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The Journal of Immunology, 2003, 170: 4506-4514.
Copyright © 2003 by The American Association of Immunologists

Homology Between an Alloantigen and a Self MHC Allele Calibrates the Avidity of the Alloreactive T Cell Repertoire Independent of TCR Affinity1

Tara M. C. Hornell2, Nancy Myers, Ted H. Hansen and Janet M. Connolly3

Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The self-restricted T cell repertoire exhibits a high frequency of alloreactivity. Because these alloreactive T cells are derived from the pool of cells selected on several different self MHC alleles, it is unknown how development of the alloantigenic repertoire is influenced by homology between a self MHC allele and an alloantigen. To address this, we used the 2C transgenic TCR that is selected by Kb, is alloreactive for Ld, and cross-reacts with Lq. Lq is highly homologous to Ld and binds several of the same peptide ligands, including p2Ca, the peptide recognized by 2C. We find that Ld/p2Ca is a high avidity agonist ligand, whereas Lq/p2Ca is a low avidity agonist ligand for 2C T cells. When mice transgenic for the 2C TCR are bred to Lq-expressing mice, 2C+ T cells develop; however, they express lower levels of either the 2C TCR or CD8 and require a higher Ld/p2Ca ligand density to be activated than 2C+ T cells selected by Kb. Furthermore, the 2C T cells selected in the presence of Lq fail to detect Lq/p2Ca complexes even at high ligand density. Thus, despite possessing the identical TCR, there is a functional avidity difference between 2C+ T cells selected in the presence of Lq vs Kb. These data provide evidence that homology between the selecting ligand and an alloantigen can influence the avidity of the T cell repertoire for the alloantigen, and suggest that thymic selection can fine tune T cell avidity independent of intrinsic TCR affinity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Through a series of well-orchestrated events during positive and negative selection, the {alpha}{beta} T cell repertoire emerges with the ability to discriminate between self and nonself with exquisite specificity. A prevailing model of T cell development posits that thymocytes with low affinity/avidity for self MHC/peptide complexes will be positively selected and mature, whereas thymocytes with high affinity/avidity for self MHC/peptide complexes are negatively selected and die (1). In this way, positive selection ensures that the selected repertoire is MHC specific and restricted, while negative selection guarantees self tolerance. However, the T cell repertoire is also degenerate in that the self-restricted T cells are capable of recognizing allogeneic MHC Ags at a high frequency relative to any one self MHC/peptide complex. It is assumed that self-restricted T cells are selected in the thymus on the same self MHC allele with which they interact, whereas alloreactive T cells are derived from the pool of T cells selected on several self MHC alleles that differ from the alloantigenic MHC. Thus, T cells selected on multiple self MHC alleles contribute to the response to a single alloantigen. However, the contribution of an individual self allele to the development of the alloreactive repertoire is unknown.

Studies addressing the impact of self MHC molecules on the alloreactive repertoire are limited. In vitro studies suggest that when allo- and self MHC alleles are more homologous, the alloreactive repertoire is more peptide specific, while the alloreactive repertoire is less dependent upon peptide when allo- and self MHC alleles are more divergent (2). In vivo, constraint of the self MHC/peptide repertoire to a single complex results in the development of T cells that exhibit heightened reactivity to foreign MHC, suggesting that the TCR repertoire may be inherently MHC reactive and selection events may limit this repertoire (3). However, the impact of self MHC molecules on the development and function of alloreactive T cells has not been systematically examined.

Of particular interest is how homology between a self and an alloantigenic MHC allele influences the quantity and quality of the positively selected T cells reactive with the alloantigen. To address this issue, we used a TCR transgenic system for which the alloantigen, the peptide ligand, and the selecting allele are known. The 2C TCR is positively selected by Kb and reacts with high affinity with Ld complexed with the p2Ca peptide (4, 5, 6, 7, 8). We show in this study that 2C T cells cross-react with Lq; however, the avidity of this interaction is much lower than that of 2C T cells with Ld/p2Ca. This low avidity interaction between 2C T cells and Lq/p2Ca suggested that Lq would allow positive selection of the 2C TCR in Lq-expressing mice. This suggestion is supported by our previous observation that the low avidity interaction between 2C T cells and cells from mice that express the agonist ligand Ld at 2% of normal levels results in positive selection of functional 2C T cells (9). In the present study, selection of the 2C TCR was compared in mice that express either Kb, Kb and Lq, or Lq. The 2C TCR+ T cells were identified using a clonotypic Ab, and thus possessed identical TCR structures and the same intrinsic TCR affinity for the cognate ligand, Ld/p2Ca. The 2C TCR+ T cells that developed in the presence of different alleles were analyzed to determine their functional avidity for Ld/p2Ca and Lq/p2Ca. Interestingly, 2C+ T cells that develop in the presence of Lq, despite having the identical TCR, have a decreased functional avidity for Ld/p2Ca compared with 2C+ T cells selected on Kb in the absence of Lq, and fail to recognize Lq/p2Ca. It is clear that the presence of Lq has a profound effect on the Ld-reactive repertoire and suggests that T cell avidity can be calibrated independent of the intrinsic TCR affinity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

