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TCR Chain That Forms Peptide-Independent Alloreactive TCR Transfers Reduced Reactivity with Irrelevant Peptide/MHC Complex¹

Fabio R. Santori^2,3,*, Zoran Popmihajlov^2,4,*, Vladimir P. Badovinac, Courtney Smith, Sasa Radoja, John T. Harty and Stanislav Vukmanovi^5,*,

^* Michael Heidelberger Division of Immunology, Department of Pathology and New York University Cancer Center, New York University School of Medicine, New York, NY 10016; Department of Microbiology, University of Iowa, Iowa City, IA 52242; and Center for Cancer and Immunology Research, Children’s Research Institute, Children’s National Medical Center, Washington, DC 20010

	Abstract

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A major feature of the TCR repertoire is strong alloreactivity. Peptides presented by allogeneic MHC are irrelevant for recognition by a subset of alloreactive T cells. To characterize peptide-independent TCRs at the molecular level, we forced the expression of a TCR chain isolated from a peptide-independent alloreactive CD8⁺ T cell line. The alloreactive TCR repertoire in the transgenic mouse was peptide dependent. However, analysis of essential TCR contacts formed during the recognition of self-MHC-restricted Ag showed that fewer contacts with peptide were established by the transgenic TCR chain, and that this was compensated by additional contacts formed by endogenous TCR chains. Thus, reduced interaction with the peptide appears to be a transferable feature of the peptide-independent TCR chain. In addition, these findings demonstrate that reactivity to peptides is preferred over the reactivity to MHC during the formation of the TCR repertoire.

	Introduction

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The TCRs recognize short peptide fragments presented by MHC molecules. Peptides are held in the groove formed by the -pleated sheets (floor) and two helices (sides) generated by the 1 and 2 domains of the class I H chain or 1 and 1 domains of MHC class II. The TCR interacts with the top surface of this structure, composed of the peptide and both helices, and, in doing so, interacts with residues of both the peptide and MHC H chain (1, 2, 3, 4). The TCR is composed of two polypeptide chains, and , both of which make contacts with peptide, and thereby jointly contribute to the Ag specificity of the TCR. All peptide/MHC-TCR interactions resolved structurally to date follow the common docking mode, differing only by up to 35 degrees in orientation (1, 2, 3, 4). The TCR chains are generated by random rearrangements at TCR and TCR loci (5, 6). Thymic selection shapes the germline TCR repertoire, whose potential complexity exceeds the number of T cells in the peripheral lymphoid organs (7). The purpose of thymic selection is to allow survival of T cells with TCRs capable of interacting with self-MHC molecules complexed to foreign peptides. This results in MHC restriction of immune responses and recognition of Ags presented by self-MHC molecules and not by other alleles of the same species.

Even though the TCR repertoire is self-MHC restricted, it is also characterized by alloreactivity. It is widely accepted that alloreactivity is a consequence of cross-reactive recognition by self-MHC-restricted T cells (3, 8). Indeed, some T cell clones specific for an Ag presented by self-MHC also showed cross-reactive recognition of alloantigens (9, 10, 11, 12). Paradoxically, although alloreactivity can be explained as a by-product of self-MHC restriction, alloreactive T cell responses are much stronger than responses to foreign Ags restricted by self-MHC. The high frequency of alloreactive T cells was proposed to be due to recognition of the diverse self-peptides presented by allogeneic MHC molecules (13), or recognition of polymorphic residues of allogeneic MHC molecules themselves (14). In the latter case, the strong alloreactivity would be a consequence of high determinant density. Experimental support for both peptide-specific (15, 16, 17, 18) and peptide-independent alloreactivity has been obtained (19, 20).

