The Journal of Immunology

Expression of the Mouse MHC Class Ib H2-T11 Gene Product, a Paralog of H2-T23 (Qa-1) with Shared Peptide- Binding Specificity

Lili Chen,* Eduardo Reyes-Vargas,* Hu Dai,* Hernando Escobar,† Brant Rudd,* Jared Fairbanks,* Alexander Ho,* Mathew F. Cusick,* Attila Kuma´novics,*,† Julio Delgado,*,† Xiao He,* and Peter E. Jensen*,†

The mouse MHC class Ib gene H2-T11 is 95% identical at the DNA level to H2-T23, which encodes Qa-1, one of the most studied MHC class Ib molecules. H2-T11 mRNA was observed to be expressed widely in tissues of C57BL/6 mice, with the highest levels in thymus. To circumvent the availability of a specific mAb, cells were transduced with cDNA encoding T11 with a substituted a3 domain. Hybrid T11D3 protein was expressed at high levels similar to control T23D3 molecules on the surface of both TAP+ and TAP2 cells. Soluble T11D3 was generated by folding in vitro with Qa-1 determinant modifier, the dominant peptide presented by Qa-1. The circular dichroism spectrum of this protein was similar to that of other MHC class I molecules, and it was observed to bind labeled Qa-1 determinant modifier peptide with rapid kinetics. By contrast to the Qa-1 control, T11 tetramers did not react with cells expressing CD94/NKG2A, supporting the conclusion that T11 cannot replace Qa-1 as a ligand for NK cell inhibitory receptors. T11 also failed to substitute for Qa-1 in the presentation of insulin to a Qa-1–restricted T cell hybridoma. Despite divergent function, T11 was observed to share peptide-loading specificity with Qa-1. Direct analysis by tandem mass spectrometry of peptides eluted from T11D3 and T23D3 isolated from Hela cells demonstrated a diversity of peptides with a clear motif that was shared between the two molecules. Thus, T11 is a paralog of T23 encoding an MHC class Ib molecule that shares peptide-binding specificity with Qa-1 but differs in function. The Journal of Immunology, 2014, 193: 1427–1439.

ajor histocompatibility complex class Ia molecules, in- H2-Q, -T, and -M (2, 5). There are ∼40 MHC class Ib genes present cludingHLA-A,-B,and-CinhumanandH2-K,-D,and in the C57BL/6 (B6) mouse genome, and only about half of them M -L in mice, are expressed on the surface of most nucleated were reported to be transcribed (3). A number of MHC class Ib cell and present mainly endogenously derived antigenic peptides to molecules have been studied, and some have been shown to have CD8+ T cells, initiating signals required for the positive selection in the specialized function, but the majority remain to be characterized. by guest on September 30, 2021. Copyright 2014 Pageant Media Ltd. thymus and activation in the periphery. The assembly and cell-surface For example, H2-M3 preferentially binds N-formylated peptides expression of MHC class I H chain with peptide and b2-microglobulin that originate from bacterial or mitochondrial proteins, and it is (b2m) is dependent on TAP and other components of the endo- not detectable on the cell surface until N-formylated peptide is plasmic reticulum (ER)–localized peptide-loading complex (1). bound (6, 7). H2-M3 has been reported to actively participate in Another group of MHC class I molecules, known as nonclassical anti–Listeria monocytogenes immune responses (8–12). By con- or MHC class Ib, is encoded by genes mostly located at the trast, TL (encoded by T18d) assembles without bound peptide (13), telomeric end of the MHC gene region (2). Class Ib molecules have and it serves as a ligand for CD8aa, regulating the function of a similar structure, at both genomic and protein levels, to class Ia asubsetofCD8aa+ intestinal intraepithelial T cells (14, 15). molecules, but class Ib molecules generally have more limited H2-T23 encodes one of the most well-studied MHC class Ib https://www.jimmunol.org tissue distribution, lower expression levels, and fewer alleles in proteins, Qa-1 (16). The T23 gene is ubiquitously transcribed (3), comparison with MHC class Ia molecules (2–4). The mouse MHC but the surface expression level of Qa-1 is lower than that of the class Ib genomic region is further divided to three subregions, MHC class Ia molecules. There are a number of identified alleles, but most laboratory mouse strains express Qa-1b or Qa-1a, and other alleles are closely related to these prototypes (17–19). Un- *Department of Pathology, University of Utah, Salt Lake City, UT 84112; and †ARUP Laboratories, Salt Lake City, UT 84112 fortunately, the genes encoding Qa-1 are not mapped in strains Downloaded from other than C57BL/6 and BALB/c; therefore, we do not know if Received for publication August 2, 2013. Accepted for publication May 29, 2014. they are allelic. Some of these Qa-1 molecules might be encoded This work was supported by National Institutes of Health Research Grants AI30554 and AI33614. by paralogous genes derived from a strain-specific gene duplica- Address correspondence and reprint requests to Dr. Xiao He and Dr. Peter E. Jensen, tion of the T23-like ancestral gene. Qa-1 appears to have a highly Department of Pathology, University of Utah, Emma Eccles Jones Medical Research selective peptide-binding specificity, predominantly loading with Building, 15 North Medical Drive East, Salt Lake City, UT 84112-5650. E-mail Qa-1 determinant modifier (Qdm; AMAPRTLLL), a peptide de- addresses: [email protected] (X.H.) and [email protected] (P.E.J.) rived from the conserved leader sequence of H-2D and H-2L class Abbreviations used in this article: CD, circular dichroism; DC, dendritic cell; DP, double-positive; EGFP, enhanced GFP; ER, endoplasmic reticulum; FP, fluorescence Ia molecules (20, 21). Despite its origin in leader sequences, polarization; LC-MS/MS, liquid chromatography-tandem mass spectrometry; b2m, loading of Qdm is dependent on TAP, as well as tapasin and b2-microglobulin; NP-40, Nonidet P-40; POLR2A, polymerase 2A; Qdm, Qa-1 presumably other component of the class I peptide-loading com- determinant modifier; qPCR, quantitative PCR; SP, single-positive. plex (4, 22). The fragment of the leader sequence that contains Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 Qdm is released into the cytoplasm after cleavage by signal

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302048 1428 A Qa-1 PARALOG ENCODED BY H2-T11

