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The Journal of Immunology, 2001, 166: 2357-2363.
Copyright © 2001 by The American Association of Immunologists

An Incremental Increase in the Complexity of Peptides Bound to Class II MHC Changes the Diversity of Positively Selected {alpha}{beta} TCRs

Rafal Pacholczyk, Piotr Kraj and Leszek Ignatowicz1

Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Positive selection of the normal repertoire of TCRs results from low-avidity interactions with a set of self-peptides bound to the MHC molecules expressed by thymic epithelial cells. The contribution of the individual peptide to positive selection remains a matter of debate. Here, for the first time, we show that two covalent class II MHC-peptide complexes positively select different TCRs expressing a common transgenic TCR{beta}-chain and endogenous TCR{alpha}-chains. Simultaneous expression of both Ab-peptide complexes changed the diversity of positively selected TCRs, indicating an additive and possibly synergistic effect of various peptides in this process.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The {alpha}{beta}TCR is inherently biased to bind MHC molecules, but only a few immature T cells survive thymic selection (1, 2, 3). The majority of immature T cells die by neglect or are actively deleted as potentially self-reactive. Only, if the thymocyte TCR weakly binds self-MHC-peptide complexes expressed on thymic epithelium, the immature T cell is rescued in the process known as positive selection (4, 5). The contribution of individual peptides to thymic selection remains unknown, although a normal repertoire of TCRs was found to be selected only when many wild-type (wt)2 peptides were bound to MHC on thymic stromal cells. Different peptides bound to MHC may be required to generate nonoverlapping sets of TCRs, which will result in an additive increase in the diversity of the selected TCR repertoire (6). Moreover, peptide diversity may have a synergistic effect on the TCR repertoire by creating an environment where the "gemish" of peptides, rather than an individual peptide, bound to MHC mediates positive selection of a substantial number of thymocytes (7). The diverse set of self-peptides may also include peptides exclusively expressed by thymic epithelium or peptides predisposed to positively select T cells (8, 9, 10, 11). Because the diversity of TCRs exceeds greatly the diversity of peptides bound to MHC molecules, it has also been postulated that peptide recognition during positive selection can be promiscuous, allowing a significant number of TCRs to be selected by a limited set of MHC-peptide ligands (12).

To examine the sensitivity of T cell selection with regard to peptide diversity, we tested whether an incremental increase in the number of peptides bound to class II MHC will have noticeable impact on the outcome of positive selection of CD4+ T cells in vivo. For that purpose, we have analyzed repertoires of TCRs found on CD4+ T cells in mice expressing MHC class II molecules (Ab) covalently bound with two peptides. We have previously found that mice with covalently bound E{alpha}52–68 peptide (Ep) had a semidiverse repertoire of TCRs specific for various antigenic peptides (13, 14). Using the same molecular strategy, we have made transgenic mice that express Ab molecules covalently bound with a close analogue of Ep peptide, in which the residue at position 58(G) was substituted with lysine (Ep58K). The altered amino acid was previously mapped as a TCR contact residue, presumably oriented toward the variable region of the TCR{alpha}-chain, which lies over the N terminus of the peptide in the orthogonal orientation of TCR and MHC class II complexes (15, 16). We tethered another Ep-like peptide to the Ab to minimize the overall conformational differences in AbEp and AbEp58K complexes, thereby favoring the same framework MHC-TCR contacts but with a single difference in the peptide-derived TCR contact residue. The new Ab{beta}Ep58K-transgenic mice were backcrossed with mice devoid of endogenous Ab{beta}-chain and invariant chain (Ii). In the following experiments, we compared the diversity of TCRs expressing common transgenic TCR{beta}-chain that are positively selected in vivo on thymic epithelium expressing exclusively AbEp, AbEp58K, or both AbEpAbEp58K complexes. Spectratyping and sequence analysis of endogenously rearranged TCR{alpha}-chains revealed that different sets of TCRs are selected in vivo by each of these MHC-peptide complexes. Additionally, we found that the TCR repertoire positively selected on both covalent Ab-peptide complexes included a number of different TCRs. These results show that two class II MHC-peptide complexes expressed separately or together positively select a number of different TCRs.