Although relatively infrequent (21), peptide-independent TCRs are of interest for two reasons. First, they are more abundant when alloreactivity is induced in vivo (20). Therefore, understanding peptide-independent recognition may be relevant for pathogenesis of allorejection and graft-versus-host disease. Second, peptide-independent allorecognition appears to violate the principles of TCR binding to its physiological ligand, peptide(s) presented by MHC. However, recognition by peptide-independent TCRs has scarcely been studied at the molecular level. A key unresolved question is whether peptide-independent interaction is an accidental consequence of cross-reactivity limited to recognition of particular alloantigen, or whether reduced reactivity with peptides is more general and, thus, potentially caused by unique structural feature(s). To address this question, we isolated the TCR chain from a peptide-independent ₂-microglobulin (₂m)^6–/–CD8⁺ alloreactive T cell line (22) and determined the contacts of the ectopically expressed TCR chain with a peptide presented by self-MHC. Transgenic expression of this chain produced a peptide-reactive TCR repertoire, with peptide contacts established mainly by endogenous TCR chains, suggesting that the reduced capacity of transgenic TCR to interact with peptides may be a structural feature of peptide-independent TCR.

	Materials and Methods

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Mice and in vivo manipulations

₂m^–/– or ₂m^+/+ mice on the C57BL/6 background and BALB/c mice were all purchased from Taconic Farms. B6.C-H2^bm12/KhEgJ (bm12), TAP1^–/–, and RAG-1^–/– mice were obtained from The Jackson Laboratory. To induce syngeneic responses to OVA_257–264, 15 x 10⁶ of wild-type (WT) or TCR-transgenic spleen cells were injected i.v. into RAG-2-deficient hosts. The recipient mice were immunized within 48 h with an actA-deficient strain of Listeria monocytogenes (LM)-expressing chicken OVA (actA-LM-OVA). Seven days later, spleen cells of immunized mice were used for intracellular staining for IFN- as described previously (23). All experiments using laboratory animals have been approved by the Institutional Animal Care and Use Committees of New York University School of Medicine, Children’s Research Institute and University of Iowa.

TCR chain cloning and transgenic mouse construction

Total mRNA was isolated from 1 x 10⁶ MD6 cells by TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer’s recommendations. The first-strand cDNA synthesis was performed using a Super Script Preamplification System for First Strand cDNA Synthesis (Invitrogen Life Technologies). cDNA was amplified by the following primers in the noncoding regions of the TCR gene: 1) C, 5'-GCGCTCAGGATGCATAAAAG-3' and 2) V2, 5'-CCCCACAGAGATAGAGAGAACCTG-3'. PCR products were cloned in the TA cloning vector kit (Invitrogen Life Technologies). Two clones were sequenced until all base calls could be assigned without ambiguity. The insert prepared by digestion using enzymes BamHI and EcoRV was cloned into the SalI (subsequently blunt-ended)-BamHI opening of the plasmid pHSE3' (24). TCR clones used for the transgenic experiment were sequenced again to eliminate any eventuality of mutations. A fragment of 6.1 kb, obtained by XhoI digestion and double purification on 0.8% agarose gel, was used for microinjection into the C57BL/6 fertilized oocytes, performed at the New York University School of Medicine transgenic facility. Transgenic progeny (designated MTB) was initially screened using the same primers used to identify the original insert. Later, screening was performed using FITC-labeled anti-V2 mAb. All experiments were performed with mice carrying the transgene in a homozygous fashion.

Reagents, cells, and flow cytometry

Isolation of peptides from P815 cells and fractionation using HPLC have been described (25). Hybridoma secreting mAb 64-3-7 specific for the open forms of H-2L^d was provided by Dr. T. Hansen (Washington University, St. Louis, MO). Indirect immunofluorescence staining was performed using a 64-3-7 hybridoma tissue culture supernatant, followed by FITC-conjugated goat anti-mouse Ig (BD Pharmingen). PE-conjugated anti-CD4, CyChrome-conjugated anti-CD8, FITC-conjugated anti-V2, anti-V5 (5.1 and 5.2), and anti-V8 (8.1, 8.2, and 8.3) were purchased from BD Pharmingen, and direct immunofluorescence assays using these reagents was performed as previously described (26). The origin of MC57G and RMA-S cell lines transfected with the H-2K^d and H-2L^d, and generation of alloreactive CD8⁺ T cell lines, was previously described (25).