B10.AKM (H-2m; KkDqLq) and BALB/c (H-2d) mice were obtained from The Jackson Laboratory (Bar Harbor, ME) or Charles River Laboratories (Wilmington, MA). The 2C TCR transgenic mice (H-2b) contain the rearranged transgenes encoding the TCR from the 2C CTL clone (6). All mice were housed and bred in the barrier animal facility at Washington University School of Medicine.

Peptides

The amino acid sequence of the murine CMV (MCMV)4 peptide corresponds to residues 168–176 (YPHFMPTNL) of the MCMV immediate early protein pp89 (10). The amino acid sequence of the tum- peptide corresponds to residues 14–22 (TQNHRALDL) of the mutant protein P91A (exon 4) from the tum- P815 tumor variant (11). The p2Ca (LSPFPFDL) and QL9 (QLSPFPFDL) peptides are derived from the endogenous mitochondrial protein {alpha}-ketoglutarate dehydrogenase (4, 5, 8). Peptides were synthesized using Merrifield’s solid-phase method (12) on a model 431A peptide synthesizer (Applied Biosystems, Foster City, CA). Peptides were purified (>98%) by reverse-phase HPLC, and purity was assessed (13).

Monoclonal Abs

28-14-8S is an IgG2a mAb specific for the {alpha}3 domain of several D region-encoded molecules, including Ld, Lq, Dq, and Db (14, 15). 30-5-7 is an IgG2a mAb that recognizes a determinant in the {alpha}2 domain of Ld that includes the amino acid residue at position 107 (14, 15, 16). 30-5-7 also recognizes Dq, but not Lq. B8-24-3 is an IgG1 mAb specific for Kb (American Type Culture Collection, Manassas, VA).

1B2 is a clonotypic mouse mAb specific for the 2C TCR (17, 18). The 1B2 mAb was purified from ascites and biotinylated using a protein biotinylation kit (Pierce Chemical, Rockford, IL). For two- and three-color flow cytometric analyses, the mAbs anti-CD8 PE, anti-CD4 FITC, and anti-CD16,32 (Fc Block) were used (all from PharMingen).

Cell lines

L-Ld and L-Lq were generated by introducing the Ld and Lq genes, respectively, into DAP-3 (H-2k), the murine Ltk- fibroblast cell line (19). R1.1-Ld, R1.1-Dq, and R1.1-Lq were generated by transfecting R1.1, a C58/J (H-2k) thymoma cell line, with the Ld, Dq, and Lq genes, respectively. The TAP-deficient RMA.S-Ld cell line was generated by transfection of the Ld cDNA into RMA.S (H-2b) cells (gift of J. Bluestone, University of California, San Francisco, CA). Cell lines were maintained at 37°C, 5% CO2, in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 2 mM L-glutamine, 10% (v/v) bovine calf serum (HyClone Laboratories, Logan, UT), 0.1 mM nonessential amino acids, 1.25 mM HEPES, 1 mM sodium pyruvate, and 100 U/ml penicillin/streptomycin. Transfected cell lines were grown in medium containing 0.6 mg/ml G418 (Geneticin; Life Technologies).

Cytotoxic T cell lines and clones

For experiments using non-TCR transgenic responder splenocytes, 7.5 x 106 responder splenocytes/well were cultured with 3.5 x 106 irradiated (2000 rad) BALB/c stimulator splenocytes/well. For experiments using 2C TCR+ transgenic responders, 3 x 106 responder splenocytes/well were cultured with 3.5 x 106 irradiated (2000 rad) BALB/c stimulator splenocytes/well, with or without exogenous p2Ca peptide (10-4 M) for 5 days in 24-well plates. Cultures were maintained in sensitization medium (RPMI 1640 medium supplemented with L-glutamine, sodium pyruvate, nonessential amino acids, 100 U/ml penicillin/streptomycin, 50 µM 2-ME, and 10% (v/v) FCS (HyClone Laboratories)). In some experiments, CTL lines generated from 2C TCR transgenic mice were used. After the first week in culture, the lines were plated at 5 x 105 cells/well and stimulated with 5 x 106 irradiated (2000 rad) BALB/c splenocytes/well in sensitization medium. After the second week, the CTL lines were restimulated in the additional presence of 10 U/ml murine rIL-2 (Biosource, Camarillo, CA).

