Molecular Identification of an MHC Class Ib (H2-Q9) Restricted T Cell Receptor Specific for a Mouse Polyomavirus Peptide VP2.139

Research Article

Austin J Clin Immunol. 2014;1(3): 1015.

Molecular Identification of an MHC Class Ib (H2-Q9) Restricted T Cell Receptor Specific for a Mouse Polyomavirus Peptide VP2.139

Zemin Zhou1, Xiao He1* and Peter E Jensen1,2

1Department of Pathology, University of Utah, USA

2ARUP Laboratories, University of Utah, USA

*Corresponding author: Xiao He, Department of Pathology, University of Utah, 15 North Medical Drive East, Suite 1200, Salt Lake City, UT 84112-5650, USA

Received: February 22, 2014; Accepted: April 29, 2014; Published: May 01, 2014


The murine major his to compatibility complex (MHC) class Ib region encodes more than 20proteins with diverse biological functions, and many have not been characterized. Evidence emerging in recent years indicates that MHC class Ib–restricted CD8 T cells can play an important role in antiviral immunity. We molecularly cloned an α⁄β T cell receptor (TCR) from a T cell hydridoma cell line, that is restricted by an MHC class Ib molecule H2–Q9, and specifically recognizes the VP2.139 peptide epitope from mouse polyoma virus (PyV). We further demonstrated that the cloned TCR, using TCRα10 and TCRβ4 genes, is functional and mediates the development of Q9⁄VP2.139–restricted CD8 T cells in vivo. The cloning of a H2–Q9–restricted, PyV–specific α⁄β TCR provides a useful tool for future study of the potential role of MHC class Ib restricted CD8 T cells in antiviral immunity.

Keywords: MHC Class Ib; Qa–2; H2–Q9; CD8+ T cell; TCR; Polyomavirus


Major his to compatibility complex (MHC) class I proteins play an important role in adaptive immunity [1,2]. They provide a mechanism of immune surveillance by presenting intracellular antigens from either self or pathogenicorigin to CD8 T cells, natural killer (NK) cells and⁄or natural killer T (NKT) cells [1,3–5]. The functions of the highly polymorphic classical MHC class I (MHC class Ia) molecules, including H2–K, –D and –L in mouse and HLA–A, –B and –C in human, are well established [2,6]; however, most of the non–classical MHC class I (MHC class Ib) molecules, with limited polymorphism, are not well studied [6,7]. The Qa–2 antigen (encoded by the H2–Q6, 7, 8, 9) is one of the best characterized class Ib molecules in mouse (possibly homologous to HLA–G in human). H2–Q9 encodes two typesof Qa–2 isoforms, membrane–bound and soluble, through alternative splicing of transcripts [8]. Multiple biological functions of Q9, as well as that of HLA–G in human, have been documented, such as participating in preimplantation embryo development [9,10], tumor immunity [11,12], and antiviral immunity [13]. However, the selection of CD8+ T cells by Q9 and the characteristics of Q9–restricted CD8+ T cells are poor understood.

Cytotoxic CD8+ T cells directly eradicate or control intracellular pathogens by killing infected cells or by releasing cytokines to inhibit further spread of infection [14]. The selection and development of antiviral CD8+ T cells by class Ia molecules has been well documented [1,2]. However, the role of class Ib–restricted antiviral CD8+ T cells needs further investigation. The potential mechanismsofcontrol of viral infection by class Ib–restricted CD8+ T cells areunclear and the development pathways for this subset of CD8+ T cells arepoorly defined. Recently, CD8+ T cells have been identified that can recognize viral epitopes presented by MHC class Ib molecules [15–17], and there is evidence that class Ib–restricted CD8+ T cells contribute to the control or eradication of virus infection [13,18,19].

Polyomaviruses are double–stranded DNA viruses, persistently infecting humans or animals, and potentially inducing tumors in immune compromised individuals [20]. Since the 1970s, at least 6 polyomaviruses, including BK virus, JC virus, KI polyoma virus, WU polyoma virus, Merkel cell polyomavirus and trichodysplasiaspinulosa virus, have been identified in humans, [20–24]. Mouse polyomavirus (PyV) was the first identified polyomavirus [20,25] and it can induce tumors in immune compromised mouse strains [25,26]. T antigens of PyV can induce class Ia–restricted CD8+ T cell responses [27,28]. Recently, Swanson et al. successfully identified MHC class Ib, Q9– restricted CD8+ T cells that responded to a PyV–derived epitopeVP2.139 [13], which provided the first solid evidence demonstrating that class Ib–restricted CD8+ T cells might play a role in controlling persistent viral infection.

In the current study, we molecularly identified a T cell receptor (TCR) from Q9⁄VP2.139–specific CD8+ T cells and confirmed its function in transducedcells as well as in retrogenic mice. To our knowledge, this was the first identified MHC class Ib–restricted TCR that responds to viral infection.