In vitro stimulation assays

Spleen cells from WT (5) or MTB mice (8 x 10⁴/well) were incubated for 5 days in flat-bottom 96-well plates in the presence of 8 x 10⁵ irradiated (2500 rad) syngeneic (C57BL/6) or allogeneic (bm12) spleen cells. During the last 16 h of culture, cells were pulsed with 0.5 µCi [³H]thymidine (Valeant Pharmaceuticals), and thymidine incorporation was subsequently measured using a beta scintillation counter 1450 MicroBeta (PerkinElmer Wallac). For generation of CTLs, 25 x 10⁶ spleen cells from WT or MTB mice were cultured for 5 days with the same number of irradiated (2,500 rad) BALB/c spleen cells or 2 x 10⁶ irradiated (25,000 rad) RMA-S or RMA-S-L^d cells. Cytotoxic T cell activity against various targets using the chromium release assay and loading of HPLC-derived peptide fractions onto RMA-S cells was performed as previously described (25).

	Results

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Transgenic expression of TCR chain from peptide-independent ₂m^–/–CD8⁺ cells

In the TCR-peptide/MHC structures resolved to date, the location and size of the CDR1, CDR2, and CDR3 footprints of the TCR chain vary less than the footprints of the TCR chain and, on average, TCR makes more contacts with MHC than TCR (1, 4). We therefore hypothesized that the TCR chain may contribute more than the TCR chain to the peptide-independent alloantigen recognition (although the contribution of TCR in peptide-independent allorecognition is not excluded). CD8⁺ T cell line MD6, originating from 2m^–/– mice, is specific for H-2K^d alloantigen irrespective of the peptide presentation, as revealed by the failure of a diverse set of peptides eluted from P815 cells (H-2^d) to increase the sensitization of MD6 cells (Ref. 22 ; see also Fig. 2A). We cloned the entire TCR chain from MD6 cells and placed it under the control of the H-2 promoter and the Ig enhancer (Fig. 1A). T cells from the transgenic mouse generated using this construct (hereafter referred to as MTB mouse) expressed high levels of V2 (Fig. 1B). The numbers of CD4⁺ and CD8⁺ T cells found in the peripheral lymphoid tissues of MTB mice were mildly lower than in the WT mice (Fig. 1C). The presence of the transgenic TCR chain prevented expression of endogenous TCR chains, as neither V5 nor V8 cell surface expression could be detected in CD8⁺ (Fig. 2) or CD4⁺ (data not shown) splenocytes. V5 and V8 were also undetectable on the surface of MTB thymocytes (data not shown), suggesting that allelic exclusion was efficiently induced in MTB mice by the transgenic TCR chain.

View larger version (39K): [in this window] [in a new window]   FIGURE 2. Transgenic TCR chain prevents expression of TCR chains encoded by endogenous BV5 and BV8 families. MTB and WT spleen cells were stained with PE-conjugated anti-CD4, CyChrome-conjugated anti-CD8, and FITC-conjugated anti-V2, anti-V5, or V8 mAbs and analyzed by flow cytometry. Shown are representative dot plots of anti-CD8 and anti-V2, anti-V5, or V8, as indicated.

View larger version (35K): [in this window] [in a new window]   FIGURE 1. Generation of the MTB transgenic mice. A, Schematic representation of the TCR construct. TCR cDNA () was placed under the control of a mouse H-2 promoter/Ig enhancer-containing DNA segment in pHSE3' vector. The human -globin gene fragment provides the cDNA with an intron and polyadenylation signal. B, Expression of the TCR transgene as revealed by immunofluorescent staining of peripheral blood cells using the V2-specific mAb. Shown are the profiles of a transgenic mouse (lower histogram) and of a nontransgenic littermate (upper histogram). C, MTB and WT spleen cells were stained with PE-conjugated anti-CD4 and CyChrome-conjugated anti-CD8 and analyzed by flow cytometry. Shown are representative dot plots.