The Ld-alloreactive CTL clone 2C was generated from a BALB.B (H-2b) anti-H-2d response (20) and was a gift from H. Eisen (MIT, Cambridge, MA). The 2C clone is V{beta}8+, 1B2+, and is specific for Ld/p2Ca (4, 20). The clone 42F3, derived in vitro from a dm2 (Ld loss mutant) anti-BALB/c response, is V{beta}8+, 1B2-, and is specific for Ld/p2Ca (21). The Ld-restricted, MCMV-specific CTL clones D7, A8, 1C6, 2C4, and 1F5 were generated, as previously described (22, 23, 24, 25). Clones D7 and A8 were generated from BALB/c splenocytes, while clones IF5, 1C6, and 2C4 were generated from DBA/2 (H-2d) splenocytes. All clones were maintained in 24-well plates in sensitization medium and stimulated weekly with 2.5 x 106 irradiated (2000 rad) BALB/c splenocytes/ml and 10 U/ml murine rIL-2. Peptide-specific clones were supplemented with 10-5 M peptide. Primary CTL cultures were tested for functional activity on day 5 after initiation of the culture. Established clones and lines were tested on day 4 or 5 after restimulation.

Flow cytometry and peptide inductions

For cell lines, the cells were washed and plated in a 96-well plate at 4 x 105 cells/well. PBMC were isolated by Ficoll (Histopaque 1083; Sigma-Aldrich, St. Louis, MO) separation. Splenocytes and thymocytes were treated with ammonium chloride lysing buffer (ACK) to remove erythrocytes. The PBMC, splenocytes, and thymocytes were plated in a 96-well plate at 1 x 106 cells/well, preincubated on ice with Fc Block for 10–15 min, and washed with 100 µl PBS + 0.2% BSA (Sigma-Aldrich). Cells were incubated on ice in PBS containing 0.2% BSA, in the presence or absence of a saturating concentration of mAb, for 30 min, washed twice with 100 µl of PBS + 0.2% BSA, and incubated with a saturating concentration of FITC-conjugated, affinity-purified F(ab')2 of goat anti-mouse IgG Fc (ICN Pharmaceuticals, Costa Mesa, CA) or streptavidin-CyChrome (BD Biosciences, Mountain View, CA) for 30 min on ice. Cells were washed twice, and the viable cells, gated by forward and side light scatter, were analyzed on a FACScan flow cytometer (BD Biosciences). Mean fluorescence values were converted from logarithmic amplification by linear regression analysis using the CellQuest 30 software (BD Biosciences). A total of 10,000 events was collected for single-color analyses, 10,000–20,000 events for two-color analyses, and 20,000–50,000 events for three-color analyses.

For peptide inductions, L-Ld or L-Lq cells were cultured overnight at 37°C in the presence or absence of peptide in RPMI 1640 medium (Life Technologies) supplemented with 10% BCS (HyClone Laboratories), 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 1.25 mM HEPES, and 100 U/ml penicillin/streptomycin. After incubation, the cells were harvested, labeled, and analyzed, as described above. The EC50 values were determined by fitting the peptide-induced Ld or Lq surface induction curves with the standard Hill function: Bmax/(1 + (EC50/[peptide])n), in which Bmax is the maximal surface induction of Ld or Lq, EC50 is the molar concentration of the peptide that produces 50% of maximal surface induction of Ld or Lq, and n is the apparent cooperativity.

51Cr release assay

A total of 1 x 106 target cells was labeled for 1 h with 150–200 µCi of 51Cr (Na 51CrO4; 1 Ci = 37 GBq; NEN, Boston, MA) in 200 µl of RPMI 1640 medium + 10% bovine calf serum at 37°C in 5% CO2. Effector cells were plated at various concentrations into 96-well microtiter plates, and 2.5 x 103 or 5 x 103 washed target cells per well were added. In some experiments, the targets were resuspended in medium containing peptide before plating. The plates were centrifuged at 50 x g for 1 min and incubated for 4 h at 37°C in 5% CO2. Radioactivity in 100 µl of supernatant was measured in an Isomedic gamma counter (ICN Biomedicals, Huntsville, AL). The mean of triplicate samples was calculated, and percentage of 51Cr release was determined, according to the following equation: percentage of 51Cr release = 100 x ((experimental 51Cr release - control 51Cr release)/(maximum 51Cr release - control 51Cr release)), in which experimental 51Cr release represents counts from target cells mixed with effector cells; control 51Cr release represents counts from target cells in medium alone; and maximum 51Cr release represents counts from target cells lysed with 5% (v/v) Triton X-100 (Sigma-Aldrich). EC50 values were determined by fitting the specific lysis curves to the function: Bmax/(1 + (EC50/[peptide])n) + B0, in which Bmax is the maximal lysis, EC50 is the molar peptide concentration that produces 50% of maximal lysis, n is the apparent cooperativity, and B0 is the lysis in the absence of added peptide.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
2C T cells cross-react with Lq/p2Ca with low avidity