Materials and Methods


C57BL⁄6J and Rag1–⁄– mice were purchased from the Jackson Laboratory. OT–I TCR transgenic mice, with specificity to OVA357– 364⁄Kb(OVA–I⁄Kb), have been previously described [29]. Kb–⁄–Db–⁄– CIITA–⁄– mice were generated in our laboratory as previously described [30]. Eight– to 12–week–old mice were used in the experiments. Mice were bred and housed in a specific pathogen–free facility at the University of Utah and were handled according to the Institutional Animal Care and Use Committee policies.

Cell lines and culture conditions

The Q9⁄VP2.139–specific C1–21 T cell clone and T cell hybridoma lines C3K and 3H6, and the recipient linesZ6.21–3B2.E7 and BWZwere provided by Dr. Aron Lukacher (Emory University).The recipient cell lines58α–⁄β– and 5KC⁄CD4were provided by Dr. Piotr Kraj (Georgia Regents University) and maintained as previously described [31]. 3H6 T cell hybridoma was further sorted into 3H6 (+) and (−), based on the presence or absence of surface expression of TCR Vα2. 3H6 (–) cells, which did not produce IL–2 upon stimulation of VP2.139 peptides were used as recipient cells in retroviral transductionexperiments. The retroviral packaging cell line Plat–E was described previously [32]. The C1–21 T cell clone was maintained in IMDM with 10% FBS (Hybrid), 10 U⁄ml of mouse IL–2, 10 uM of VP2.139 peptides and irradiated Kb–⁄–Db–⁄–CIITA–⁄– mouse splenocytes as antigen presenting cells (APCs).C3K and 3H6 T cell hybridomas, recipient cell line sand Plat–E packaging cell lineswere maintained in DMEM with 10% FBS, supplemented with 100 U⁄ml of penicillin, 100 mg⁄ml of streptomycin, 292 mg⁄ml of L–glutamine, 100 mM of nonessential amino acids, 1 mM of sodium pyruvate, and 55 uMof 2–ME (Invitrogen),as complete DMEM as previously described [18]. Cells were maintained in a humidified 37oC incubator supplied with 5% CO2.

Generation of soluble H2–Kb, H2–Q9 proteins and Q9⁄VP2.139 tetramer

The peptides L19 (ILMEHIHKL), VP2.139 (HALNVVHDW), OVA–I (SIINFEKL) were synthesized, and substitutedL19–H5C, VP2.139–V5C and OVA–I–N4C peptides were synthesized and biotinylatedat the core facility ofUniversity of Utah. Soluble H2– Kb and H2–Q9 proteins were purified from E. coli (BL21)–derived proteins and refolded with the appropriated peptides in a 1:1 molar ratioto produceQ9⁄L19, Q9⁄VP2.139 or Kb⁄OVA–I monomers, using ourpreviously describedmethod [33]. To generate Q9⁄VP2 tetramers, refolded Q9⁄VP2.139 monomer was biotinylated and tetramerized with streptavidin–APC (Invitrogen) in a 4:1 molar ratio as previously described [13,34]. Peptide dissociation rates and MHC–peptide complex half–liveswere measured as previously described [33,35]. Briefly, Q9⁄L19, Q9⁄VP2.139 or Kb⁄OVA–I complex (50 nM) was incubated with biotin–L19, biotin–VP2.139 or biotin–OVA–I (1 μM) peptide, respectively, in PBS with 0.01% Nonidet P–40 at RT for overnight. Then, unlabeled L19, VP2.139 or OVA–I peptide (200 μM), respectively, was added and incubated at 37°C for the indicated lengths of time. The remained biotin–peptide that bound to the MHC class I molecules was measured by Eu–ELISAusing an anti–β2m capture mAb [35].

Abs and flow cytometry

Fluorophore–conjugated mAbs to mouse CD4 (H129.19), CD8 (RPA–T8), CD62L (MEL–14), Vα2 (B20.1), Vα3.2 (B21.14), Vα8.3(RR3–16), Vα11(RR8–1), isotype–matched control mAbs,and a mouse Vβ TCR Screening Panel (557004) were purchased from BD Pharmingen. Cell surface staining and Q9⁄VP2 tetramer staining were performed according to the standard procedures. Stained cells were analyzed on a FACS can flow cytometer (BD Bioscience) and data were analyzed using Flow Jo 8.4 software.

Cloning of the TCR α chain and β chain gene and generation of expression constructs

Total mRNA of C3K hybridoma cells was extracted using the RN easy Kit (Qiagen) and the cDNA was reversely transcribed, with the Super ScriptTM III Reverse Transcriptase Kit (Invitrogen) following the manufacture’s instructions, for further cloning.The TCRExpress™ mouse TCR Vα screening kit was purchased from BioMed Immunotech. TCR variable region prediction and open reading frame analysis were performed using the IMGT website for mouse TCR analysis (http:⁄⁄⁄IMGT_vquest⁄), as well as the sequences previously published [36–38]. The primers used for cloning of the full length TCRα10 and TCRβ4 cloning were listed as below:

TCRα10 forward (TCRVα10f–F): 5’–GTGCGGCCGCGTTT A A A C A T G A A G A G G C T G C T G T G C T C T C T G C – 3 ’ , TCRα10 reverse (TCRCαf–R): 5’–TTCCTGCAGGGTT TA AACTCAACTGGACCACAGCCTCAG–3’; TCRβ4 forward (TCRVb4f–F): 5’– GCTTAATTAAGTTTAAACATGGGCTCCA TTTTCCTCAGTTG–3’, TCRβ4 reverse (TCRCb1fR–R): 5’– ATGGCGCGCCGTTTAAACTCATGAATTCTTTCTTTTGA CCATAGCC–3’. To co–express TCRα10 and TCRβ4, we fused full–length TCRα10 (excluding stop codon) and TCRβ4 genes with T2A (GAGGGCAGAGGAAGTCTTCTAACATGCGGTG ACGTGGAGGAGAATCCCGGCCCT) sequence [39], using primers TRA10–2A–R (5’–CTCCTCCACGTCACCGCATGTT AGAAGAC T T C C T C TGC C C T CGT TAACAC TGGAC CACAGCCTCAGCG–3’) and 2A–TRB4 –F (5’–CTTCTAAC ATGCGGTGACGTGGAGGAGAATCCCGGCCCTATGG GCTCCATTTTCCTCAGTTGC–3’), paired with TCRVα10f–F or TCRCβ1fR–R, respectively, in two rounds of PCR amplification,to generate a TCRα10–T2A–β4 (C3K) construct. All of these PCR products were amplified with the Platinum® Taq DNA Polymerase High FidelityPCR Kit (Invitrogen) following the manufacture’sinstruction and cloned into the pCR2.1 vector using the TOPO® TA CloningKit (Invitrogen).The DNA sequences were determined by sequencing(the Gen Bank accession number: KJ755332). Then, the TCRα10–T2A–β4 construct in the pCR2.1 vector were digested with PmeI (Roche) and further cloned into a retroviral vector MigR1 as previously described [40,41]. The OT–I TCR construct TCRα2–T2A–β5.2 in MigR1,used as a positive control in generation of the retrogenicmice, was a gift from Dr. KateVignali (St. Jude Children’s Research Hospital).

Retroviral transfection and retrogenic mouse generation

Retroviral supernatants were generated by transfection of Plat–E cells with MigR1–TCRα10–T2A–β4 (C3K) or MigR1–TCRα2– T2A–β5.2 (OT–I) as previously described [32,41,42]. The C3K and OT–I retrogenic mice were generated as previously described [43]. Briefly, 5x107bone marrow cells were harvested from 6–week–old Rag1–⁄–mouseand cultured in 15 ml of complete DMEM with 20% FBS, supplemented with IL3 (20 ng⁄ml), IL–6 (50 ng⁄ml) and SCF (50 ng⁄ml) (R&D systems, Inc) for 48 hours., The infected bone marrow cells were re–infected by adding 5 ml of fresh C3K or OT–I retroviral supernatants produced from Plat–E transfectants. Four days post–transduction, semi–lethally irradiated (450 rad) Rag1–⁄– recipient micewerereconstituted with the transduced bone marrow cells, following the previously described protocol [43]. Five–weeks post reconstitution, peripheral bloodsamples werecollected and analyzed for GFP expression and cell surface staining. Six weeks postreconstitution, retrogenic mice were sacrificed for analysis.

T cell activation assay and measurement of IL–2 production

To stimulateC1–21 T cells, 3H6 and C3K T cell hybridoma cells, or the TCR transductant, 2x105 cells (per well in a 96–well plate), with 2x105of irradiated Kb–⁄–Db–⁄–CIITA–⁄–splenocytes, which only expresses MHC Ib [30], as APCs, and 0, 1, or 10 uM of VP2.139,OVA–I or L19 peptides, were added. For retrogenic mice T cell activation assay, 4x105 cellsper well of C3K retrogenic or OT–ITCR transgenic mouse splenocytes were cultured with 0, 0.5, 1.0, 2.0, or 4.0 uM of VP2.139,OVA–I or L19 peptides. Eighteenhourslater, the supernatants were harvested and measured for mouse IL–2 production by the standard IL–2 ELISA procedure (eBioscience).


Characterization ofQ9⁄VP2.139complex stability andT cell specificity

It has been shown previously that VP2.139 peptide bound to Q9 canstimulate specific CD8+ T cells during mouse polyomavirus infection [13]. In order to further characterize the VP2.139 peptide binding capacity with Q9 and to generate a Q9⁄VP2.139 tetramer reagent for staining of T cells, soluble recombinant Q9 protein (α1 toα3 domains without the transmembrane and cytoplasmic domains) was expressed in E.coli and the inclusion body was purifiedand refolded with VP2.139 peptide or L19 peptide(as acontrol), a known Q9 bound endogenous peptide derived from mouse 60S ribosomal protein [44,45]. In addition, soluble Kb [33]was prepared using the same method (Figure 1A). Interestingly, the Q9⁄VP2.139 complex has relatively lower complex stability with a half–life of 248 min, compared with the complex of Q9⁄L19 or Kb⁄OVA–I with half–life’s of 1732 min or 1155 min, respectively (Figure 1B), which is consistent with previously observations [46].