View larger version (12K): [in this window] [in a new window]   FIGURE 3. Characterization of allogeneic responses and TCR expression in MTB mice. WT or MTB spleen cells (H-2b) were stimulated for 5 days in vitro with irradiated BALB/c (H-2d) spleen cells. Cytotoxic activity of cultured cells was tested for lytic activity against 51Cr-labeled parental (P) MC57G (left) or RMA-S (right) cells, or their H-2Kd- or H-2Ld-transfected variants, as indicated.

To address the issue of the peptide dependence of alloreactive MTB CD8⁺ T cells, RMA-S-K^d cells loaded with distinct peptide fractions eluted from P815 cells (H-2^d) were offered as targets to MTB or WT effector cells generated by stimulation with BALB/c spleen cells (H-2^d). Peptide fractions increased the lysis mediated by MTB effector cells (Fig. 4A), suggesting that the alloreactivity of MTB CD8⁺ T cells was peptide dependent. Even reactivity against RMA-S-K^d and RMA-S-L^d cells in the absence of any exogenously added fractions most likely reflects interactions with peptides, because MTB CD8⁺ T cells stimulated with RMA-S-L^d cells were unable to lyse the TAP-proficient P815 targets (Fig. 4B). TAP-proficient cells in general have more peptide-free MHC class I molecules at the cell surface than the TAP-deficient cells, even though the proportion of peptide-free molecules is larger in TAP-deficient cells (27). This is also true in our model, because staining of P815 cells with an Ab specific for peptide-free H-2L^d (28, 29) is brighter than that of RMA-S-L^d (Fig. 4C). Therefore, the most likely explanation for selective lysis of RMA-S cells by MTB T cells is the recognition of the peptide(s) presented in a TAP-independent manner that is competitively displaced in the presence of TAP. Peptide-dependent allorecognition also applies to MTB CD4⁺ T cells. H-2^bm12 differs from H-2A^b by three amino acids buried in the peptide-binding cleft and unavailable for direct contact with the TCR. MTB spleen cells proliferated to irradiated bm12 stimulator cells (Fig. 4D), suggesting that alloreactive responses of CD4⁺ T cells are directed either to different self-peptides presented by H-2^bm12 and/or to peptide-induced conformations unique to H-2^bm12. It therefore appears that alloreactive T cells in MTB mice are directly and/or indirectly peptide specific.

View larger version (27K): [in this window] [in a new window]   FIGURE 4. MTB TCR repertoire is dominated by peptide-dependent alloreactivity. A, Lysis of H-2Kd-transfected RMA-S targets by the original MD6 cell line or the effector MTB cells stimulated by H-2d splenocytes. The assay was performed in the presence or absence of peptide-containing fractions obtained from P815 (H-2d) cells. RMA-S-Kd cells were cultured overnight at room temperature to up-regulate MHC class I, labeled with 51Cr, and then incubated with various peptide-containing fractions for 1 h at room temperature. E:T ratios were 3 for MD6 and 20 for MTB or WT effector cells. B, MTB spleen cells were stimulated for 5 days in vitro with irradiated BALB/c spleen cells (left) or RMA-S-Ld cells (right). Cultured cells were then tested for CTL activity against indicated targets. (RMA is a TAP-sufficient, parental version of RMA-S cells and is of the H-2b haplotype). C, Immunofluorescence analysis of P815, RMA-S-Ld, and parental RMA-S cells was performed using anti-H-2Ld-specific mAb 64-3-7, followed by FITC-anti-mouse Ig () or secondary Ab alone (). D, WT or MTB spleen cells were cultured for 5 days in the presence of irradiated B6 or bm12 spleen cells. Proliferation of responder cells was measured by tritiated thymidine uptake. Shown are the mean and SDs of triplicate cultures.