To determine how homology between MHC class I alleles influences T cell repertoire development, we used the 2C TCR transgenic system. We initially sought to determine whether the Ld-specific 2C T cell clone can recognize the highly related class I MHC molecule Lq. H-2Ld and H-2Lq are highly homologous class I molecules with 98% identity in amino acid sequence (26). Fig. 1 displays an amino acid sequence comparison of Ld, Lq, and several other murine MHC class I alleles. Ld and Lq differ by only 6 aa in the {alpha}2 domain: positions 95, 97, and 116 in the peptide-binding groove, positions 155 and 157 on the {alpha}-helix, shown to be important for interaction with TCR, and a serologic determinant at position 107 on a loop between the {alpha}-helix and {beta}-sheet (16). We previously showed that when the residues at 155/157 of Ld were changed to those found in Lq (LdY155H/R157K), the 2C T cell clone recognized this Ld mutant at high, but not low, peptide ligand density. This indicates that increased determinant density can compensate for poor TCR interaction (25), and suggested that 2C T cells may also cross-react with Lq.



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  		FIGURE 1. Amino acid sequence comparison of MHC class I alleles. The alleles are listed in order of their homology to Ld. Only the polymorphic {alpha}1 and {alpha}2 domains are shown. Conservative differences are highlighted in gray, and nonconservative differences are highlighted in black.

 
To assess whether 2C and other Ld-reactive T cells can also recognize Lq in the presence of high peptide ligand density, we initially established how well several Ld peptide ligands bind to Lq. Addition of exogenous peptide to Ld- and Lq-expressing L cells causes a concentration-dependent up-regulation of cell surface class I from which relative binding affinities can be determined. Such peptide induction experiments demonstrate that p2Ca and QL9 bind equivalently to Ld and Lq, MCMV binds 6-fold better to Lq than to Ld, and tum- binds Ld well and Lq poorly (Table I). Because p2Ca is derived from a ubiquitous protein and binds equally well to Ld and Lq, it is likely that Lq-expressing cells endogenously display Lq/p2Ca complexes. The functional avidity of 2C for Ld/p2Ca and Lq/p2Ca was compared by titering the amount of peptide required for target cell lysis in a cytolysis assay. As shown in Fig. 2, the EC50 for 2C recognition of Ld/p2Ca is 1.4 nM, while the EC50 for 2C recognition of Lq/p2Ca is 2 µM, a difference of three orders of magnitude (Fig. 2) (27). Thus, 2C cross-reacts with the homologous MHC molecule Lq, but with a much lower functional avidity than its recognition of Ld. Because the effector T cells are identical and the target cells only differ in expression of Ld vs Lq, this functional avidity difference is most likely due to a difference in the 2C TCR affinity for Ld/p2Ca vs Lq/p2Ca.


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  		Table I. Peptide binding to Ld vs Lq

 


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  		FIGURE 2. 2C recognizes Lq in the presence of exogenous peptide. A, 2C recognition of R1.1, R1.1-Ld, and R1.1-Lq targets in the presence (open symbols) or absence (closed symbols) of continuous 10-5 M p2Ca peptide. B, Peptide titration of 2C recognition of R1.1, R1.1-Ld, and R1.1-Lq targets. The assay was performed at an E:T ratio of 2:1 in the presence of continuous p2Ca peptide.

 
Ld-reactive clones, other than 2C, do not cross-react with Lq

Because 2C recognizes Lq (Fig. 2B) only at high peptide ligand density, we analyzed other Ld-reactive clones to determine whether they also could recognize Lq at high peptide ligand density. Seven Ld-restricted, MCMV-specific clones; one Ld-alloreactive, p2Ca-specific clone; and one Ld-alloreactive clone of unknown peptide specificity were tested for recognition of R1.1-Lq targets. None of the MCMV-specific clones, five of which are shown in Fig. 3, was able to cross-react with Lq, even at high ligand density. Of interest is the fact that the MCMV peptide binds better to Lq than to Ld (Table I). Although clones D7 and 1C6 were previously shown to recognize the Ld->Lq mutant, LdY155H/R157K at a high concentration of peptide (25), they do not recognize Lq even in the presence of continuous 10-5 M MCMV peptide. Thus, the observed difference in recognition cannot be overcome by increased peptide binding and is therefore due to a conformational difference between Ld/MCMV and Lq/MCMV. Either the differences in the peptide-binding groove of Lq are sufficient to induce a conformation of the MCMV peptide that cannot be recognized by D7 or 1C6, or the combination of amino acid differences in both the {alpha}-helices and the peptide-binding groove of Lq prevents recognition. In addition, the two Ld-alloreactive clones tested, 1C2 and 42F3, do not recognize Lq (Fig. 4A). 42F3 also fails to recognize Lq at high ligand density (Fig. 4B). 42F3 is one of several Ld/p2Ca-specific clones derived from a dm2 anti-BALB/c response (21) that were previously observed to possess a lower avidity for Ld/p2Ca than the 2C clone (not shown). Although 2C T cells cross-react with Lq, the other Ld-reactive T cells tested do not, even at high ligand density. Thus, the ability to interact with Lq at high peptide ligand density is unique to the 2C clone and is possibly due to the high affinity of the 2C TCR for Ld/p2Ca (8). This low avidity interaction between 2C and Lq/p2Ca suggested to us that the presence of Lq could influence the selection of the 2C TCR.



