain and a dominant but not exclusive TCR β-chain. Thus, the QFL-specific T cells contain a major subset of invariant αβ TCRs as well as a minor more diverse set of αβ TCRs. Both TCRs recognize the same QFL ligand, but whether the receptor contacts with the ligand similarly is not yet known.

Interesting parallels emerge when the QFL-specific αβ TCRs are compared with other TCRs that are also restricted by different nonclassical MHC Ib molecules. The iNKT and MAIT cells also bear semi-invariant receptors with invariant TCR α-chains and a limited set of TCR β-chains (38, 43). These TCRs recognize MHC Ib molecules CD1d or MR1 presenting glycolipids or vitamin B metabolites, respectively (23, 44, 45). Structural analysis has revealed that the orientation of type I iNKT TCRs positioned on CD1d-Ag surface is distinct from the conventional diagonal TCR-binding mode for classical pMHC I–TCR recognition (46). Because of the positioning of iNKT TCRs, the invariant TCR α-chain predominantly contacts the CD1d ligand and determines ligand recognition with variable TCR β-chains. The interaction between the invariant TCR α-chain and the ligand is mainly mediated by the CDR3 region, with emphasis on the Jα segment (4749). Alternatively, the MAIT TCRs, although positioned in the classical diagonal TCR-binding mode, contact the MR1 ligands mainly through the invariant TCR α-chain (50, 51). The structure of the αβ TCR complexed with its QFL ligand is not yet known. Nevertheless, we found that the CDR3 regions of both the TCR α- and β-chains were essential for QFL binding. Furthermore, the Jα segment was important for ligand specificity because amino acid substitutions in this segment resulted in complete loss of ligand recognition. Thus, the QFL-specific TCRs appear structurally related to the iNKT TCRs.

The rearrangement of particular TCR α segments has revealed an unusual developmental pathway for iNKT and MAIT cells (38, 52). In both lineages the canonical TCR α-chain uses a distal Vα (TRAV11 or TRAV1) and a proximal Jα (TRAJ18 or TRAJ33) segment relative to the constant α gene segment. This configuration of Vα-Jα gene segments suggested that the invariant TCR α-chains could arise by secondary TCR α rearrangements in immature T cells (53). Indeed, development of the iNKT and MAIT cell lineages depends on the master transcriptional regulators (HEB and RORγt) that prolong the time window available for continuing TCR α rearrangements by inducing expression of the antiapoptotic factors such as Bcl-xL (5456). Remarkably, the invariant TCR α-chain in QFL-specific T cells is also generated by rearrangement of another distal TRAV9d-3 Vα segment to the proximal TRAJ21 Jα segment. Thus, it is likely that the TCR α-chains in QFL-specific T cells would also occur late in development. Furthermore, it is well established that iNKT, MAIT, as well as CD8+ T cells restricted by H2-M3 MHC Ib molecules are positively selected by recognizing CD1d, MR-1, or H2-M3 expressed by double-positive cells in the thymus and mature into cells that can rapidly secrete cytokines when stimulated in the periphery (54, 55, 57). Whether the QFL-specific T cells develop in a similar manner or function as cytokine producers in discrete anatomical locations is unknown.

In addition to the semi-invariant TCRs, the QFL-specific T cells are similar in some but not all characteristics of the iNKT and MAIT cells. We found that a large fraction of the QFL-specific T cells was CD44hiCD122hi and expressed higher levels of CD25 and CD127 markers, which indicates that these cells are readily activated and Ag experienced. Interestingly, unlike the innate-like NKT cell subset that is comprised of cell populations that express each of the three master regulators T-bet, PLZF, or RORγt, the CD44hi QFL-specific T cells expressed T-bet+ but essentially lacked expression of PLZF or RORγt. Taken together with our previously reported observation that these cells produce IFN-γ and are cytotoxic toward ERAAP-deficient target cells, we speculate that the QFL-specific T cells are likely innate-like cytotoxic CD8+ T cells. Despite the clear functional differences in cytokine secretion profiles versus cytotoxicity, the iNKT, MAIT, and QFL-specific T cells may nevertheless share similarities in their developmental pathways.

In conclusion, we have identified a novel CD8+ T cell population whose specificity is restricted by the nonclassical Qa-1 MHC Ib molecule. These cells express semi-invariant αβ TCRs similar to those expressed by the iNKT and MAIT cells restricted by other nonclassical CD1d and MR1 MHC Ib molecules. Note that the nonpolymorphic MHC Ib molecules predate the classical, highly polymorphic MHC Ia molecules in evolution and bridge the innate and adaptive immune systems (58). Thus, unraveling the developmental pathway and functional properties of QFL-specific T cells in immune surveillance of ERAAP deficiency should be interesting.

We thank the late Chulho Kang, Hector Nolla, and Alma Valeros from the Cancer Research Laboratory for help in generating the transgenic mice and help with flow cytometry.

This work was supported by grants from the National Institute of Allergy and Infectious Diseases and the National Institutes of Health (to N.S.). J.G. was supported by a grant from the Chinese Scholarship Council.

The sequences presented in this article have been submitted to GenBank (https://www.ncbi.nlm.nih.gov/genbank/) under accession numbers KY271957 and KY271958.

The online version of this article contains supplemental material.

Abbreviations used in this article:

ER

endoplasmic reticulum

ERAAP

ER aminopeptidase associated with Ag processing

iNKT

invariant NKT

MAIT

mucosal-associated invariant T

MHC I

MHC class I

PLZF

promyelocytic leukemia zinc finger

pMHC I

peptide–MHC class I

ROR

retinoic acid–related orphan receptor

βTg mice

transgenic mice expressing the BEko.β-chain.

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The authors have no financial conflicts of interest.

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