Peptide reactivity of the MTB TCR repertoire could be imposed by endogenously rearranged TCR chains. Alternatively, the transgenic TCR may be capable to interact with peptides in general, despite its involvement in the peptide-independent recognition of H-2K^d. To determine the extent to which the transgenic TCR chain is able to interact with peptides, we compared the major TCR contacts in MTB and WT CD8⁺ T cells. The TCR chain interacts with the N-terminal, whereas the TCR chain interacts with the C-terminal peptide portion (1). We chose to evaluate the CD8⁺ T cell response to OVA_257–264 as a model epitope, because the TCR contacts for this Ag in WT CD8⁺ T cells are well established (30), and because the TCR contacts are less degenerate during recognition of foreign Ag presented by self-MHC (31). WT or MTB spleen cells were injected i.v. into RAG-1-deficient hosts, which were subsequently immunized with actA-deficient LM-OVA (32). The use of the actA-deficient strain allows high-dose immunization, even of immunocompromised strains (33). Seven days after infection, spleen cells were stimulated in vitro with OVA_257–264 or with alanine-replacement variant peptides. The proportion of T cells secreting IFN- was determined. As expected, the major TCR contacts for WT TCRs were at positions 4, 6, and, to a lesser extent, 7, because mutations of these positions resulted in 90, 98, and 62% reduction of the response to original OVA_257–264, respectively (Fig. 5). In addition, contact at position 3 was important for about one-half of the WT CD8⁺ T cells (47% reduction). In contrast, major TCR contacts for MTB CD8⁺ T cells were at positions 1, 3, 4, and 6 (95, 90, 97, and 97% reduction, respectively). Therefore, there is a shift of major TCR contacts in MTB CD8⁺ T cells toward the N-terminal portion of the antigenic peptide, suggesting greater, but not exclusive, involvement of TCR in peptide interactions. This finding suggests that reduced interaction with the peptide is a fixed feature of the transgenic TCR not limited to the recognition of alloantigen.

View larger version (35K): [in this window] [in a new window]   FIGURE 5. Shift of essential TCR contacts with peptide in MTB T cells. WT or MTB spleen cells were transferred to RAG-1–/– mice that were subsequently challenged with actA-LM-OVA. Seven days later, spleen cells from recipient mice were stimulated with OVA257–264 or individual variant peptides replacing original amino acid with alanine. Stimulated cells were stained for cell surface CD8 and Thy1.2 and intracellular IFN- content. A, Individual contour plots with numbers indicating percentage of cells stained positive for both markers (left, WT; right, MTB). B, Cumulative data (means of triplicate samples and SDs) displaying responses to variant peptides as percentage of the response obtained with the original OVA257–264.

	Discussion

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The MTB TCR chain does not intrinsically favor reactivity for the original Ag T cells from MTB mice display similar levels of reactivity to the original alloantigen (H-2K^d) and an alloantigen not recognized by the original T cell line (H-2L^d). Consistent with this notion, no deletion of MTB CD4⁺CD8⁺ thymocytes was evident when MTB mice were bred to the H-2^d (BALB/c) background (data not shown). This phenotype distinguishes the MTB mice from various TCR-transgenic mice generated in the past that demonstrated selectively enhanced T cell responses and/or precursor frequency of T cells specific for the original Ag (34, 35, 36, 37, 38, 39). The difference between these and the present studies is in the specificity of the original TCR, which in the earlier studies were individual peptide-MHC complexes, while in the present case, is the H-2K^d allogeneic MHC molecule irrespective of its association with specific peptides. Consequently, TCR chains in the earlier studies transferred to the recipient cells’ propensity to react with the original Ag, whereas the MTB TCR chain appears to have transferred reduced reactivity with peptides. It should be noted that the successful transfer of reduced reactivity with the peptide does not necessarily imply that the TCR chain is the only or the critical component predisposing peptide-independent alloreactivity in MD6 cells. The possibility of transfer of reduced reactivity with peptides using the MD6 TCR chain cannot be ruled out at present, although the room for potential compensation in reactivity with peptides (at least in the case of OVA_257–264) by endogenous TCR chains appears to be limited.