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

Mice expressing the transgenic AbEp complex were generated at the National Jewish Medical and Research Center (Denver, CO) as previously described (13). The same cloning strategy was used to generate transgenic mice expressing the AbEp58K complex. Both types of mice expressing covalent Ab-peptide complexes were further backcrossed with mice deficient for the invariant chain (Ii-), endogenous A{beta}b chain (Ab-), and {beta}2-microglobulin ({beta}2m-) as indicated. Mice transgenic for the TCR V{beta}14J{beta}26 chain were generated in our laboratory (P. Kraj, data not shown) and were crossed once with mice that lack endogenous TCR{alpha}-chain (TCR{beta}Tg{alpha}+/-). Radiation chimeras were generated by irradiating 6- to 10-wk-old hosts with 1000 rad, followed by reconstitution with 3 x 106 T cell-depleted bone marrow from TCR{beta}Tg{alpha}+/- mice. Mice deficient in expression of endogenous TCR{alpha}-chain or {beta}2m were purchased from The Jackson Laboratory (Bar Harbor, ME).

T cell hybridomas

T cell hybridomas {beta}2m4.2 and IgM77.1, specific for {beta}2m48–58 and IgM377–392 peptides, respectively, were kindly provided to us by Dr. A. Y. Rudensky (Seattle, WA).

Fetal thymic organ cultures (FTOC)

Thymic lobes from 16-day gestational fetuses were placed onto nitrocellulose filters (Millipore, Bedford, MA) supported by a gelatin sponge (Gelofoam; Pharmacia, Piscataway, NJ) in a 24-well plate and were incubated for 7–8 days at 37°C in DMEM high glucose medium (Cellgro, Herndon, VA) supplemented with 10% FBS (Life Technologies, Rockville, MD), 2 mM L-glutamine, 50 µM 2-ME, nonessential amino acids, penicillin, and streptomycin. The Abs and media were replenished every day where indicated.

Isolation and purification of CD4+V{beta}14+-transgenic T cells

Single-cell suspensions from the pooled axillary, inguinal, mesenteric, and paraaortic lymph nodes were incubated with MACS MicroBeads (Miltenyi Biotec, Bergisch Gladsbach, Germany) conjugated to anti-CD8 for 15 min at 4°C, washed, and sorted out using an autoMACS cell separator (Miltenyi Biotec). The negative fraction, depleted of CD8+ T cells, was incubated with anti-V{beta}14-biotin (PharMingen, San Diego, CA) for 15 min at 4°C and were washed and incubated again with MACS MicroBeads conjugated to streptavidin for 15 min at 4°C. The cells were then washed and sorted using an autoMACS cell separator. The positive fraction contained CD4+V{beta}14+ T cells of >97% purity.

RNA extraction and cDNA synthesis

Total RNA was extracted from 2 x 105 CD4+V{beta}14+ T cells using Ultraspec RNA reagent (Biotecx Laboratories, Houston, TX) and was converted to cDNA using the reverse transcription system (Promega, Madison, WI).

Spectratyping analysis of V{alpha}1-V{alpha}4 repertoires

The technique used in this study was described elsewhere (14). In brief, cDNA was amplified in a standard PCR (35 cycles) with V{alpha}(1, 2, 3, 4)-specific sense primers and an antisense primer complementary to the C{alpha} region (17) using 1/50 of the cDNA previously generated per reaction. One to 5 µl of PCR product was used as a template for a runoff reaction with a nested fluorescent C{alpha} gene-specific primer TCRCA57 (6FAM, 5'-GCT GTCCTGAGACCGAGGATCT-3'). The denatured runoff products were separated on a 6% polyacrylamide gel, and the bands were analyzed with an ABI Prism 377 DNA sequencer (PE Applied Biosystems, Foster City, CA) using GeneScan software. The bands are expressed in graphic form as peaks, with the area of each peak corresponding to the intensity of the band. The relative intensity of the band was calculated by comparing it to the combined intensity in the particular VJ rearrangement.

Sequencing of V{alpha}3-J{alpha} rearrangements with a given length

This approach was described elsewhere (14). In brief, after 35 cycles of standard PCR with V{alpha}(1, 2, 3, 4)-specific sense primers and an antisense primer complementary to the C{alpha} region, bands corresponding to the complementarity-determining region 3 (CDR3) of 9 and 10 aa in length and were excised from a 6% polyacrylamide gel after ethidium bromide staining. DNA was extracted from each band and was amplified by PCR and cloned into the pCR2.1 vector with the TOPO-TA cloning system (Invitrogen, San Diego, CA). Twenty randomly picked colonies were sequenced with V{alpha}3 primer using the ABI Prism 377 DNA sequencer. The CDR3 sequences reported here were obtained from three independent experiments for each chimera.

