Definition of a novel binding site on CD8 cells for a conserved region of the MHC class Ib molecule Qa1 that regulates IFN-γ expression

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Definition of a novel binding site on CD8 cells for a conserved region of the MHC class Ib molecule Qa1 that regulates IFN-γ expression
  0014-2980/01/0101-87$17.50+.50/0© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001 Definition of a novel binding site on CD8 cells for aconserved region of the MHC class Ib moleculeQa-1 that regulates IFN-   q  expression Rijian Wang 1, 2 , Sridhar Ramaswamy  3, 4 , Dan Hu 1, 2 and Harvey Cantor 1, 2 1 Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, USA  2 Department of Pathology, Harvard Medical School, Boston, USA  3 Department of Adult Oncology, Dana-Farber Cancer Institute, Boston, USA  4 Department of Medicine, Harvard Medical School, Boston, USA  Natural killer (NK) cells and activated CD8 cells both express cytotoxic activity and producesubstantial levels of IFN-   +  in response to viral and bacterial infections. In the case of NKcells, cellular activation and IFN-   +  expression are regulated by an interaction between NKreceptors and MHC class Ib molecules, including HLA-E/Qa-1. We have used soluble tetra-meric complexes of the murine class Ib molecule Qa-1 to define the significance of this inter-action for CD8 cells. We find that all CD8 cells express a receptor for Qa-1 and that ligationof this receptor by Qa-1 results in up-regulation of IFN-   +  production. Key words:  CD8 / Qa-1 / IFN-   +  / MHC / T cell developmentReceived 26/6/00Revised 15/9/00 Accepted 6/10/00 [I 21008] 1 Introduction CD8 cells play a critical role in the immune response tointracellular pathogens and tumors after TCR ligation byMHC class I / peptide complexes and differentiation tocytotoxic T cells (CTL). CD8 cell activation is alsomarked by production of large amounts of IFN-   +  thatcontributes to the inflammatory component of type 1immunity. Although CD8 cells efficiently produce IFN-   +  insettings that fail to stimulate significant IFN-   +  responsesby CD4 cells [1], the molecular basis of this differencebetween these two major T cell subsets is poorly under-stood.One approach to this problem comes from the similaritybetween the phenotype of NK cells and CD8 cells. Bothexpress cytotoxic activity and produce substantial levelsof IFN-   +  in response to viral and bacterial infections. Inthe case of NK cells, cellular activation and IFN-   + expression are regulated by an interaction between NKreceptors and MHC class Ib molecules including HLA-E/ Qa-1 [2]. We have used soluble tetrameric complexes ofthe murine class Ib molecule Qa-1 to examine whetherthere is a specific interaction between CD8 cells andQa-1. We define a receptor for Qa-1 that is expressed onall CD8 cells and show that ligation of this receptor byQa-1 up-regulates production of IFN-   +  . 2 Results and discussion 2.1 Soluble Qa-1a and Qa-1b proteins refold withQdm and preproinsulin II peptides  in vitro  toproduce soluble tetramers Both Qa-1a and Qa-1b molecules refolded with the Qdmpeptide form soluble tetramers (Fig. 1a, b) with the samerefolding efficiency of about 25%. In addition, both Qa-1alleles refold with a 9-amino acid peptide derived fromthe leader sequence of preproinsulin [3] (Fig. 1c, d) withan efficiency of approximately 10%. In contrast, neitherQa-1 allele refolded with a control peptide derived fromovalbumin (amino acids 257–264) [4] (Fig. 1g, h). 2.2 Binding of Qa-1a and Qa-1b tetramers to DPthymocytes and CD8 cells Thymocytes, splenocytes and lymph node cells fromC57BL/6 (Qa-1b) and C57BL/6.Tla (Qa-1a) congenicmice were incubated with PE-conjugated Qa-1a or Qa-1b tetramers and fluorescein-conjugated anti-CD3, anti-CD4, anti-CD8, anti-B220, and anti-NK1.1 antibodies.FACS analysis revealed that virtually all DP and CD8 SPthymocytes, but not CD4 SP thymocytes, bound tetra-mers formed by both Qa-1 alleles (a and b) (Fig. 2A).Moreover, Qa-1 tetramers bound to all peripheral CD8cells but not to CD4 cells (Fig. 2B). Tetramer binding wasnot restricted by the Qa-1 allele, since Qa-1a and Qa-1btetramers bound equally well to CD8 cells from bothC57BL/6 (Qa-1b) or C57BL/6.Tla (Qa-1a) mice. As Eur. J. Immunol. 