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  		FIGURE 3. MCMV-specific, Ld-restricted CTL clones fail to recognize Lq. MCMV-specific CTL clones were tested for recognition of R1.1, R1.1-Ld, and R1.1-Lq target cells in the presence of continuous 10-5 M MCMV peptide at E:T ratios of 5:1 (A8), 2:1 (D7), 2.5:1 (1F5), 2:1 (2C4), and 2.5:1 (1C6).

 


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  		FIGURE 4. Ld-alloreactive clones 1C2 and 42F3 do not cross-react with Lq. A, Clones 1C2, specific for Ld, and 42F3, specific for Ld/p2Ca, were assayed for recognition of R1.1, R1.1-Ld, and R1.1-Lq in the presence (for 42F3 open symbols) or absence (closed symbols) of 10-5 M p2Ca peptide. B, p2Ca peptide titration of recognition of R1.1, R1.1-Ld, and R1.1-Lq by 42F3 at an E:T ratio of 5:1.

 
The presence of Lq does not inhibit the ability of Kb to positively select 2C+ T cells

The lower avidity interaction between 2C and Lq/p2Ca compared with the high avidity interaction between 2C and Ld/p2Ca, which negatively selects the 2C TCR (6), led us to hypothesize that Lq may alter the selection of 2C TCR-expressing T cells. If the avidity of 2C for this weak agonist is sufficiently low, positive selection of 2C+ T cells would occur in the presence of Lq, whereas too high an avidity would cause negative selection. To address these possibilities, we examined positive selection of the 2C TCR by Kb in the presence or absence of Lq. 2C TCR transgenic mice (H-2b) were bred with the Lq+ mouse strain B10.AKM (H-2m, KkDqLq). Progeny were intercrossed, and the PBMC from the resulting F2 generation mice were screened by flow cytometry for expression of Lq, Kb, and the 2C TCR. The results of this analysis are shown in Table II for the 20 F2 mice. The proportion of each genotype of the F2 mice corresponds with predictions based on Mendelian genetics.


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  		Table II. MHC haplotypes and TCR specificity of the 20 (2C x B10.AKM)F2 mice

 
To determine the effect of Lq on positive selection of the 2C TCR by Kb, we used 1B2, the clonotypic mAb specific for the 2C TCR. The percentage of CD8+1B2+ T cells was compared between mice that express Kb alone and mice that express both Kb and Lq. Two-color flow cytometric analysis was performed on PBMC from two Kb+Lq-2C+ mice, four Kb+Lq+2C+ mice, and the two Kb-Lq+2C+ mice. Representative dot plots are shown in Fig. 5A. Although the percentage of CD8+1B2+ T cells is reduced in Kb+ mice that also express Lq, clearly a significant number of 2C TCR+ T cells are positively selected in the presence of Lq (mean of 27% for H-2b vs 19% for H-2bxm for all mice tested). Three-color analyses of thymocytes from H-2bxm mice show a large number of CD8 single-positive (SP) cells that express high levels of the 2C TCR (Fig. 5B). Thus, the expression of Lq does not interfere with the ability of Kb to positively select 2C+ T cells. However, the mean fluorescent intensity of CD8 expressed by PBMC was lower on T cells from Kb+Lq+ mice compared with Kb+ mice (1898 vs 2745). This decrease in both the number of CD8+1B2+ T cells and the level of expression of CD8 in the peripheral blood was confirmed with additional 2C+ H-2bxm mice. Although positive selection of 2C+ T cells does occur in the presence of Lq, the 2C T cells that develop are both quantitatively and qualitatively different from 2C T cells positively selected by Kb in the absence of Lq.