Cell preparations and flow cytometry analysis

Single-cell suspensions were prepared from thymi and spleen by mechanical disruption. Spleen cell suspensions were additionally incubated with buffered ammonium chloride to remove RBC. To analyze stromal epithelial cells in suspension, thymi were incubated for 30 min at 37°C with Collagenase (1 mg/ml, type IV; Sigma, St. Louis, MO) and DNase (0.02 mg/ml, bovine pancreatic DNase I; Sigma) followed by incubation with EDTA (0.01 M) for 5 min, then filtered to remove debris and used for FACS analysis. The following Abs were used for flow cytometry analysis: anti-V{beta}14-FITC, anti-CD4-PE, anti-CD4-APC, anti-CD8-PerCP, BP-1-PE (all from PharMingen), anti-TCR-FITC, Y3P-FITC, and B220-biotin (prepared in our laboratory). Biotinylated Ab was detected with streptavidin-PE (PharMingen). Staining was done on ice in 1x wash buffer (balanced salt solution containing 2% FBS and 0.1% NaN3). All FACS analysis was performed using FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA) and CellQuest software (Becton Dickinson). Dead cells were excluded by gating of forward and side scatter.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The covalent AbEp and AbEp58K complexes contribute to the positive selection of the majority of CD4+ T cells in AbEpIi and AbEp58KIi- mice

To determine the role of peptide in positive selection of CD4+ T cells, we produced two lines of transgenic mice that expressed Ab molecules covalently bound with two peptides. Expression of Ab on peripheral APCs was similar in both lines of transgenic mice but was reduced >10-fold in comparison to wt mice. However, expression of Ab on thymic epithelial cells was similar in the transgenic and wt mice (Fig. 1Go, A and B). The new covalent AbEp58K complex was detected by the YAe mAb, which was originally described as specific for the AbEp complex (18, 19). The AbEp58K complex was also recognized by some T cell hybridomas specific for the AbEp complex (data not shown). Staining of the AbEp58K complex by the peptide-independent Y3P mAb was abolished in the presence of the YAe mAb (data not shown), implying that the detectable Ab molecules remain covalently bound with Ep58K peptide. Moreover, splenic APCs expressing the AbEp58K complex did not present a detectable amount of endogenous peptides to two T cell hybridomas specific for {beta}2m48–58 or IgM377–392 peptide (20) (Fig. 1Go, C and D). These results showed that both covalent Ab-peptide complexes have common conformational domains that are recognized by Abs and specific T cells and that both peptides are firmly attached to Ab.



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  		FIGURE 1. Ab is firmly bound with the covalently attached Ep and its variant. Expression of I-Ab complexes in Ab- (filled histogram), AbEp (dotted line), AbEp58K (dashed line), AbEpEp58K (dashed and dotted line), and Abwt (thin line) mice on (A) gated cortical epithelial cells (BP-1+) and (B) B cells (B220+). Thymocytes or splenocytes from mice with the indicated genotypes were stained with mAb BP-1 or B220, respectively, and Y3P. APCs expressing covalent Ab-peptide complexes did not present endogenous (C) {beta}2m48–58 and (D) IgM377–392 peptides to the specific T cell hybridomas. Indicated amounts of splenocytes from Ab{beta}- ({blacktriangleup}), Ii- ({blacksquare}), AbEp58K ({circ}), AbEp ({diamond}), and AbEpAbEp58K ({square}) mice were cocultured with 105 specific hybridoma cells for 24 h. IL-2 production was measured with the IL-2-sensitive HT-2 cell line by MTT assay (36 ). Data are presented as units of IL-2.

 
To prove that the covalent Ab-peptide complexes select CD4+ T cells in AbEpIi- or AbEp58KIi- mice, we cultured 16-day-old fetal thymi from each transgenic line in the presence of YAe or Y3P mAbs. Although complete inhibition of the differentiation of CD4+ thymocytes using mAb in FTOCs is difficult to obtain (21), both YAe and Y3P mAbs comparably blocked the development of CD4+ thymocytes in FTOCs (Fig. 2Go.) Blocking with YAe mAb was better in FTOCs derived from AbEpIi- fetuses. This was most likely due to the reduced affinity of this Ab for the AbEp58K complex (an exchanged residue is included in the epitope recognized by YAe) and the significantly higher expression of the AbEp58K complex on bone marrow-derived thymic stromal cells (Fig. 1GoA). The number of CD4+ thymocytes was around 50% or less in FTOCs incubated with YAe, indicating that the covalent Ab-peptide complexes selected a substantial population of CD4+ T cells. Collectively, the results obtained from in vitro peptide presentation and FTOC experiments suggested that the covalent peptides remain bound to Ab and significantly, if not exclusively, select CD4+ T cells.