2001.  31:  87–93 Definition of a novel binding site on CD8 cells 87  Fig. 1.  Refolding of soluble Qa-1a and Qa-1b molecules withQdm and preproinsulin-derived peptides. Soluble Qa-1aand Qa-1b proteins were purified and refolded in the pres-ence of either Qdm or a nine amino acid peptide derivedfrom the leader sequence of preproinsulin II (ins-II) asdescribed in Sect. 3. Thepeak retentionprofiles of thegel fil-tration results for the refolding reactions are shown. Insertsshow Coomassie blue-stained SDS polyacrylamide gels forpurified monomeric Qa-1 molecules. Panels  a  and  b  showQa-1a and Qa-1b proteins after refolding with the Qdm pep-tide;  c  and  d  show Qa-1a and Qa-1b after refolding with theins-II peptide;  e  and  f  show the purified and biotinylated Qa-1a and Qa-1b molecules formed tetramers in the presenceof streptavidin; and  g  and  h  show that the Qa-1a and Qa-1bmolecules did not refold with the control peptide derivedfrom chicken ovalbumin. Arrow 1 indicates the 13-min reten-tion peaks with molecular mass of about 200 kDa, the pre-dicted molecular mass for Qa-1 tetramers; arrow 2 indicatesthe 18-min retention peaks with molecular mass of about 38kDa, the predicted molecular mass for Qa-1 monomers; andarrow 3 indicates the 24-min retention peaks with molecularmass of about 23 kDa, the predicted molecular mass for thedimerized human   g    2 microglobulin. expected, a proportion of splenic NK1.1 + cells (   ˚  30%),but not B220 + B cells, also bound the Qa-1 tetramers(Fig. 2C, D). Incubation of thymocytes or T cells withheat-denatured Qa-1 tetramers or PE-streptavidin alonedid not result in detectable binding (data not shown). Thelineage-specific binding activity of the Qa-1a and Qa-1btetramers is summarized according to fluorescenceintensity of binding to different T cell subpopulations(Fig. 2E). 2.3 Tetramer binding kinetics and independencefrom CD8 The CD8 coreceptor did not contribute to Qa-1 tetramerbinding because (a) the addition of an anti-CD8 mAb at50-fold excess to tetramer did not inhibit binding to0805B cells (not shown) and (b) the mouse lymphomaS49.33 cell line which expresses both CD8   §    and   g   chains according to FACS analysis (Fig. 3A) did not bindQa-1a/b tetramers and (c) loss of surface CD8 expres-sion secondary to genetic mutation did not affect Qa-1binding: the same proportion of CD3 + thymocytes fromC57BL/6 and C57BL/6 CD8   §    -/- mice bound Qa-1b/Qdmtetramer (not shown).Tetramer binding was greatly reduced after preincuba-tion of T cells with unlabelled tetramer indicating that theinteraction was Qa-1 tetramer-specific and was inde-pendent of the Qa-1 allele: Qa-1a binding was efficientlyblocked by cold Qa-1b tetramer and vice versa (notshown). We examined the kinetics of the interaction withQa-1 tetramer using the 0805B BALB/c CD8 + CD4 - CTLline specific for a  lacZ  -derived peptide [5]. 0805B cells(Fig. 3A, panels 1–3) bound Qa-1a/b tetramers contain-ing either Qdm (Fig. 3A, panel 2) or preproinsulin pep-tides (Fig. 3A, panel 3). Fluorescence-conjugated mAbstaining showed that virtually all 0805B cells expressCD3 and CD8, but not CD4 or NK1.1 (data not shown).Double staining with FITC-conjugated mAb and PE-labeled Qa-1 tetramer confirmed that 0805B cellsexpress CD8 but not CD4; like CD4 + 8 + thymocytes, bind-ing was not inhibited by anti-CD8 antibody (data notshown). To determine whether binding of Qa-1 tetramersto CD8 + thymocytes and T cells is a receptor-mediatedprocess, 0805B CD8 cells were incubated with coldQa-1 tetramers before addition of PE-conjugated Qa-1tetramer. Qa-1 tetramer binding to CD8 T cells displayedtypical receptor-ligand kinetics, including saturation ofbinding at high concentration of ligand and specific inhi-bition by cold ligand. Tetramer binding increased untilthe concentration of PE-conjugated tetramer was 200ng/ml and then plateaued, indicating saturable binding(Fig. 3C, D). Cross-blocking experiments indicated thattetramer binding by cold ligand was neither Qa-1 allele-specific nor peptide dependent. 