The finding that recognition of OVA_257–264 by the population of MTB CD8⁺ T cells does not depend on contacts with peptide position 7, whereas the same contact is required by the majority of OVA_257–264-specific WT CD8⁺ T cells suggests that transgenic TCR indeed has generally reduced the ability to interact with peptides. Although the requirement for the original amino acid at position 6 may be due to direct interactions of TCR chains or indirect effects of the peptide on the MHC class I molecule conformation, the most likely implication is the direct contact of the transgenic TCR with peptide. Thus, the interactions of the TCR chain with peptide are not completely prevented, suggesting that the designation of alloreactive TCRs as "peptide independent" does not necessarily imply complete absence of contacts with peptides. In contrast, the ability of TCRs in MTB mice to productively interact with MHC-presented peptides suggests that the transgenic TCR does not prevent interactions with peptide-filled MHC complexes. Hence, peptide-independent recognition is not necessarily based on interactions with "empty" MHC molecules that would involve interaction with an MHC determinant covered or destroyed by the bound peptide(s) or protrusion of peptide-independent TCRs into the peptide-binding cleft, thereby preventing the TCR interaction when peptides are bound. Rather, peptide-independent recognitions appear to involve interactions with peptide-filled MHC molecules, where the energy of binding is most likely primarily drawn from contacts with the MHC molecule itself. It will be interesting to uncover the structural basis of reduced peptide reactivity. Perhaps a depression in the central portion of the TCR chain surface disables some contacts with peptides, but not with the MHC molecules.

In conclusion, we demonstrated in this study that the TCR chain, with a reduced ability to contact the irrelevant self-MHC-restricted epitope, is a part of peptide-independent alloreactive TCR, suggesting that peptide-independent interaction, at least in some cases, may be due to structural limits. TCR chains with extra contacts with peptides are paired with this TCR to achieve the essential goal of T cell immunity, to specifically interact with peptides presented by self-MHC. Peptide-independent alloreactivity in the light of these findings is likely a chance matching of a "peptide interaction-impaired" TCR (or TCR) and a TCR (or TCR) that is generally peptide reactive, but specifically unable to interact with peptides presented by a given allogeneic MHC molecule. Thus, despite the variability of individual TCR chains in the extent of peptide contacting, the bias of the TCR repertoire for contacting peptides is ensured through the compensatory selection of the TCR chains (that rearrange later than the TCR) from the opposite end of the spectrum of peptide reactivity.

	Acknowledgments

 
We thank Steve Jameson for donation of the OVA_257–264 mutant peptides, Ted Hansen for donation of the 64-3-7 hybridoma cell line, Ed Palmer for providing the pHSE3' vector, and John Hirst for the FACS analysis.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grants AI48837 and AI41573 (to S.V.) and AI42767 (to J.T.H.).

² F.R.S. and Z.P. contributed equally to this work and should both be considered first authors.

³ Current address: Skirball Institute for Molecular Medicine, Department of Pathology and New York University Cancer Center, New York University School of Medicine, 540 First Avenue, New York, NY 10016.

⁴ Current address: Department of Medicine, Division of Immunology, Weill Medical College of Cornell University, 515 East 71st Street, S-222, New York, NY 10021.

⁵ Address correspondence and reprint requests to Dr. Stanislav Vukmanovi, Center for Cancer and Immunology Research, Children’s Research Institute, Children’s National Medical Center, 111 Michigan Avenue NW, Washington, DC 20010. E-mail address: svukmano{at}cnmc.org

⁶ Abbreviations used in this paper: ₂m, ₂ microglobulin; WT, wild type; LM, Listeria monocytogenes.

Received for publication December 19, 2006. Accepted for publication March 5, 2007.

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