peptidase and signal peptide peptidase, thus requiring TAP for AGCACCTCAGGGTGACTTCAT-39. PCR was performed using TaKaRa rTaq transport into the ER lumen. Qa-1–Qdm complexes function as polymerase from Clontech. the sole ligand for CD94/NKG2 inhibitory and activating recep- Murine RNA polymerase 2A (POLR2A) was used as the reference gene for quantitative PCR (qPCR) analysis of T11. The primers for tors on NK cells and recognition by CD94/NKG2 is highly spe- qPCR were: T11 forward, 59-TAAACCTGAGGACCCTGCTC-39;T11 cific for the sequence of bound Qdm peptide (23, 24). The reverse, 59-TAGGCCTCCTGACAATACCC-39; POLR2A forward, 59- expression of Qa-1–Qdm serves as a quality-control system, such GACAAAACTGGCTCCTCTGC-39; and POLR2A reverse, 59-GCTT- that cells lacking components of the peptide loading machinery GCCCTCTACATTCTGC-39. The mouse tissues were collected and stored + in RNAlater solution (Ambion) for less than a week at 4˚C before RNA required for generation of Qa-1–Qdm are killed by CD94/NKG2A was extracted. The total RNA was extracted using the RNeasy mini kit NK cells (25). (Qiagen). The cDNA was synthesized using QuantiTect Reverse Tran- Although Qdm is the dominant peptide presented by Qa-1 scription kit (Qiagen). The qPCR kit Absolute QPCR SYBR Green Mix molecules, it is evident that Qa-1 has a capacity to present other (Thermo Scientific) was used in the qPCR analysis, which was performed peptides to CD8+ T cells. Qa-1–specific T cells have been reported on a Lightcycler 480 system (Roche). to participate in immune responses to L. monocytogenes (9, 26) Generation of hybrid H2-T11 and H2-T23 molecules and Salmonella tryphimurium (27, 28), and Qa-1–restricted T cells The a3 domain of H2-T11 or H2-T23 cDNA was replaced with the a3 with specificity for proinsulin (29) and insulin (30, 31) have been domain of the H2-Db cDNA, and the hybrid molecules were named as characterized. A number of studies have reported a role for Qa-1– H2-T11D3 or H2-T23D3, respectively. The H2-T11D3 and H2-T23D3 restricted CD8+ T cells in regulating immune responses and self- cDNAs were synthesized at Biomatik. The synthesized cDNAs were verified tolerance (32–35) and in immune surveillance of TAP-deficient by sequencing before being cloned into expression vectors. The cDNAs were tumors (36, 37). Recently, Nagarajan et al. (38) have demon- cloned into a retroviral vector MigR1 for expression in mammalian cells b (45). The soluble forms (lack of the transmembrane and cytoplasmic strated a role for Qa-1 –restricted T cells in monitoring the func- domains) of H2-T11D3 or H2-T23D3 were generated by PCR and cloned tion of ERAAP, an aminopeptidase that mediates trimming of into a bacteria expression vector pTCF (the NIH Tetramer Core Facility), peptides presented by MHC class I molecules in the ER. Cytotoxic and the cloned products were verified by restriction enzyme digestion effector cells were shown to recognize a self-peptide (FL9) that is and sequencing. All restriction enzymes were from New England Biolabs. The plasmids were purified using the Plasmid Mini Kit (Qiagen) for di- selectively presented by Qa-1 in ERAAP-deficient cells. gestion and cloning and the EndoFree Plasmid Maxi Kit (Qiagen) for The MHC is shaped by successive rounds of segmental dupli- transfection. cations. The mouse H2-T region, where Qa-1 is encoded, contains ∼20 class I genes. This number varies greatly among haplotypes Abs, flow cytometry, and cell sorting due to strain-specific deletions/duplications. The H2-T region of Purified anti–Qa-1b (6A8, mouse IgG1, k), anti-Db a3 domain (28-14-8s, C57BL/6 and BALB/c contains two and A/J mice contain three mouse IgG2a, k), and anti-human b2m (BB7.7) Abs were purified from highly similar segments (39–42). These duplicated segments were hybridoma supernatants using protein A affinity chromatography. FITC- labeled anti-mouse CD3ε (145-2C11), PerCP-Cy5.5–labeled anti-mouse further modified by monogenic duplications, deletions, and single B220 (RA3-6B2), PE-labeled anti-mouse NKp46 (29A1.4), and PE- nucleotide changes, leading to strain-specific class I gene/pseudogene Cy7–labeled anti-mouse NK1.1 (PK136) Abs were purchased from content. This process led to variable numbers of T23/T11-, T22/ eBioscience or BioLegend. The Ab was diluted in a buffer composed of T10-, T25-, and T18/T3-like paralogous genes, pseudogenes, and PBS, 0.5% BSA, and 2 mM EDTA. The suspended cells were incubated gene fragments (42). For example, the TL Ag (43), which is ex- with the Abs and/or the tetramer for 20 min at 4˚C. The stained cells were washed twice with the above buffer and fixed with 1% paraformaldehyde. by guest on September 30, 2021. Copyright 2014 Pageant Media Ltd. pressed on intestinal epithelium and thymocytes, can be encoded The fluorescence was detected on an FACSCanto II (BD Biosciences). The by one (H2-T3), two (H2-T3 and -T18), or three genes (in A/J), data were analyzed by FlowJo (Tree Star). allophycocyanin-labeled TCRb depending on the strain. Qa-1b isencodedinBALB/cbytheH2-T23d (H57-597), PE- or allophycocyanin-labeled anti-mouse B220 (RA3-6B2), gene (originally gene D37), whereas H2-T11d (originally named FITC- or PerCP-Cy5.5–labeled anti-mouse CD4 (GK1.5), FITC- or PE- labeled anti-mouse CD8a (53-6.7), FITC-labeled anti-mouse CD11c T10c) from BALB/c is a pseudogene due to a one-base deletion (HL3), and PE-labeled anti-mouse NK1.1-PE (PK136) were purchased leading to a frame-shift and early stop in exon 2 (44). In this study, from BioLegend, eBioscience, or BD Biosciences and used for sorting of we show that in C57BL/6, both H2-T23b and H2-T11b are func- CD42CD82 double-negative, CD4+CD8+ double-positive (DP), CD4+ + tional genes. single-positive (SP), CD8 SP, B cells, and dendritic cell (DC) subpopu- lations from thymus as well as T cells, CD4+ T cells, CD8+ T cells, B cells,

https://www.jimmunol.org DC, NK, and NKT subpopulations from spleen. The cell sorting was Materials and Methods performed using a BD FACSAria III (BD Biosciences). Mice Cell culture b2/2 C57BL/6 mice (B6) were purchased from The Jackson Laboratory. Qa-1 Phoenix-GP, Hela, and T2 cells were cultured in DMEM complete media mice were a gift from Dr. Harvey Cantor (Harvard Medical School) (35). supplemented with 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, All of the mice were maintained in the University of Utah specific 292 mg/ml L-glutamine, 100 mM nonessential amino acids, 1 mM sodium pathogen-free animal facility and used according to the protocol approved

Downloaded from pyruvate, and 55 mM 2-ME (all from Invitrogen). Splenocytes were cul- by the Institutional Animal Care and Use Committee of University of tured in RPMI 1640 complete media supplemented the same way as the Utah. DMEM complete media. To stimulate splenocytes, cells were cultured Molecular cloning of the B6 genomic locus of H2-T11 with plate-bound anti-CD3 (145-2C11) and anti-CD28 (37.57.1) Abs in RPMI 1640 complete media. All cells were maintained at 37˚C in a hu- Based on the genomic DNA sequence of the H2-T11 locus from 129 mice midified cell-culture incubator containing 5% CO2. (A. Kuma´novics, unpublished observations), specific oligonucleotide pri- mers were designed and synthesized from Integrated DNA Technologies. Retroviral transduction The whole region of the H2-T11 locus region from B6 mice was cloned, Phoenix-GP cells were added at 4 3 106 cells/dish to 6-cm collagen using TA cloning kits (Invitrogen), and subsequently sequenced. All DNA I–coated cell-culture dishes (BD Biosciences). Eighteen hours later, 10 mg products were examined using 1 or 2% agarose gel. MigR1 empty vector or MigR1 containing the T11D3 or T23D3 cDNA Gene expression analysis was cotransfected with 5 mg Env plasmid into the Phoenix cells by calcium-phosphate precipitation as previously reported (46). Two days The primers used for amplifying H2-T11 and H2-T23 from the B6 mouse later, the supernatant containing the packaged retrovirus was harvested genome and total RNA were as follows: T11 forward, 59-CGGTATTTCCA- and filtered through 0.45-mm sterile filters (BD Biosciences) before CACCGTCGTA-39;T11reverse,59-TAGAGATATGCGAGGCTAAGTTG-39; transduction. Half of a milliliter of 1 3 106/ml Hela or T2 cells was T23 forward, 59-AGTATTGGGAGCGGGAGACTT-39;andT23reverse,59- mixed with 0.5 ml retroviral supernatant in the presence of 5 mg/ml The Journal of Immunology 1429