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  		FIGURE 2. The Ep and Ep58K peptides significantly contribute to positive selection of CD4+ T cells in single peptide mice. FTOCs from (A) AbEp58K or (B) AbEp mice cultured in the presence of the indicated Abs are shown as CD4 vs CD8 plots of TCR intermediate and high gated thymocytes. One lobe from each day 16 fetal thymus was cultured in medium only (control) and the other lobe was cultured with either Y3P or YAe at the final concentration of 40 µl/ml. For each set of conditions, three thymi were analyzed. A representative experiment is shown. In a control experiment, YAe Ab did not block positive selection of CD4+ T cells in FTOCs from Abwt mice (data not shown).

 
Covalent Ab-peptide complexes positively select a number of CD4+ T cells expressing a transgenic TCR{beta}-chain and endogenously rearranged TCR{alpha}-chains

An initial analysis of the TCR V{beta} segment distribution in AbEpIi- or AbEp58KIi- mice revealed that, despite the limited number of CD4+ T cells, both repertoires consist of many different TCRs (data not shown). Furthermore, in mice expressing each of the covalent Ab-peptide complexes, positive and negative selection of T cells was mediated by different peptide species, so an imprint of the particular peptide on positive selection was difficult to investigate. Hence, we lethally irradiated AbEpIi-, AbEp58KIi-, and double peptide AbEpAbEp58KIi- mice and separately reconstituted them with bone marrow from mice transgenic for TCRV{beta}14+, which had only a single functional TCR{alpha} locus (TCR{alpha}+/-) and expressed Ab molecules loaded with wt peptides (Abwt). Expression of the transgenic TCRV{beta}14+ chain forced all T cells to express the same TCR{beta}-chain (22), while the preservation of only one functional, endogenous TCR{alpha} locus ensured that the expressed TCR{alpha}-chain was used in thymic selection. Therefore, in radiation chimeras, thymocytes will survive only if their TCRs, composed of a single transgenic TCR{beta}-chain coupled to the rearranged endogenous TCR{alpha}-chain, weakly bind covalent Ab-peptide complex(es). To minimize restraints that might occur from the arbitrarily mismatched transgenic TCR{beta}-chain and covalent Ab-peptide complex, we used transgenic TCR{beta}-chain derived from the TCR originally positively selected in vivo on the AbEp complex (23). Furthermore, reconstitution of the AbEpIi- or AbEp58KIi- mice with bone marrow cells bearing Abwt normalized negative selection to the same set of endogenously derived peptides and emphasized the role of the two covalent peptides bound to Ab on the positive selection of CD4+ T cells. Finally, the possibility that one of the covalently attached peptides could negatively select some of the TCRs was controlled by the use of double Ab-peptide chimeras, where both covalent peptides were simultaneously expressed.

In AbEp58KIi- and AbEpIi- chimeras reconstituted with the Abwt TCR{beta}Tg{alpha}+/- bone marrow, a few V{beta}14+CD4+ T cells were positively selected (Fig. 3Go). Roughly three times more transgenic CD4+ thymocytes was found positively selected by the AbEp58K complex than by the AbEp complex, implying that 58K residue may be important for low-affinity interactions between the covalent MHC-peptide complex and multiple {alpha}{beta}TCRs. Staining with mAbs specific for different V{alpha} segments indicated multiple rearrangements of endogenous TCR{alpha}-chains (data not shown), proving that the transgenic V{beta}14+ chain associates with different endogenous TCR{alpha}-chains.



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  		FIGURE 3. Selection of TCR{beta}Tg{alpha}+/- CD4+ T cells on thymic epithelium expressing AbEp or AbEp58K complexes. The left column shows CD4 vs CD8 plots, the right column shows V{beta}14 expression on gated CD4 single-positive thymocytes. Data are representative of analyses on at least three chimeras of each group.