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  		FIGURE 5. 2C+, CD8+ T cells are positively selected in Lq-expressing mice. A, Representative two-color flow cytometric analysis of PBMCs from (left to right) 2C+ H-2b, 2C+ H-2bxm, 2C+ H-2m, and 2C- mice. Cells were stained with CD8 PE, biotinylated 1B2, and streptavidin-CyChrome. The percentage of each cell type is indicated in the quadrants. B, Representative three-color flow cytometric analysis of thymocytes from (left to right) 2C+ H-2b, 2C+ H-2bxm, 2C+ H-2m, and 2C- mice. Cells were stained with CD4 FITC, CD8 PE, biotinylated 1B2, and streptavidin-CyChrome. Expression of CD4 and CD8 is displayed in the top panels. Expression of the 2C TCR (1B2 staining) was determined after gating on CD4- CD8+ cells, and the histograms are shown below each CD4/CD8 dot plot. Each plot shows data from a single animal. Analyses from additional animals were performed.

 
The 2C+ T cells that develop in Kb-Lq+ (H-2m) mice express lower levels of either CD8 or TCR compared with 2C+ T cells that develop in Kb+Lq- (H-2b) mice

To examine whether 2C TCR-expressing T cells can develop in mice that express Lq and not Kb, thymocytes from the mice homozygous for H-2m were analyzed. As shown in Fig. 5B, 2C TCR+ T cells do develop in mice that are Kb-Lq+. However, there are fewer CD8 SP thymocytes in H-2m 2C+ mice compared with either H-2b or H-2bxm 2C+ mice, with the reduction paralleled by an increase in the number of CD4CD8 double-positive cells in the H-2m thymuses. The CD8 SP cells in H-2m 2C+ mice express high levels of the 2C TCR, indicating that positive selection of 2C+ T cells does occur.

Analysis of PBMC from the 2C+ H-2m mice revealed that, as in the thymus, there are fewer CD8+1B2+ cells in H-2m vs H-2b 2C+ mice (mean of 9 vs 27%, respectively, for all mice tested). Interestingly, these CD8+1B2+ cells fall into two populations. One population expresses high levels of the 2C TCR and a pronounced decrease in CD8 staining (mean fluorescence intensity of 1000 for H-2m vs 2745 for H-2b), whereas the other population expresses high levels of CD8 and severely reduced levels of the 2C TCR (mean fluorescence intensity of 451 for H-2m vs 1727 for H-2b). Thus, 2C+ T cells can develop in Lq-expressing mice, but they express lower levels of either CD8 or the 2C TCR. This suggests that these T cells possess a lower avidity than 2C T cells positively selected on Kb.

The 2C+ T cells that develop in H-2m mice recognize Ld, but not Lq, even at high ligand density

To determine whether functional 2C+ T cells were present, splenocytes from the 2C+ H-2b, 2C+ H-2bxm, or 2C+ H-2m, or 2C- mice were stimulated in vitro with BALB/c splenocytes in the presence or absence of exogenous p2Ca peptide. Primary T cell lines from Lq-expressing mice (either 2C+ H-2bxm or 2C+ H-2m) stimulated with BALB/c splenocytes in the absence of peptide recognized Ld/p2Ca-expressing targets well (Fig. 6). However, the response was much weaker than that generated by 2C+ H-2b anti-BALB/c in that ~8- to 32-fold more effector cells from 2C+ H-2m anti-BALB/c and 2C+ H-2bxm anti-BALB/c were required to obtain equivalent levels of lysis to that seen with 2C+ H-2b anti-BALB/c effectors. Primary T cell lines stimulated with BALB/c splenocytes and exogenous p2Ca peptide all showed similar high levels of lysis of Ld/p2Ca-expressing targets. This indicates that the 2C+ T cells that develop in the presence of Lq are functional. However, the 2C+ T cells selected in H-2m mice require a higher Ag dose to be activated in primary culture and for target cell recognition than 2C+ T cells selected in the exclusive presence of H-2b. This indicates that they possess a lower functional avidity. Consistent with this, none of the CTL lines generated from 2C+ H-2m or 2C+ H-2bxm mice recognizes Lq-expressing targets even in the presence of 5 x 10-6 M exogenous p2Ca, whereas the CTL lines from 2C+ H-2b mice recognize Lq/p2Ca well (Fig. 7).



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  		FIGURE 6. The 2C+ T cells that develop in the presence of Lq are low avidity. Splenocytes from 2C+ H-2b, 2C+ H-2bxm, 2C+ H-2m, and 2C- mice were stimulated with BALB/c splenocytes in the presence or absence of 10-4 M p2Ca. Each pair of graphs represents data from a single mouse. Cytotoxicity assays were performed using RMA-S-Ld target cells in the presence or absence of 2 x 10-5 M p2Ca.

 


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  		FIGURE 7. The 2C+ T cells that develop in the presence of Lq do not recognize Lq/p2Ca. CTL lines generated from 2C+ H-2b, 2C+ H-2bxm, or 2C+ H-2m mice were tested for recognition of R1.1, R1.1-Ld, R1.1-Lq, and R1.1-Dq. Assays were performed at an E:T ratio of 20:1, and for some of the targets continuous p2Ca peptide was present at a concentration of 5 x 10-6 M.