polybrene (Sigma-Aldrich), and the cells were distributed evenly on Circular dichroism assay collagen I–coated 6-cm dishes. Four hours later, 4 ml fresh DMEM complete media was added. After at least 3 d, the cells were harvested, The circular dichroism (CD) spectrum was measured by an AVIV 410 CD and the enhanced GFP (EGFP) expression was examined by flow instrument (AVIV Biomedical). The folded MHC monomer concentration cytometry. The transduced cells were sorted at least twice according to was determined by OD280 and diluted to 250 mg/ml in PBS. The far-UV the EGFP expression level for cells stably expressing high levels of CD spectrum was recorded in a cuvette with 1-mm path length at 25˚C. target genes. The sample was scanned from 200–260 nm with a step of 1 nm. The averaging time was 3 s, and every step was scanned three times. The PBS Expression of soluble recombinant proteins background data were recorded in the same cuvette and subtracted from the sample data. The pTCF vector containing the T11D3 or T23D3 genes was transformed The thermal stability of the folded MHC was measured by detecting the into BL21 (DE3) Escherichia coli (Invitrogen). T11D3- or T23D3-positive CD signal at 222 nm. The temperature was increased by a step of 2˚C from clones were verified for producing recombinant proteins before they were 25 to 79˚C. At each temperature point, the sample was equilibrated for 30 s expanded in 2 l Luria broth media. Isopropyl b-D-thiogalactoside (1 mM) before collecting the data. The averaging time was 30 s. was added to the culture when the OD600 reached ∼0.6. The bacteria were harvested 4 h later. The bacteria were pelleted by centrifugation, resus- MHC class I tetramer preparation pended in resuspension buffer (50 mM [pH 8] Tris-HCl, 25% [w/v] su- crose, 1 mM EDTA, 0.1% [w/v] Na azide, and 10 mM DTT), and stored at T11D3 and T23D3 MHC tetramers were prepared as described before (49). 280˚C. The frozen bacteria were thawed, and 1 mg/ml lysozyme, 5 mM The S300 purified MHC monomer was buffer exchanged to 10 mM Tris MgCl ,33mg/ml DNase I, 3.3% (v/v) Triton X-100, and 10 mM DTT were buffer (pH 8) and concentrated to 2 mg/ml. One milligram MHC monomer 2 3 added. The bacteria were stirred and lysed at room temperature for 1 h (8/10volumes)wasmixedwith1/10volumeof10 BiomixA, 1/10 3 before sonication. The inclusion body was washed multiple times with volume of 10 BiomixB, and 5 mg BirA enzyme (GeneCopoeia). The wash buffer (50 mM [pH 8] Tris-HCl, 0.5% Triton X-100, 100 mM NaCl, reaction was kept at 25˚C overnight. The product was further purified by 1 mM EDTA, 0.1% Na azide, and 1 mM DTT) until the protein pellet was a MonoQ anion exchange column (GE Healthcare). The biotinylated MHC 2 white and the supernatant was clear. The pellet was washed one more time was snap-frozen and stored at 80˚C. When tetramer was generated, the with the same wash buffer without DTT. The recombinant inclusion body biotinylated MHC monomer was thawed and tetramerized by gradually was solubilized in 6 M guanidine chloride, analyzed using 12% SDS- adding APC-labeled streptavidin to it. The final molar ratio of bio- PAGE, aliquoted, and stored at 280˚C until use. tinylated MHC to streptavidin was 4:1. One tenth of the total required allophycocyanin-labeled streptavidin was added to the sample each time, In vitro MHC class I folding and the sample was incubated in dark at room temperature for 10 min after each addition, so that each streptavidin was saturated. The tetramers were Soluble recombinant T11D3 and T23D3 proteins were folded in vitro as stored at 4˚C and used within 6 mo. described before (25). Briefly, 18 mg inclusion body of T11D3 or T23D3 H chain together with 6 mg human b2m L chain was diluted in 100 mM Tris Peptide elution and identification folding buffer (pH 8) containing 400 mM L-arginine, 2 mM EDTA, 0.5 mM oxidized glutathione, 5 mM reduced glutathione, and 0.2 mM PMSF in The T11D3 and T23D3 bound peptides were eluted and identified using the absence or presence of Qdm peptides. The gram ratio of H chain/L liquid chromatography-tandem mass spectrometry (LC-MS/MS) as de- chain/peptide was 3:1:1. The folding reaction was performed at 10˚C for scribed (50). Briefly, Hela-MigR1, Hela-T11D3, and Hela-T23D3 cells 2 d before it was harvested and concentrated by Amicon ultrafiltration cells were cultured in 20–25 T150 cell-culture flasks, and the cells were har- ∼ (Millipore). The concentrated sample was filtered through a 0.45-mm filter vested at 80–90% confluent. The total cell numbers collected for each . 3 9 and purified by an S300 gel filtration column. The purified folded products cell type were 1 10 . The cells were pelleted, washed twice with cold 2 were concentrated by Amicon Ultra centrifugal filters (Millipore), buffer Dulbecco’s PBS, and stored at 80˚C until lysing within 2 mo. The cells exchanged with PBS, and stored at 280˚C. The MHC folding products were lysed in NP-40 buffer (0.5% NP-40, 500 mM Tris-HCl [pH 8], 150 were analyzed using 4–20% gradient Tris–HCl PAGE (Bio-Rad). mM NaCl, and protease inhibitors). The lysate was centrifuged, and cell by guest on September 30, 2021. Copyright 2014 Pageant Media Ltd. debris was removed. The supernatant was passed through a Tris-blocked Ag presentation assay Sepharose column to preclear the lysate. The hybrid MHC was immuno- precipitated by 28-14-8s cross-linked on Protein A beads. The beads then 4 6 Transduced Hela cells (5 3 10 ) or B6 splenocytes (1 3 10 ) were used as were washed with four different buffers sequentially: 1) 0.005% NP-40, 50 5 APCs and cocultured with 1 3 10 6C5 T cell hybridoma cells (31) mM Tris-HCl (pH 8), 150 mM NaCl, and 5 mM EDTA; 2) 50 mM Tris- overnight in the presence of different doses of bovine insulin (Sigma- HCl (pH 8) and 150 mM NaCl; 3) 50 mM Tris-HCl (pH 8) and 450 mM Aldrich) in 96-well plates. The supernatant was harvested, and the pro- NaCl; and 4) 50 mM Tris-HCl (pH 8). The MHC–peptide complexes were duction of IL-2 was measured by Eu-based immunoassay (47). eluted from the beads by 10% acetic acid, lyophilized, resuspended in 100 ml DMSO, loaded, and fractionated by HPLC (Beckman Coulter) into Eu-based peptide binding assay 27 fractions. Each fraction was concentrated to ∼10 mlbeforemass spectrometry. The sequences of the eluted peptides were gained by https://www.jimmunol.org The peptide binding capacity of folded recombinant T11D3 or T23D3 was examined using an Eu-based immunoassay (47). Briefly, a 96-well ELISA LC-MS/MS. plate was coated with 50 ml5mg/ml anti-human b2m mAb (clone BB7.7) Mass spectrometry analysis of peptide eluted from T11D3 or T23D3 was at 37˚C for 2 h; the plate was blocked by 200 ml MTB (5% powdered skim performed as previously reported. Briefly, 1 to 2 ml each HPLC fraction was milk, 1% BSA, and 0.01% Na azide in TTBS buffer [50 mM Tris, 150 mM analyzed by MS/MS (Agilent 6510 quadrupole time-of-flight instrument NaCl, and 0.1% Tween-20] [pH 7.5]) for 30 min at room temperature; 1 mg with Chip Cube electrospray ionization; Agilent Technologies). The m samples were injected using nanospray protein chip number 1 (40-nl trap, folded MHC monomers was diluted in 100 l MTBN and incubated in the 3 Ab-coated plate for .2 h at 4˚C; the biotin-labeled peptides were diluted 75 43 mm, C-18SB-ZX chip, 5-mm particles) at a flow rate of 400 nl/min. Downloaded from in PBS with 0.01% Nonidet P-40 (NP-40) and incubated the plate over- Data acquisition was done using MassHunter (version B.01.03) in a night at room temperature. The plate was washed extensively using Tween 2-GHz extended dynamic range at a rate of three scans per second followed 20 and Tris-buffered saline before the addition of the next reagent. Finally, by data-dependent MS/MS fragment scans of the three most intense ions. the plate was developed using Europium reagent, and the fluorescence Precursor ion exclusion was set for 12 s after two consecutive MS/MS signal was recorded by a Victor3V plate reader (PerkinElmer). scans. Before each experiment, the quadrupole time-of-flight analyzer was tuned to a resolution of .12,000, and mass accuracy was calibrated to ,2 Fluorescence polarization assay ppm. Acquired MS/MS spectra were searched with no enzyme specificity using Spectrum Mill (Agilent Technologies) against the UniProt human The folded MHC monomers were incubated with Alexa Fluor 488–labeled FASTA protein database (August 2007 download). Raw peptide data files Qdm-4C (ANACRTLLL) peptides in citrate/phosphate buffer (200 mM generated were converted into Excel format (Microsoft) and sorted citric acid and 200 mM Na2HPO4 [pH 7]). The parallel and perpendicular according to their corresponding mass-to-charge ratio values, charge state, fluorescence signals (III and I’) were recorded at 60-s intervals for a total retention time, and intensity (50, 51). A user-defined intensity threshold of 60,000 s by an Infinite F200 microplate reader (Tecan) at 37 or 25˚C. (7.0) above the background noise was fixed to limit false-positive identi- Pure MHC monomer, Alexa Fluor 488–labeled peptide, or buffer alone fication. All identified peptides sequences above this score were manually was also detected independently to record the background signals. After verified (51). In addition, peptides found in the fractions from control the background signals were subtracted, the anisotropy was calculated Hela-MigR1 lysates were considered contaminants and subtracted from the according to the following formula: A ¼ III 2 I’ (48). IIIþ2I’ final list of peptides. 1430 A Qa-1 PARALOG ENCODED BY H2-T11