88 R. Wang et al. Eur. J. Immunol. 2001.  31:  87–93  Fig. 2.  Lineage-specific binding of Qa-1a and Qa-1b tetramers to T cells. Flow cytometric analysis of C57BL/6 and C57BL/6.Tlathymocytes, lymph node and spleen cells after incubation with Qa-1a/Qdm, Qa-1b/Qdm, Qa-1a/ins-II, and Qa-1b/ins-II tetra-mers. (A) and (B) show the FACS histogram of the thymocytes after incubation with Qa-1a/Qdm and Qa-1b/Qdm tetramers. (C)and (D) show FACS scatter plot data of peripheral lymphocytes stained with Qa-1a/Qdm and Qa-1b/Qdm tetramers and differentmAb. Panel E summarizes tetramer binding of different cell populations of Qa-1a (     Æ  ) and Qa-1b (     |     ), according to fluorescenceintensity. 2.4 Qa-1a tetramer binding co-stimulates IFN-   q expression by CD8 cells We tested the functionalconsequences of the interactionbetween Qa-1 and CD8 cells. Addition of Qa-1b/Qdmnatural, but not denatured, tetramer to anti-CD3-stimulated 0805B CD8 + cells increased the IFN-   +   response by 4–6 fold (Fig. 4A). Addition of Qa-1b/Qdmtetramer, but not denatured tetramer or monomer, alsoenhanced IFN-   +    response of CD8 cells from female HYTCR transgenic mice to the HY antigen four to fivefold(Fig. 4B). Qa-1-dependent enhancement of IFN-   +    pro-duction was antigen specific since soluble Qa-1 tetra-mer, but not monomer, enhanced the IFN-   +    response offemale HY-TCR CD8 cells to HY peptide presented APCby five to sixfold (Fig. 4C). These findings suggest thatexpression of Qa-1 on activated APC might normallyserve to enhance IFN-   +    expression by CD8 cells. Thishypothesis received additional support from the obser-vation that addition of anti-Qa-1b mAb (but not anisotype-matched control) to cultures of CD8 cells and(Qa-1 +  ) activated B cells inhibited IFN-   +    production bymore than 90% (Fig. 4D).Previous findings have indicated that Qa-1 can regulateinnate immune responses through interaction with inhibi-tory and activating receptors on NK cells [6, 7]. Our find-ings confirm binding of Qa-1/peptide tetramers tosplenic NK cells. However, we also find that thymocytesacquire binding activity for a conserved portion of Qa-1/ peptide complexes at the DP stage of thymic develop-ment and maintain this binding site during CD8 but notCD4 cell development in the thymus and peripheral tis-sues. Binding of Qa-1/peptide tetramer to CD8 cells is asaturable and inhibitable process that displays kineticstypical of a receptor-ligand interaction. Since binding toQa-1 is neither peptide- nor Qa-1 allele-specific anddoes not depend on the CD8-coreceptor, it is likely to bemediated by a conserved (non-clonal) receptor that isexpressed during development of CD8 cells that mayregulate the development and/or function of this T cellsubset. Indeed, one functional consequence of the inter-action between this receptor and Qa-1/peptide com-plexes expressed on the surface of APC is enhancedproduction of IFN-   +    .The molecular identity of the Qa-1/peptide receptor onCD8 cells is not established in these studies. Although Eur. J. Immunol. 2001.  31:  87–93 Definition of a novel binding site on CD8 cells 89  Fig. 3.  Binding kinetics of Qa-1 tetramer to CD8 cells. 0805B cells (CD8 +  ) and S49 (CD8 +  ) cells were stained with Qa-1b/Qdm tet-ramers.(A) Immunofluorescenceof 0805Bcellsand S49cells after incubationwithQa-1b tetramers: fluorescencecontrol (panels1 and 4); Qa-1b/Qdm tetramer (panels 2 and 5); Qa-1b/ins-II tetramer (panels 3 and 6). (B) Double staining of the cells with Qa-1b/Qdm tetramer and mAb against TNP,CD3,CD4, or CD8.