 
To further test this hypothesis and to quantitate the functional avidity difference, 2C T cells from H-2b and H-2m mice were analyzed to determine the amount of p2Ca peptide required to obtain half-maximal lysis of RMAS.Ld target cells. The 2C+ T cell lines generated from H-2m mice show a shifted response to peptide, in that they require more peptide for equivalent lysis, compared with 2C+ T cell lines from H-2b mice (Fig. 8). The EC50 for recognition of Ld/p2Ca by the 2C+ H-2b lines is 2 nM, while for the 2C+ H-2m lines the EC50 is on the order of 80 nM. These data indicate that the H-2m and H-2bxm 2C+ T cells differ from 2C+ T cells that develop in the exclusive presence of Kb in that they possess a lower functional avidity and do not detectably recognize self MHC, despite expressing the identical TCR. Together these data provide an example of fine tuning of T cell avidity during development independent of TCR affinity.



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  		FIGURE 8. Peptide titration of Ld/p2Ca recognition by 2C+ CTL generated from H-2b vs H-2m mice. CTL lines generated from 2C+ H-2b (A) or 2C+ H-2m (B) mice were tested for recognition of RMA-S-Ld target cells at an E:T ratio of 20:1 in the continuous presence of p2Ca peptide.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The allogeneic response is a secondary manifestation of the primary role of the MHC in self vs nonself discrimination. Disparity at the MHC leads to rapid rejection of transplanted tissue, and thus the alloreactive response continues to be a bane for transplantation. Despite recent progress delineating the molecular interactions involved in self-restricted responses, the molecular basis for cross-reactivity on allogeneic MHC remains poorly understood. We demonstrate in this study that homology between a self MHC allele and an alloantigen has a profound influence on the development of T cells reactive with the alloantigen. Using TCR transgenic mice, we show that this homology influences the avidity of the repertoire independent of intrinsic TCR affinity. Selection of the transgenic Ld-alloreactive 2C TCR by Kb leads to development of high avidity 2C TCR+ T cells, whereas selection in the presence of the homologous allele, Lq, leads to the development of low avidity 2C TCR+ T cells.

In the present study, the low avidity interaction of 2C T cells with Lq and the structural homology between Ld and Lq led us to question the impact of the presence of Lq on the development of 2C T cells that are, by the specificity of their TCR, reactive with the homologous alloantigen Ld, yet must maintain self tolerance to Lq. In an earlier study, the strong Ld-alloreactive response generated in Lq+ mice suggested that the T cell repertoire reactive with Ld was derived from the pool of T cells selected by Lq (21). This led us to hypothesize that Lq could positively select the Ld-alloreactive 2C TCR+ T cells. Due to the presence of other MHC alleles besides Lq in B10.AKM mice (H-2m; Kk, Dq, Lq), we cannot definitively determine whether Lq is responsible for the positive selection of 2C T cells in H-2m mice. Regardless, 2C T cells that develop in the presence of Lq are functionally and phenotypically distinct from those selected in the absence of Lq, despite expression of the identical TCR. We find that the presence of Lq has a very strong impact on selection of the 2C TCR by the positively selecting allele, Kb. In the presence of both Kb and Lq, 2C, T cells do develop; however, there are fewer 2C T cells selected and they express lower levels of CD8 than 2C T cells selected by Kb alone. Thus, in Kb-expressing mice, the presence of Lq most likely results in increased negative selection and the positively selected 2C T cells possess a lower avidity for ligand. In the presence of Lq and not Kb, even fewer T cells are positively selected. The 2C+ T cells that emerge into the periphery of H-2m mice have down-regulated either TCR or CD8, indicating that they also possess a lower avidity for ligand. In both cases, the 2C T cells that develop are functional in that they are capable of lysing Ld/p2Ca-expressing targets. However, they are not capable of lysing Lq-expressing targets even at high p2Ca ligand density. This observation is in contrast to the 2C T cells selected by Kb that cross-react with Lq/p2Ca at high ligand density. Thus, although the TCRs selected in the presence or absence of Lq are structurally identical and possess the same intrinsic affinity, the functional avidities of the positively selected 2C T cells differ. Only the low avidity T cells incapable of reacting with self MHC have matured. These results suggest that T cells can calibrate avidity, independent of intrinsic TCR affinity, as a means of establishing and maintaining tolerance.