Results Furthermore, CD4-2CD82 double-negative, CD4+CD8+ DP, Transcription of T11 gene in B6 and T23/Qa-1b knockout mice CD4+ SP, CD8+ SP, B cells, and DC subpopulations as well as T cells, CD4+ T cells, CD8+ T cells, B cells, DC, NK, and NKT H2-T11 and H2-T23 genes appear to have been duplicated from 2 2 subpopulations were sorted from thymus or spleen of Qa-1b / a common ancestor gene in evolution (42). Because the genomic mice, respectively. Expression levels of H2-T11 of those sub- sequence of the T11 from B6 mice is not available from the Na- populations were compared by qPCR and shown in Fig. 2D tional Center for Biotechnology Information database, we cloned (thymus, top panel, and spleen, bottom panel). Expression levels and sequenced the T11 genomic region from B6 mice using the are similar across all tested cell subpopulations with the exception H2-T11 locus genomic DNA sequence from 129 mice as the tem- of DP thymocytes, which express relatively high levels of T11 plate (A. Kuma´novics, unpublished observations). The H2-T11 and mRNA. Upon activation by TCR engagement with CD3 and CD28 H2-T23 cDNA sequences showed a high degree of homology with Ab, surface Qa-1 expression on splenocytes of B6 mice was in- a 95% identity (data not shown). At the amino acid level, the T11 creased, as reported previously. Similarly, using qPCR, T23 putative protein is 91% identical to H2-T23 (Qa-1b)(Fig.1). transcripts were also increased after activation. T11 transcripts PCR analysis was performed using oligonucleotide primers 2 2 from Qa-1b / splenocytes were increased by activation as well, specific for T11 or T23 gene, respectively, as reported previously but with delayed kinetics, peaking on days 3 and 4 (Fig. 2E). (42). Genomic DNA analysis showed both T11 (628-bp band) and T23 (677bp band) genes were detected in wild-type B6 mice, Expression of H2-T11 encoded protein on the surface of +/+ 2/2 whereas only T11 but not T23 was detected in T23/Qa-1b TAP and TAP cells 2 2 knockout (Qa-1b / ) mice, because the amplified region (exons Our PCR and sequencing results indicated that T11 might encode 2 2 1–3 of T23) is deleted in Qa-1b / mice (35) (Fig. 2A). RT-PCR a functional protein. We generated cDNA expression constructs analysis showed that the T11 gene was transcribed, as represented encoding a chimeric T11 protein substituted with the a3 domain as a 415-bp band in the spleen and thymus. The T23 transcript (a of H2-Db to circumvent the absence of an mAb that recognizes 438-bp band) was also detected in the spleen and thymus as ex- T11 (37). The H2-Db a3 domain is recognized by mAb 28-14-8s. pected (Fig. 2B). All of the amplified PCR products were con- Chimeric Qa-1 molecules were generated as a control, and these firmed by sequencing, and the different sizes of the PCR products hybrid molecules were designated H2-T11D3 and H2-T23D3, from genomic DNA and cDNA reflected correctly spliced prod- respectively. The hybrid T11D3 and T23D3 were transduced in- ucts. Therefore, T11 is not a pseudogene in B6 mice and has the to Hela (TAP+) and T2 (TAP-deficient) cells using the MigR1 potential to encode a functional protein. retroviral transduction system, which has an EGFP reporter gene Next, qPCR analysis was performed to compare the mRNA following an internal ribosome entry site (45). expression levels of T11 in multiple tissues. Because of a high High levels of T23D3 were detected on the surface of Hela cells similarity of T23 and T11, we were not able to design T11-specific with mAb 28-14-8s demonstrating that the chimeric Qa-H1 chain primers to selectively amplify the short amplicon needed in qPCR. efficiently assembles with endogenous human b2m to form a sta- A pair of primers flanking T11 exons 2 and 3 was used in qPCR to ble complex (Fig. 3A, HeLa-T23D3 panel). As previously re- produce a 127-bp amplicon, but they also cross-amplified a product ported for wild-type Qa-1 (23, 25, 53, 54), cell-surface T23D3 can 2 2 from the T23 gene. To circumvent this problem, we used Qa-1b / also be expressed at high levels in TAP-deficient cells (Fig. 3A,

by guest on September 30, 2021. Copyright 2014 Pageant Media Ltd. mice to evaluate the T11 expression levels. Murine RNA POLR2A T2-T23D3 panels). Presentation of the dominant Qa-1–associated was used as the reference gene (52). The qPCR result showed that peptide Qdm is strictly TAP dependent (20, 53). However, Qa-1 T11 was expressed at relatively high level in spleen, thymus, and has been reported to assemble with alternative non-Qdm peptides intestine and at lower levels in kidney, heart, and pancreas. Among in TAP-deficient cells (37, 55). Cell-surface T23D3 was also eight different tissues examined, the highest T11 expression level detected with mAb 6A8, which recognizes an epitope in the a2 was detected in the thymus (Fig. 2C). domain of Qa-1b (Fig. 3A, T2-T23D3 panels). https://www.jimmunol.org

FIGURE 1. Alignment of amino b a Downloaded from acid sequences of Qa-1 , Qa-1 , and T11 ectodomains. The amino acid sequences of Qa-1b, Qa-1a, and T11 ectodomains were aligned, using CLUSTALW, SDSC Biology Work- Bench 3.2 (http://workbench.sdsc.edu). a 1, 2, and 3 domains are boxed. *, fully conserved; :, strongly con- served; ., weakly conserved. The Journal of Immunology 1431

FIGURE 2. Expression of T11 gene. (A) PCR amplification of T11 and T23 genes from the wild-type C57BL/6 (B6) and Qa-1b2/2 (knockout [KO])

by guest on September 30, 2021. Copyright 2014 Pageant Media Ltd. mouse genomic DNA. The T11 amplicon size was 628 bp, and the T23 amplicon size was 677 bp. (B) RT-PCR amplification of T11 and T23 transcripts from B6 mouse spleen and thymus. The amplicon sizes are 415 and 438 bp for T11 and T23, respectively. (C) qPCR to determine the T11 transcription level in different tissues. The total RNA was extracted from Qa-1b2/2 mice. The reference gene was RNA POLR2A. The ratio of T11 to POLR2A was calculated according to the Pfaffl method. The qPCR was done in triplicates (n = 3), and the experiment was repeated twice with similar results. One of them is shown. (D) qPCR to determine the T11 transcription level in different cell subpopulations of thymus and spleen. The total RNA was extracted from thymus and spleen of Qa-1b2/2 mice. The qPCR was done as above, at least in triplicates. (E) Qa-1/T23 and T11 expression after stimulation. Splenocytes of B6 or Qa-1b2/2 mice were stimulated with plate-bound anti-CD3/28 Ab. Surface expression of Qa-1 on B6 splenocytes was determined by FACS. The total RNA was extracted from unstimulated or stimulated B6 or Qa-1b2/2 splenocytes. The qPCR for T23 used RNA from B6 or for T11 used RNA from Qa-1b2/2 splenocytes, respectively. The qPCR was done as same as above, in triplicates (n = 3), and the experiment was repeated twice with similar results. One surface staining of Qa-1 is shown. DN, double-negative. https://www.jimmunol.org

The chimeric T11D3 protein was expressed at levels similar to insulin to 6C5 T cells, with even greater function than B6 T23D3 on the surface of both Hela and T2 cells, as determined by splenocytes (Fig. 3B). Thus, the substituted a3 domain does not mAb 28-14-8s staining (Fig. 3A, top panel). Significantly reduced affect the Ag presentation function of Qa-1 from these T cells. By staining was observed with the anti-Qa-1b mAb 6A8, suggesting contrast, no T cell response was observed in experiments with that the epitope recognized by this mAb is not fully preserved in Hela cells expressing similar levels of T11D3. We concluded that Downloaded from T11. These results suggest that T11 can efficiently assemble and T11 cannot substitute for Qa-1 in Ag presentation to insulin- be expressed as a stable cell-surface protein, with the caveat that specific 6C5 T cells. the a3 domain has been substituted in our experiments. Like Folding of soluble T11D3 with or without Qdm peptide in vitro T23D3, high levels of T11D3 were expressed in TAP-deficient T2 cells. It is possible that T11, like TL (T18d), assembles without The class Ia leader sequence-derived peptide Qdm has an optimal bound peptide Ag (13, 56). Alternatively, like Qa-1, T11 may sequence for binding to Qa-1 (24, 57), and it is the dominant assemble with peptides through a TAP-independent mechanism. peptide bound to Qa-1 molecules in TAP-expressing cells (21). As previously demonstrated for wild-type Qa-1b (23, 24), T23D3 H T11 cannot substitute for Qa-1 in T cell Ag presentation chain assembles efficiently under standard folding conditions Previous findings from our laboratory showed that a subset of CD8+ in vitro in the presence of Qdm peptide and b2m based on size- T cells with specificity for insulin is selected by Qa-1b in mice (30, exclusion chromatography (Fig. 4A). No folded protein was de- 31). The Qa-1b–restricted CD8+ T cell hybridoma 6C5 recognizes tected in the absence of Qdm, indicating that the in vitro folding an epitope in the B chain of insulin. Hela cells expressing the reaction is strictly dependent on the presence of an appropriate chimeric Qa-1 T23D3 molecules were able to efficiently present peptide ligand. Given the high degree of sequence similarity with 1432 A Qa-1 PARALOG ENCODED BY H2-T11

FIGURE 3. Expression of hybrid T11D3 and T23D3 molecules and test of the function of hybrid molecule– expressing cells as APCs. (A) FACS analysis of hybrid T11D3 and T23D3 on the surface of Hela and T2 cells. Transduced Hela cells were stained with 28-14-8s (a-Db a3), and T2 cells were stained with 28-14-8s and 6A8 (a-Qa-1b a2), respectively, shown as marked (red lines). The staining of isotype control Ab is blue. (B) Ag presentation assay to test capability of the hybrid MHC class Ib molecule–expressing cells to present insulin to 6C5 T hybridoma cells specific to bovine insulin (bIns). The assay was set up in triplicates (n =3), and the experiment was repeated twice with similar results. One of them is shown. spl, spleen.