(C)0805Bcells wereincubated withincreasing concentrations of un-labeled Qa-1b/Qdm tetramer before addition of a fixed amount of PE-conjugated Qa-1b/Qdm tetramer. (D) 0805B cells wereincubated with a fixed amount of unlabeled Qa-1b/Qdm tetramer before addition of increasing concentrations of PE-conjugatedQa-1b/Qdm tetramer: saturation binding, kinetics, non-specific binding and specific inhibition are shown. Qa-1 binds to the NK receptor CD94/NKG2A and CD94/ NKG2C, it is unlikely that these receptors account forbinding activitysince onlya smallproportionof CD8cells(less than 10%) express these receptors before or afteractivation [8, 9]. Another candidate receptor is theNKG2D-DAP10 receptor complex, which serves asreceptor for the stress-inducible MHC-like moleculeMICA [10, 11]; however, COS7 cells successfully trans-fected with murine NKG2D do not acquire binding activ-ity for Qa-1/peptide tetramers (data not shown). It islikely that the CD8:Qa-1 interaction documented in thestudies reported here may reflect the activity of an as yetundefined receptor(s) expressed during CD8 develop-ment. Definition of this receptor should allow additionalinsight into the molecular basis of the CD8 phenotypeand the developmental relationship of these cells tothose of the NK lineage. 3 Materials and methods 3.1 Animals and cell lines C57BL/6J (Qa-1b), C57BL/6-Tla (Qa-1a), A.J. (Qa-1a),BALB/c (Qa-1b), and HY transgenic (C57BL/6 background)mice were purchased from The Jackson Laboratory (BarHarbor, ME) and maintained in the Dana-Farber CancerInstitute Animal Facility. 0805B cells (a BALB/c   g    -galactosidase-specific CD8 + cytotoxic T cell line), p815 (aDBA/2 mastocytoma cell line), CPAG60 (p815 cells stablytransfected with pSP control vector) and p13.1 (p815 cellsstably transfected with pSP vector expressing   g    -galactosidase) [5] were incubated in RPMI 1640 mediumsupplemented with 10% fetal bovine serum, 100 U/ml peni-cillin, 50   ?  g/ml streptomycin, and 50   ?  M   g    -merca-ptoethanol.90 R. Wang et al. Eur. J. Immunol. 2001.  31:  87–93  Fig. 4.  Effect of Qa-1 tetramer binding on IFN-   +   production by CD8 cells. (A) IFN-   +   production of 0805B CD8 cells in the presenceof anti-CD3 and Qa-1/Qdm tetramer. 0805B cells (2×10 4  ) were incubated with 12.5 ng/ml anti-CD3 mAb for 48 h in the absenceor presence of Qa-1b/Qdm un-denatured or denatured tetramer before supernatants were assessed for IFN-   +   production byELISA. (B) IFN-   +   production of CD8 cells from female anti-HY TCR tg mice incubated with syngeneic female LPS-activated Bcells with the indicated preparation of Qa-1/Qdm tetramer. (C) IFN-   +   production by CD8 cells from female anti-HY TCR tg miceincubated with syngeneic female LPS-activated B cells with or without HY peptide and Qa-1/Qdm tetramer or monomer. (D) CD8cells from C57BL/6 mice incubated with syngeneic LPS-activated B cell blasts in the presence of mAb against Qa-1b or anisotype-matched control mAb (anti-TNP) before assessment of IFN-   +   production by ELISA. 3.2 Antibodies and reagents Cychrome-conjugated 135–2C11 (anti-CD3  4   ), fluoresceinisothiocyanate (FITC)-conjugated GK1.5 (anti-CD4), FITC-anti CD3, Ly-2 (anti-CD8), PK136 (anti-NK1.1), DX5 (anti-NK), M1/70 (anti-Mac-1), RA3–6B2 (anti-B220) mAb andsodium azide-free, endotoxin-free anti-Qa-1b, anti-TNP(isotypic control) and 17A2 (anti-CD3 complex) mAb werepurchased from PharMingen (San Diego, CA). Fetal calfserum,   g  -mercaptoethanol, L-arginine, aprotinin, pepstatin A, glutathione reduced form, glutathione oxidized form, dis-odium EDTA and urea were purchased from Sigma ChemicalCompany (St. Louis, MO). Qdm and preproinsulin peptideswere synthesized by the Biopolymers laboratory, HarvardMedical School. 3.3 Generation of Qa-1a and Qa-1b tetramers The Qa-1a expression vector pET-23a/Qa-1a was con-structed using RT-PCR to amplify a truncated Qa-1a mole-cule (amino acids 1–279) from a cDNA library prepared fromC57BL/6-Tla mouse splenocytes. The oligonucleotide prim-ers (5’-gcg cat atg AGT TCA CAT TCG CTG-3’ and 5’-actaag ctt GGA TGG AGG AGG CTC-3’) were used for theamplification of the Qa-1a cDNA (Nde I and BamH I sitesunderlined). After digestion with Nde I and BamH I, the Qa-1a PCR fragment was cloned into Nde I/BamH I digestedpET-23a/Qa-1b vector [12]. The integrity of the Qa-1a cDNA and the biotinylation site in the pET-23a vector was verifiedby DNA sequencing. The pET-23a/Qa-1a and pET-23a/Qa-1b vectors [6] were transformed into  Escherichia coli   strainEur. J. Immunol. 2001.  31:  87–93 Definition of a novel binding site on CD8 cells 91
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