T cell tuning during development is supported by earlier studies such as that by Sebzda et al. (28), in which they used TCR transgenic T cells specific for LCMV p33 presented by Db. The authors found that T cells positively selected in fetal thymic organ culture by the variant peptide A4Y, a moderate agonist, react strongly with the native p33, but are unable to react with A4Y. Thus, the transgenic thymocytes are positively selected and modified so that they are tolerant to A4Y. In a study from our lab, we showed that 2C T cells positively selected on very low levels of the agonist, Ld, were unable to recognize endogenous levels of Ld/p2Ca, but elicited functional CTL when exogenous p2Ca was added (9). Along with our data presented in this work, these studies using transgenic TCR systems demonstrate that T cells are capable of modifying their activation thresholds during thymic maturation, such that the overall avidity is calibrated to permit positive selection and to avoid autoimmunity. Our data suggest that one way in which avidity can be regulated is by down-regulating either CD8 or TCR expression. A question that remains is whether this is the result of active regulation of receptor expression or the result of specific selection of cells within a narrow range of avidities.

Earlier studies have shown that expression levels of either CD8 or TCR influence the outcome of thymic selection. TCR up-regulation normally accompanies thymic development and is thought to be important for negative selection of T cells reactive with abundant self peptides (1). Increased expression of CD8 during development through transgenic expression of CD8 results in enhanced negative selection of transgenic T cells (29, 30). However, in these cases, the functional effect on the activation threshold was not examined. In the present study, we observe down-regulation of expression of TCR or CD8, in this case by varying the selection environment of the 2C TCR to include an allele homologous to Ld. Selection in the presence and absence of the homologous allele led to the development of naive peripheral T cells that possess the identical TCR, but different functional avidities and therefore different activation thresholds. Of interest is the fact that these avidity differences remained constant during several months of culture. Furthermore, following activation, the functional avidity of 2C T cells selected in the presence of Lq never increased to the level achieved by 2C T cells selected in the absence of Lq. Thus, the self environment has imprinted a limit to the functional avidity that a T cell can acquire, apparently to avoid the development of autoreactivity.

It will be interesting to determine how our findings correlate with recent reports demonstrating that peripheral Ag-activated CD8+ T cells can undergo increases in avidity without significant changes in intrinsic TCR affinity (31, 32, 33). In these cases, the increase in avidity was not accompanied by differences in levels of CD8 or TCR, but was associated with modulation of the TCR and associated signaling machinery on the cell surface. Using a TCR transgenic system, Fahmy et al. (32) demonstrated that the TCR avidity for MHC-peptide complexes is 20- to 50-fold higher on activated T cells than on naive T cells. The activated T cells bound more MHC-peptide dimers compared with naive T cells, and this binding was sensitive to the cholesterol content of the cell membrane. This suggested that during activation TCRs are redistributed into cholesterol-rich lipid rafts. In other studies, it has been shown that engagement of TCR results in recruitment of TCR and signaling molecules to rafts, thus reducing the activation threshold and increasing avidity (34, 35, 36). Similarly, Slifka and Whitton (31) found that virus-specific T cells analyzed directly ex vivo underwent a >50-fold increase in their functional avidity early in virus infection. This was accompanied by an increase in Lck expression, which led these authors to speculate that the increase in functional avidity results from optimization of the T cell’s signal transduction machinery.

These studies on the molecular reorganization of cell surface receptors that accompanies the transition from naive to activated T cells complement earlier work, demonstrating the reorganization of cell surface molecules into a supramolecular activation complex, or immunological synapse, following exposure of T cells to APC (37, 38, 39, 40). It will be of interest to determine whether these findings that compare functional avidities between naive and activated peripheral T cells can be extended to explain the different functional avidities observed when T cells with identical TCRs develop in different thymic environments. Furthermore, this system can be used to ascertain whether differences exist in the topological rearrangements of cell surface molecules in activated 2C T cells that are reactive with Lq compared with those that are tolerant to Lq. Thus, we have identified a unique system that will allow us to address questions regarding the influence of thymic education on T cell activation requirements and how tolerance is maintained in peripheral T cells. Understanding how the self environment influences the functional avidity differences between mature T cells is of importance for understanding the development and maintenance of tolerance to self as well as for designing approaches to establish allograft tolerance.


   Acknowledgments
 
We thank Dr. Lonnie Lybarger for critical review of the manuscript, Tina Primeau and Shiloh Martin for technical assistance, and Eva-Marie Wormstall for maintenance and breeding of the mice.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI27568 and AI19687. T.M.C.H. was supported by National Institutes of Health Training Grant AI07163. Back

2 Current address: Department of Pediatrics, Stanford University Medical School, Stanford, CA 94305. Back

3 Address correspondence and reprint requests to Dr. Janet M. Connolly, Department of Genetics, Box 8232, Washington University School of Medicine, 4566 Scott Avenue, St. Louis, MO 63110. E-mail address: connolly{at}genetics.wustl.edu Back

4 Abbreviations used in this paper: MCMV, murine CMV; SP, single positive. Back

Received for publication November 22, 2002. Accepted for publication February 26, 2003.


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 Introduction
 Materials and Methods
 Results
 Discussion
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