by guest on September 30, 2021. Copyright 2014 Pageant Media Ltd. Qa-1, we set up folding reactions with T11D3 under identical increasing temperature, T11D3 displayed evidence of denatur- conditions. A large peak corresponding to folded MHC class I ation, as shown by an increased molar ellipticity, but the dena- molecules was observed with T11D3 in the presence of Qdm. In turation was not complete at 80˚C. Instead, the molar ellipticity contrast to T23D3, a peak corresponding to folded T11D3 protein stabilized at ∼50˚C, possibly reflecting entry into a relatively was also observed in the absence of Qdm peptide, although the stable misfolded conformation. yield was lower than that observed in the presence of Qdm. The appropriate peaks from size-exclusion chromatography were con- The capacity of chimeric T11 and T23 proteins to bind Qdm centrated and examined by SDS-PAGE. Both H and L chains of peptide MHC class I were detected, indicating the assembly of T11D3 The peptide binding capacity of folded T23D3 and T11D3 was https://www.jimmunol.org with b2m, even in the absence of Qdm (Fig. 4B). The products evaluated using biotin-labeled Qdm in Eu-streptavidin–based were also analyzed using an Eu-based fluorescence immunoassay immunoassays. Folded proteins were incubated for 18 h at room (Fig. 4C). In this assay, the folding product was captured with an temperature in microtiter wells coated with anti-b2m capture mAb anti-b2m mAb (BB7.7), washed, and detected by 28-14-8s (anti in the presence or absence of biotin–Qdm or the control Kb- Dba3) or 6A8 (anti Qa-1b) mAbs. The results showed that folded binding peptide biotin-SIINFEKL. After washing, bound peptide T11D3 and T23D3 products can be recognized by both 28-14-8s was detected with Eu-streptavidin. As shown in Fig. 5A, wild-type Downloaded from and 6A8 Abs, further confirming assembly with b2m. Qa-1b and T23D3 bind Qdm but not SIINFEKL through a peptide- To further characterize the structure of the folded T11D3 and exchange reaction. T11D3 that had been folded in the presence of T23D3 proteins, CD was used to analyze secondary structure. The Qdm was also able to bind biotin-Qdm. No binding activity, CD results showed that all three folded proteins—T11D3-b2m, however, was observed with T11D3 that was generated by folding T11D3-b2m-Qdm, and T23D3-b2m-Qdm—displayed wavelength in the absence of peptide. This suggests that the “empty” T11D3 spectrums (Fig. 4D) similar to those previously published for may assume a conformation (acquired before or during the binding MHC class I molecules (13, 58–60). The CD spectrums each assay) that is not receptive to peptide binding. showed a single maximum signal at ∼220 nm, which was in Peptide binding was further analyzed in fluorescence polariza- conformity with a b-sheet–dominated structure in the MHC H tion (FP) assays. Alexa Fluor 488–labeled Qdm peptide was in- chain. Thermal stability studies showed that folded T11D3 prod- cubated with the folded MHC proteins, and peptide binding as ucts had abnormal stability profiles (Fig. 4E). For most MHC class measured by fluorescence anisotropy was recorded in real time. I proteins, the molecule loses its regular secondary structure, Peptide binding to Qa-1–Qdm and T23D3-Qdm was rapid at 37˚C, forming random coils, as the temperature increases, such that the approaching saturation in ∼1 h (Fig. 5B, left panel). The kinetics molar ellipticity gradually approaches zero (58, 59, 61). With is consistent with an exchange reaction in which unlabeled Qdm is The Journal of Immunology 1433

FIGURE 4. In vitro folding of the hybrid T11D3 and T23D3 molecules. (A) S300 spectrum of the folding products. Arrows indicate the correct folding product peak. Top panel: black line, T23D3-b2m folding; gray line, T23D3-b2m-Qdm folding; bottom panel: black line, T23D3-b2m folding; gray line, T11D3-b2m-Qdm folding. (B) SDS-PAGE analysis of the purified folding products. The MHC H chain is ∼33 kD, and the b2m L chain is ∼14 kD. (C) Eu- based immunoassay to examine the folding products. Folding MHC monomer was captured by the anti-b2m mAb and detected by biotinylated 28-14-8s and 6A8 (b-28-14-8s and b-6A8). The assay was set up in triplicates (n = 3), and the experiment was repeated twice with similar results. One of them is shown. (D) In vitro folded MHC monomers were analyzed using far-UV CD. Each spectrum curve was the average of three independent scans, and

by guest on September 30, 2021. Copyright 2014 Pageant Media Ltd. a representative one is shown. (E) Thermal denaturation curves were generated from CD signals recorded at 222 nm, and the temperature was increased at 2˚C intervals from 25 to 79˚C. Each curve presented the average of three independent experiments.

replaced by the labeled peptide. Previous results have indicated expressed on a major fraction of NK cells, NKT cells, as well as + that Qdm dissociates from Qa-1 with a t1/2 of 40–100 min at 37˚C a subpopulation of CD8 T cells. CD94/NKG2 receptors display (25), a relatively rapid rate of dissociation despite having an op- a high degree of specificity for the sequence of Qa-1–bound timal sequence for binding to Qa-1 (24). The observed peptide peptide, specifically recognizing Qdm (24). Several Qa-1 alleles association rates probably reflect rate of dissociation of Qdm, have been identified, and they appear to share the capacity to bind a step necessary for binding of labeled peptide. https://www.jimmunol.org Qdm and serve as ligands for CD94/NKG2 receptors (19). Given The kinetics of binding of labeled Qdm to T11D3-Qdm were the similarity of T11 to Qa-1 and the capacity of T11D3 to bind highly unusual, displaying a rapid initial kinetics followed by Qdm peptide, we were interested in determining whether T11 a rapid decay in binding signal. This pattern suggests that T11D3- could serve as an alternative ligand for CD94/NKG2. Qdm complexes are unstable at 37˚C, rapidly undergoing dena- CD94/NKG2 ligand binding can be demonstrated by flow turation or conversion to a peptide unreceptive conformation. cytometry using tetramers generated from in vitro–folded Qa-1– Consistent with the results of Eu–streptavidin peptide binding

Downloaded from Qdm complexes to stain NK cells (23, 24). Similar to wild-type experiments, T11D3 that was folded in the absence of peptide Qa-1, tetramers generated with chimeric T23D3-Qdm molecules showed little or no peptide binding activity. This protein may were observed to stain a major fraction of CD3-NKp46+ NK cells initially bind some labeled Qdm, but it very rapidly assumes from the spleen and liver of B6 mice (Fig. 6). Thus, substitution of a peptide unreceptive state. Complexes formed with labeled the T23 a3 domain does not disrupt receptor recognition. A peptide and T11D3-Qdm were considerably more stable at 25˚C substantial fraction of CD3+ lymphocytes from liver was also relative to 37˚C, yet the unusual decay in signal was still observed + at this temperature (Fig. 5B, right panel). Overall, the results in- tetramer positive, reflecting the large number of CD94/NKG2 dicated that T11D3 can bind Qdm peptide, but T11D3-Qdm NKT cells present in liver. By contrast, no staining was observed complexes are highly unstable and subject to denaturation or with tetramers generated from T11D3-Qdm molecules. The T11D3 conversion to a peptide unreceptive conformation. molecules used to generate the tetramers were confirmed to contain b2m and Qdm, and they were appropriately biotinylated and tet- Evidence that T11 is not a ligand for CD94/NKG2 receptors ramerized with streptavidin (data not shown). Nevertheless, it is Qa-1 plays a key role in regulating NK cell activation as the ex- possible that a fraction of T11D3 tetramers had loss of function clusive ligand for CD94/NKG2 signaling receptors, which are from dissociation of Qdm. Despite this caveat, our results support 1434 A Qa-1 PARALOG ENCODED BY H2-T11

FIGURE 5. Qdm binding capability of T11D3. (A) Eu-based immunoassay to test the ability of folded T11 and T23 monomers of binding Qdm peptide. Folded MHC class Ib monomers were captured on plates by the coated the anti-b2m mAb and incubated with the biotin-labeled peptides at room tem- perature overnight. The assay was set up in triplicates (n = 3), and the experiment was repeated twice with similar results. One of them is shown. (B and C) FP assay. Folded MHC class Ib monomers were incubated with Alexa Fluor 488–labeled Qdm peptides, and the FP signal was recorded every 60 s at 37 or 25˚C, respectively. The experiments were repeated twice (37˚C) and three times (25˚C) with similar results. One of each is shown.

the conclusion that T11 cannot substitute for Qa-1 as an alter- peptide-derived sequences VMAPRTLIL and VMAPRTLVL from by guest on September 30, 2021. Copyright 2014 Pageant Media Ltd. native ligand for CD94/NKG2 receptors. In addition, no T11D3 HLA-C and HLA-A together represented ∼11% of the validated tetramer-positive lymphocytes were identified in lymphocytes peptide hits. These well-established ligands for HLA-E and CD94/ from thymus, inguinal lymph node, mesenteric lymph node, NKG2A in humans have previously been shown to bind Qa-1 with Peyer’s patches, or bone marrow (data not shown). affinities similar to Qdm. This result is consistent with the dom- inant presentation of Qdm (or related peptides) in TAP-expressing Analysis of peptides eluted from T11D3 and T23D3 expressed cells. However, it is noteworthy that a large variety of other in Hela cells peptides were also identified. Peptides ranged in length from 8–12 T11D3 (like T23D3) can be expressed at high levels on the surface aa, with a strong predominance of 9-mers (Fig. 7A). The T23D3- of TAP-deficient T2 cells, and some degree of folding of T11D3 H bound peptides shared a clear motif, with strong preference for https://www.jimmunol.org chain and b2m was observed in vitro in the absence of peptide. leucine at P9, as well as a preference for alanine at P2 (Fig. 7C). The T11D3-Qdm folding product was observed to bind labeled Proline is prominent at P4, and this amino acid is conserved in the Qdm peptide, yet the resulting complexes appeared to be unstable mouse and human class Ia leader peptides recognized by CD94/ (Fig. 5). These results leave the unanswered question of whether NKG2A receptors. Hydrophobic amino acids are present in the C- T11 normally assembles with peptides in cells and, if so, would it terminal position in all of the peptides, regardless of length, with be relevant to determine the nature of the peptides and the extent similar representation of amino acids as shown for 9-mers. The Downloaded from to which the peptide-loading specificity differs from T23. In ad- peptides identified are derived from proteins with broad intracel- dition, there is limited information on the nature of peptides other lular distribution, including slightly .50% from the cytoplasm than Qdm that can be presented by T23 (Qa-1). To directly address and/or nucleus, and the remainder with predominant localization these questions, T11D3- and T23D3-bound peptides were eluted in plasma member, ER, Golgi, or mitochondria. The two class Ia from Hela-T11D3 and Hela-T23D3 cells and identified by LC- leader sequence-derived peptides were the only peptides identified MS/MS. Background peptides identified in eluates from control from ER leader sequences. These results indicate that even in Hela-MigR1 cells were subtracted from the eluted peptide pools. TAP-expressing cells, Qa-1b can load with a diverse repertoire of Acquired MS/MS spectra from peptide pools eluted from T23D3 peptide sequences sharing a common motif. were initially searched using Spectrum Mill Proteomics Work- Eighty-nine peptide sequences were identified on initial analysis bench (Agilent Technologies), yielding 190 peptide sequences. of samples from T11D3, with 32 confirmed by manual validation, These were further culled through a stringent validation process, including 23 unique sequences (Table I). The length distribution involving expert manual inspection of the LC-MS/MS fragmen- was similar to that observed with T23D3, with dominant repre- tation spectra as previously described (51), resulting in 82 peptides sentation of 9-mers (Fig. 7A). There was a striking degree of with 41 unique sequences (Table I). The two HLA class Ia leader overlap in peptides isolated from T11 and T23 (Fig. 7B), including The Journal of Immunology 1435

FIGURE 6. MHC class Ib–Qdm tetramer staining of lymphocytes. B6 spleen and liver lymphocytes were stained with surface markers for B cells (B220), T cells (CD3ε), NK cells (NKp46), and the hybrid T23D3-Qdm or T11D3-Qdm tetramers. allophyco- cyanin-labeled streptavidin was used as a negative control. The lympho- cytes were gated out for FACS anal- ysis. The staining was repeated twice with similar results, and one of each is shown.

17 sequences shared between the two samples (Table I). The motif detection by an existing mAb. The chimeric T11D3 protein was identified with peptides eluted from T11 is very similar to that expressed at high levels on the surface of both TAP-expressing obtained with T23 (Fig. 7C, 7D), and there was no discernable Hela and TAP-deficient T2 cells, at levels very similar to those difference in the subcellular localization of the source proteins. In observed with the control T23D3 cDNA. Confirmation that the contrast to T23, class Ia leader peptides were not as prominent natural T11 protein is assembled and expressed on the surface of by guest on September 30, 2021. Copyright 2014 Pageant Media Ltd. among the validated peptides, with only one hit representing 3% primary tissues will require the generation of an appropriate mAb. of the total peptides. These results provide direct evidence that Our study does not exclude the possibility that the T11 a3 domain T11 normally assembles with peptides, and it shares a peptide- contains substitutions that interfere with protein assembly. T11 binding motif very similar to that of Qa-1. differs from the known Qa-1 alleles at four amino acid positions in the N-terminal segment of the a3 domain (residues 191, 195, Discussion 197, and 198). However, the amino acid present at each of these A large number of MHC class Ib genes remain to be characterized positions in T11 is also present in other mouse class I molecules. with respect to expression and potential function. Analysis is chal- For example, 191R is present in H-2Kk, and 195P/197G/198D are lenging because of the high level of sequence homology among d d

https://www.jimmunol.org present in H-2K and H-2D . In addition, these residues are class I genes and the limited availability of specific mAbs. In the largely surface exposed. Thus, it is likely that the T11 a3 domain current study, we investigated the potential for expression and is functional and that the T11 protein is expressed at the surface of function of the H2-T11 gene from C57BL/6 mice, a gene with a cells in tissues. high degree of homology to the Qa-1b–encoding H2-T23 gene. Given the high degree of sequence similarity between T11 and After cloning and sequencing the T11 gene, RT-PCR was used to Qa-1, we were interested in determining whether Qa-1 function is demonstrate that correctly spliced T11 mRNA is expressed widely conserved in T11. In addition to its function in regulating NK cell Downloaded from in tissues and leukocyte subpopulations and that expression is activation, Qa-1 has been demonstrated to function in T cell Ag particularly high in thymus. Expression was also prominent in presentation. Control experiments demonstrated that Hela cells spleen, intestine, lung, and liver. The highest expression of T11 expressing chimeric T23D3 molecules were highly functional in was observed in the DP subpopulation of the thymocytes. The presenting Ag to insulin-specific, Qa-1b–restricted 6C5 T cells. possibility that T11 may have a special function in thymic se- Thus, substitution of the a3 domain did not disrupt that Ag- lection remains to be explored. Previous studies have demon- presentation function of T23. By contrast, no responses were strated that, in contrast to conventional class Ia–restricted T cells, observed in Ag-presentation experiments with cells expressing class Ib–restricted T or NKT cells can be positively selected by T11D3, indicating the T11 cannot substitute for Qa-1 in Ag pre- thymic hematopoietic cells (including DP thymocytes) expressing sentation to 6C5 T cells. In addition to two substitutions in the Qa-1, H2-M3, or CD1 (31, 62, 63). b-sheet floor of the peptide-binding groove, T11 has six sub- Further support for the conclusion that H2-T11 encodes a stitutions in surface-exposed positions of the a1a2 helices as functional MHC protein was obtained using cDNA encoding a compared with Qa-1b. Any of these substitutions could impact chimeric T11 protein with a substituted a3 domain that allowed TCR recognition. Although it is possible that some T cells may 1436 A Qa-1 PARALOG ENCODED BY H2-T11

Table I. Peptide eluted from T23 and T11

Start Sequence Amino Acid Length T23 Hits T11 Hits Accession No. Name VMAPRTLIL 3 9 6 0 P04222 HLA class I histocompatibility Ag, Cw-3 a-chain VMAPRTLVL 3 9 3 1 P01892 HLA class I histocompatibility Ag, A-2 a-chain precursor KLFGSTSSF 195 9 4 0 P08174 Complement decay-accelerating factor SAIPHPLIM 226 9 4 0 P61201 COP9 signalosome complex subunit 2 VFGPILASLL 166 10 3 0 O43147 Small G protein signaling modulator 2 ETYPDAVKI 182 9 3 0 Q8WV22 Nonstructural maintenance of chromosomes element 1 homolog FVPAEKIVI 301 9 3 0 Q9UHF4 IL-20 receptor subunit a AKYPEIKSL 28 9 2 0 O15121 Sphingolipid d (4)-desaturase DES1 AAFHEEFVV 217 9 2 0 P51114 Fragile X mental retardation syndrome-related protein 1 AQLPEKVEY 986 9 2 0 P51532 Transcription activator BRG1 LPMFIIVV 308 8 2 0 P53794 Sodium/myoinositol cotransporter GQLPGLHEY 561 9 2 0 Q8TAT6 Nuclear protein localization protein 4 homolog FGFHKPKMY 2 9 2 0 Q9NP50 Protein FAM60A YAYDGKDYIA 140 10 1 0 P01892 HLA class I histocompatibility Ag, A-2 a-chain YAYDGKDYIAL 140 11 1 0 P01892 HLA class I histocompatibility Ag, A-2 a-chain RKLEAAEDIAY 239 11 1 0 P35232 Prohibitin FAYPAIRYL 117 9 1 0 P51398 28S ribosomal protein S29, mitochondrial HSAEILAEI 75 9 1 0 Q6FI81 Anamorsin HDLIRVVY 155 8 1 0 Q8IXS8 Protein FAM126B AAFAYTVKY 243 9 1 0 Q8N2K1 Ubiquitin-conjugating enzyme E2 J2 HTANIQTLI 313 9 1 0 Q8NG31 Protein CASC5 LAAQILAVL 202 9 1 0 Q96IK0 Transmembrane protein 101 SKLPIGDVATQY 291 12 1 0 Q99832 T-complex protein 1 subunit h SLINEFKL 147 8 1 0 Q9NYW6 Taste receptor type 2 member 3 RAFDQGADAIY 34 11 1 0 Q9UDW1 Cytochrome b-c1 complex subunit 9 GKAPLNVQF 876 9 3 1 O75369 Filamin-B AAFLKAIGY 744 9 3 1 O75533 Splicing factor 3B subunit 1 SAIDRIFTL 403 9 4 2 Q86TU7 Histone-lysine N-methyltransferase setd3 TASPLVKSV 866 9 3 1 Q6P4F7 Rho GTPase-activating protein 11A NIFRNVEV 80 8 3 2 Q7L523 Ras-related GTP-binding protein A AAFDKIQQL 121 9 3 2 Q9P2I0 Cleavage and polyadenylation specificity factor subunit 2 by guest on September 30, 2021. Copyright 2014 Pageant Media Ltd. SAKTPGFSV 143 9 2 1 Q9NY61 Protein AATF ISTPVIRTF 989 9 2 1 Q9NZB2 Constitutive coactivator of PPAR-g-like protein 1 MTPEIIQKL 168 9 2 2 Q9UMS4 Pre–mRNA-processing factor 19 SAVPFKILY 1030 9 1 1 P10586 Receptor-type tyrosine-protein phosphatase F ALIEFIRSEY 355 10 1 1 P26639 Threonine–tRNA ligase, cytoplasmic ETFNTPAMYV 825 10 1 1 Q6S8J3 POTE ankyrin domain family member E AAMPRPVSY 576 9 1 1 Q6TFL4 Kelch-like protein 24 EDDNISVTI 239 9 1 1 Q9NWH9 SAFB-like transcription modulator RQADFVQVL 414 9 1 1 Q9Y2V7 Conserved oligomeric Golgi complex subunit 6

https://www.jimmunol.org EIFADPRTV 228 9 1 2 Q92871 Phosphomannomutase 1 AQRMTTQLL 2 9 0 1 P15328 Folate receptor a AASSIQRVL 124 9 0 1 Q5BKT4 Dol-P-Glc:Glc(2)Man(9)GlcNAc (2)-PP-Dol a-1,2-glucosyltransferase QAVAKCAQLL 336 10 0 1 Q6ZU64 Coiled-coil domain-containing protein 108 DVIYPMAVV 177 9 0 2 P78406 mRNA export factor GAFGKPSSL 442 9 0 2 Q8IVT2 Uncharacterized protein C19orf21 VAAPQVQQV 231 9 0 3 Q12772 Sterol regulatory element-binding protein 2 Downloaded from

cross-recognize Qa-1 and T11, there are enough amino acid in peptide exchange reactions with folded T11D3-Qdm protein substitutions in the a1a2 domain to make it unlikely that T cell and labeled Qdm peptide. The possibility that T11 can serve as an cross-recognition is common. alternative ligand for CD94/NKG2 receptors was investigated Qa-1 plays a major role as the exclusive ligand for CD94/NKG2 using MHC tetramers and flow cytometry. Control T23D3-Qdm inhibitory and activating receptors on NK cells. Receptor recog- tetramers were observed to stain large populations of NK cells and nition is highly specific for the sequence of the Qa-1–bound NKT cells from spleen and liver, demonstrating that a3 domain peptide. Thus, it was important to determine whether the capacity substitution does not prevent binding of Qa-1 to CD94/NKG2 to bind to the canonical class Ia leader sequence-derived peptide receptors. By contrast, no staining was observed with compara- Qdm is conserved in T11. Recombinant T11D3 H chain was ob- ble T11D3-Qdm tetramers, supporting the conclusion that T11 served to fold in vitro in the presence of Qdm peptide and b2m cannot serve as an alternative ligand for CD94/NKG2. with an efficiency similar to that observed with the control T23D3 The cocrystal structure of HLA-E bound to human CD94/ H chain. The capacity of T11 to bind Qdm was further confirmed NKG2A demonstrates that the inhibitory receptor binds to the The Journal of Immunology 1437

LC-MS/MS analysis of peptides eluted from T11D3 isolated from transduced Hela cells demonstrated that T11 loads with a diversity of predominantly 9-mer peptides that share a common motif, with dominant preference for aliphatic and hydrophobic amino acids at P9 and preference for alanine in P2. This motif is very similar if not identical to that obtained with T23D3. Indeed, a large majority of the peptide sequences identified from T11D3 were also identified in the T23D3 peptide sample. T11 differs from Qa-1b in only two positions inside the peptide-binding groove, T9H and A11V. These residues are located in the floor of the peptide-binding groove, interacting primarily with the a1 helix but not with bound peptide. A11V is a conservative substi- tution, and 9H is present in the a, c, and d alleles of Qa-1 that, like the b allele, bind Qdm and serve as functional ligands for CD94/ NKG2 receptors. Based on the crystal structure of Qa-1b-Qdm, none of the amino acids that differ between T11b and all Qa-1 alleles directly contact peptide (57). Thus, it appears that the peptide-binding specificity of Qa-1 is conserved in T11. Early work demonstrated that Qa-1 predominantly loads with FIGURE 7. Peptide elution from Hela-T11D3 and Hela-T23D3 cells. Qdm, in striking contrast to that large diversity of peptides that A ( ) Length distribution of the peptides eluted from Hela-T11D3 and Hela- assemble with class Ia molecules (21). Crystal structures of Qa-1 T23D3 cells. (B) The unique peptides from the T23- and T11-eluted and its human ortholog HLA-E with bound leader peptides have peptide pool were analyzed. Numbers of 8-, 9-, and 10-mer peptides are shown in the Venn diagram. Sequence logo of the total eluted unique demonstrated five primarily hydrophobic anchor sites distributed peptides from T23 (C) and T11 (D). Each column represents 1 aa position throughout the length of the groove that accommodate side chains in the peptide. Amino acids with different properties were labeled with from peptide positions P2, P3, P6, P7, and P9, with P2, P7, and P9 different colors. being relatively deep (57, 66). This distribution of pockets may in part account for the exceptionally restricted peptide-binding specificity of these MHC molecules, favoring sequences closely same general surface of the MHC molecule as do TCRs (64). related to Qdm. It is also important to note that, despite having Examination of this structure suggests T11 position 65 as a can- ideal sequences for binding Qa-1 and HLA-E, the Qdm-related didate receptor contact residue that might preclude recognition by peptides dissociate relatively rapidly from the MHC molecules CD94/NKG2A (57). This position contains a nonconservative (25). Thus, only a relatively small fraction of Qa-1–binding pep- arginine substitution in T11 replacing tryptophan in all Qa-1 tides with near optimal affinities may form complexes that sur- alleles. It is noteworthy that, like T11 and unlike Qa-1, arginine vive long enough to be present in appreciable quantities at steady is also present at this position in HLA-E. It has previously been state. by guest on September 30, 2021. Copyright 2014 Pageant Media Ltd. demonstrated that although peptide-binding specificity is con- An important finding in the current study is the identification served between Qa-1 and HLA-E, CD94/NKG2 receptors do not of a relatively large number of different Qa-1 (T23D3) –bound cross-recognize species-mismatched MHC class Ib molecules, peptides. Qdm-related HLA-C– and HLA-A–derived leader even if they are bound to a species-matched leader peptide (65). sequences were readily detected in the eluted peptide samples, Thus, there has been coevolution of CD94/NKG2, its MHC class representing ∼11% of the identified peptides, yet many other Ib ligand, and the MHC class Ia leader sequences that provide the peptide sequences were also present. A number of studies have source of Qdm and related peptides. characterized T cells with specificity for alternative peptide Ags Our results demonstrate that recombinant T11D3 has the ca- presented by Qa-1, demonstrating that Qa-1 can bind and present pacity to bind Qdm peptide. Size-exclusion chromatography, peptides other than Qdm. An Hsp60-derived peptide was identified https://www.jimmunol.org immunoassays, and far-UV CD analysis indicated that recombinant by mass spectrometry from Qa-1 isolated from cells lacking a T11D3, generated in vitro by the folding of H chain with b2m and source of Qdm (55). In the current study, the peptides isolated Qdm, assembles similarly to conventional MHC class I molecules. from T23D3 were predominantly 9-mers, and a clear motif was In addition, this protein can bind labeled Qdm through a peptide- identified, with dominant preference for hydrophobic amino acids exchange reaction. However, an atypical thermal denaturation at P9 and preference for alanine at P2 and P3. Some degree of profile was observed in CD measurements with recombinant selectivity was also observed at the other anchor positions (P6 and Downloaded from T11D3-Qdm consistent with entry into a relatively stable mis- P7). The anchor positions in Qa-1 have been defined by binding folded conformation with preservation of some b-sheet secondary experiments with substituted peptides (24) and from the Qa-1b– structure at increasing temperatures. In addition, the kinetics of Qdm crystal structure (57). The results are generally consistent binding of labeled Qdm to T11D3-Qdm was unusual, displaying with findings from a previous study analyzing the relative pref- very rapid association kinetics followed by decay in the binding erence for specific amino acids at each anchor position based on signal consistent with rapid conversion to a peptide-unreceptive in vitro folding reactions with pools of substituted Qdm peptides conformation at 37˚C. Given these findings, one must consider the randomized at individual positions (24). In the latter study, the possibility that T11 is in general unstable at physiological tem- greatest observed specificity was for leucine at P9. Previously perature. This possibility, however, is strongly countered by the described Qa-1–restricted T cell epitopes, as well as the Qdm- observation that high levels of T11D3 are expressed at steady state related leader peptides from mice and humans, contain leucine on the surface of transduced cells, and these molecules were ob- at P9 and hydrophobic residues at P2. These include the ERAAP- served to be associated with bound peptides. Thus, we favor the sensitive self-peptide FL9 (FYAEATPML) (38), a Salmonella pep- hypothesis that T11 can function as a stable peptide presentation tide (GMQFDRGYL) (27), peptides from proinsulin (ALWMRFLPL) molecule. (29), influenza (FYAEATPML) (24), and an epitope recognized 1438 A Qa-1 PARALOG ENCODED BY H2-T11

by T cells with specificity for TAP-deficient tumor cells 8. Xu, H., T. Chun, H. J. Choi, B. Wang, and C. R. Wang. 2006. Impaired response to Listeria in H2-M3-deficient mice reveals a nonredundant role of MHC class (FAPLPRLPTL) (37). Ib-specific T cells in host defense. J. Exp. Med. 203: 449–459. Recently, Oliveira et al. (37) reported a large number of peptides 9. Seaman, M. S., C. R. Wang, and J. Forman. 2000. MHC class Ib-restricted CTL identified by LC-MS/MS isolated from TAP-deficient EC7.1 cells provide protection against primary and secondary Listeria monocytogenes in- b b fection. J. Immunol. 165: 5192–5201. expressing a chimeric Qa-1 protein containing the H-2D a-3 10. Cho, H., H. J. Choi, H. Xu, K. Felio, and C. R. Wang. 2011. Nonconventional domain, analogous to the constructs used in our experiments. The CD8+ T cell responses to Listeria infection in mice lacking MHC class Ia and distribution of peptide lengths was greater as compared with our H2-M3. J. Immunol. 186: 489–498. 11. Gulden, P. H., P. Fischer, III, N. E. Sherman, W. Wang, V. H. Engelhard, results. The frequent presence of Leu at the C terminus as well as J. Shabanowitz, D. F. Hunt, and E. G. Pamer. 1996. A Listeria monocytogenes Ala at P2 was consistent with our current results with TAP- pentapeptide is presented to cytolytic T lymphocytes by the H2-M3 MHC class expressing cells. By contrast, a very strong signature for Asn at Ib molecule. Immunity 5: 73–79. 12. Lenz, L. L., B. Dere, and M. J. Bevan. 1996. Identification of an H2-M3- P5 was present in their peptides but absent from our results (which restricted Listeria epitope: implications for antigen presentation by M3. Immu- showed no specificity at the P5 position). As noted above, P5 is nity 5: 63–72. not an anchor position in Qa-1b. EC7.1 is a Kb- and Db-negative 13. Weber, D. A., A. Attinger, C. C. Kemball, J. L. Wigal, J. Pohl, Y. Xiong, b E. L. Reinherz, H. Cheroutre, M. Kronenberg, and P. E. Jensen. 2002. Peptide- variant of the TAP-deficient H-2 RMA-S cell line. Given that the independent folding and CD8 alpha alpha binding by the nonclassical class I anti-Db mAb 28-14-8S was used to isolate chimeric Qa-1 mole- molecule, thymic leukemia antigen. J. Immunol. 169: 5708–5714. b 14. Leishman, A. J., O. V. Naidenko, A. Attinger, F. Koning, C. J. Lena, Y. Xiong, cules in the Oliveira et al. study (37), it is possible that D -asso- H. C. Chang, E. Reinherz, M. Kronenberg, and H. Cheroutre. 2001. T cell ciated peptides were also present in the samples analyzed in that responses modulated through interaction between CD8alphaalpha and the non- study from low-level surface or intracellular pools of residual Db classical MHC class I molecule, TL. Science 294: 1936–1939. b 15. Huang, Y., Y. Park, Y. Wang-Zhu, A. Larange, R. Arens, I. Bernardo, molecules. The D peptide-binding motif (Asn at P5 and Met, Ile, D. Olivares-Villago´mez, D. Herndler-Brandstetter, N. Abraham, B. Grubeck- or Leu at the C terminus) overlaps with Qa-1b at the C terminus, Loebenstein, et al. 2011. Mucosal memory CD8⁺ T cells are selected in the but the dominant Asn anchor at P5 is not observed with Qa-1. periphery by an MHC class I molecule. Nat. Immunol. 12: 1086–1095. 16. Jensen, P. E., B. A. Sullivan, L. M. Reed-Loisel, and D. A. Weber. 2004. Qa-1, It is interesting to note that there appears to be selectivity for Pro a nonclassical class I histocompatibility molecule with roles in innate and at the solvent-exposed nonanchor P4 position in T23D3-bound adaptive immunity. Immunol. Res. 29: 81–92. peptides. It is possible that Pro at this position may constrain the 17. Connolly, D. J., L. A. Cotterill, R. A. Hederer, C. J. Thorpe, P. J. Travers, J. H. McVey, J. Dyson, and P. J. Robinson. 1993. A cDNA clone encoding the conformation of the peptide, favoring optimal positioning of anchor mouse Qa-1a histocompatibility antigen and proposed structure of the putative residues. Alternatively, cellular mechanisms impacting peptide pro- peptide binding site. J. Immunol. 151: 6089–6098. 18. Hermel, E., A. J. Hart, R. Miller, and C. J. Aldrich. 1999. CTL and sequence cessing or loading might introduce bias favoring Pro at P4. analyses of MHC class IB antigens Qa1(c) (H2-T23(r)) and Qa1(d) (H2-T23(f)). Overall, our results establish a common peptide-binding motif Immunogenetics 49: 712–717. shared by Qa-1b and T11b. It appears very likely that T11 is a 19. Hermel, E., A. J. Hart, I. Gunduz, H. Acton, C. Kim, M. Wurth, S. Uddin, C. Smith, K. Fischer Lindahl, and C. J. Aldrich. 2004. Polymorphism and functional MHC class Ib molecule with a capacity for peptide conservation of the genes encoding Qa1 molecules. Immunogenetics 56: 639–649. binding and cell-surface expression. T11 differs from all Qa-1 20. Aldrich, C. J., A. DeCloux, A. S. Woods, R. J. Cotter, M. J. Soloski, and alleles through substitutions at a number of positions on the J. Forman. 1994. Identification of a Tap-dependent leader peptide recognized by alloreactive T cells specific for a class Ib antigen. Cell 79: 649–658. a-helical receptor-contact surface of the peptide-binding domain 21. DeCloux, A., A. S. Woods, R. J. Cotter, M. J. Soloski, and J. Forman. 1997. that may prevent T11 from serving as an alternative ligand for Dominance of a single peptide bound to the class I(B) molecule, Qa-1b. J. CD94/NKG2 or Qa-1–restricted TCRs. We cannot rule out the Immunol. 158: 2183–2191. 22. Li, L., B. A. Sullivan, C. J. Aldrich, M. J. Soloski, J. Forman, A. G. Grandea, III, by guest on September 30, 2021. Copyright 2014 Pageant Media Ltd. possibility that T11 might have specialized function, serving as P. E. Jensen, and L. Van Kaer. 2004. Differential requirement for tapasin in the a ligand for a yet to be identified receptor. Given that T11 shares presentation of leader- and insulin-derived peptide antigens to Qa-1b-restricted CTLs. J. Immunol. 173: 3707–3715. a very similar peptide-binding specificity with Qa-1, it is also 23. Vance, R. E., J. R. Kraft, J. D. Altman, P. E. Jensen, and D. H. Raulet. 1998. + interesting to consider the possibility that T11-restricted CD8 Mouse CD94/NKG2A is a natural killer cell receptor for the nonclassical major T cells might have regulatory functions that parallel those iden- histocompatibility complex (MHC) class I molecule Qa-1(b). J. Exp. Med. 188: 1841–1848. tified for Qa-1–restricted T cells. These possibilities will require 24. Kraft, J. R., R. E. Vance, J. Pohl, A. M. Martin, D. H. Raulet, and P. E. Jensen. further investigation. 2000. Analysis of Qa-1(b) peptide binding specificity and the capacity of CD94/ NKG2A to discriminate between Qa-1-peptide complexes. J. Exp. Med. 192: 613–624. Acknowledgments 25. Kambayashi, T., J. R. Kraft-Leavy, J. G. Dauner, B. A. Sullivan, O. Laur, and https://www.jimmunol.org We thank Xiaomin Wang for strong technical support and Matthew Weinstock P. E. Jensen. 2004. The nonclassical MHC class I molecule Qa-1 forms unstable for the help with the circular CD experiments. peptide complexes. J. Immunol. 172: 1661–1669. 26. Bouwer, H. G., M. S. Seaman, J. Forman, and D. J. Hinrichs. 1997. MHC class Ib-restricted cells contribute to antilisterial immunity: evidence for Qa-1b as a Disclosures key restricting element for Listeria-specific CTLs. J. Immunol. 159: 2795–2801. The authors have no financial conflicts of interest. 27. Lo, W. F., H. Ong, E. S. Metcalf, and M. J. Soloski. 1999. T cell responses to Gram-negative intracellular bacterial pathogens: a role for CD8+ T cells in immunity to Salmonella infection and the involvement of MHC class Ib mole-

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