 
Different repertoires of TCRs are selected in mice expressing similar Ab-peptide complexes

To further assess the diversity of TCR{alpha}-chains selected in vivo by AbEp or AbEp58K complexes, we used a high-resolution RT-PCR method that visualizes the spectrum of sizes of the TCR{alpha} CDR3 (14). We amplified rearrangements of four randomly picked TCR{alpha} families (V{alpha}1-V{alpha}4). The size distribution of PCR products was visualized as bands of different intensity after separation on the sequencing gel. To exclude the possibility that different peaks reflect random PCR amplification, the reproducibility of the profiles was verified in four separate PCRs, which yielded identical results (Fig. 4GoA). The analysis of the CDR3 of four related V{alpha}-chains associated on CD4+ T cells with transgenic V{beta}14 chain revealed significant differences between the TCRs positively selected by the AbEp or AbEp58K complex. For example, the pattern of V{alpha}1 selected on the AbEp58K complex was bell shaped, indicating scattered rearrangements, while the same V{alpha}1 family selected on AbEp complex was dominated by a CDR3 of 11 aa. The CDR3s containing 8–9 aa contributed significantly to the repertoire selected in AbEp58K chimeras (8.8 ± 2.4% for 8 aa; 15.9 ± 4.8% for 9 aa), but their contribution was diminished in the repertoire amplified from AbEpAbEp58K chimeras (4.7 ± 1.2% for 8 aa; 5.6 ± 1.3% for 9 aa) due to the lack of these CDR3s in the repertoire selected in AbEp chimeras. The CDR3 that was 12 aa long in AbEpAbEp58K chimeras gained from the coexpression of both covalent Ab-peptide complexes (13.9 ± 3.3% in AbEp58K; 18.0 ± 7.8% in AbEp; 27.3 ± 2.8% in AbEpAbEp58K). The TCR V{alpha}2 profile selected on AbEp58K complex was dominated by the 9-aa-long CDR3 over the 10-aa-long CDR3 (39.7 ± 4.5% for 9 aa; 25.1 ± 2.0% for 10 aa), while the opposite result was obtained for the profile from the chimeras expressing the AbEp complex (28.9 ± 1.7% for 9 aa; 41.2 ± 6.6% for 10 aa). Due to the anticipated additive effect, the V{alpha}2 profiles from mice expressing both covalent Ab-peptide complexes had these two peaks almost balanced (31.3 ± 3.0% for 9 aa; 27.3 ± 0.6% for 10 aa). The V{alpha}3-bearing TCRs, with CDR3 length of 9 aa, were overrepresented in AbEp58K mice but were barely detectable in AbEp mice. The length of CDR3s in the V{alpha}4 family of TCRs selected on the AbEp58K complex was dominated by CDR3s that were 10 and 11 aa long, of which only the former was substantially represented in the repertoire selected by the AbEp complex. Instead, in these latter chimeras, the TCRs with a CDR3 containing 9 aa prevailed. Importantly, in chimeras expressing both covalent Ab-peptide complexes, the plot depicting the polymorphism of the CDR3s of endogenous TCR{alpha}-chains results from superimposing peaks found in the single Ab-peptide mice. This suggests an additive effect of each of the Ab-peptide complexes on positive selection. Also, as shown in Fig. 4GoB, the TCR{alpha} CDR3 polymorphism was the same in AbEp58KIi- mice expressing or devoid of nonclassical class I MHC molecules, indicating that the observed diversity of the TCR{alpha}-chains is derived from recognition of the peptides bound to Ab.



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  		FIGURE 4. Strong influence of peptide on profiles of analyzed CDR3s of the TCR{alpha}-chains positively selected in single or double peptide mice. The peaks corresponding to CDR3 with a length of 10 aa are indicated with a vertical dotted line. A, Results of two representative experiments displaying CDR3 polymorphism in the four analyzed TCR V{alpha} families are shown. B, Similar profiles of the TCR{alpha}-chains positively selected on AbEp58KIi- complex in the absence or presence of {beta}2m-dependent, nonclassical molecules. All profiles shown are representative of at least four independent experiments.

 
To determine the degree of diversity of endogenous TCR{alpha}-chains selected by AbEp, AbEp58K, or AbEpAbEp58K complexes, we excised gel fragments containing amplified CDR3 sequences that were 9–10 aa long and were derived from V{alpha}3+ TCRs. The CDR3 sequences of this length were commonly represented in the TCRV{alpha}3+ profiles obtained from mice expressing the AbEp58K or both AbEpAbEp58K complexes, but were relatively underrepresented in the TCRV{alpha}3+ profile amplified from mice expressing the AbEp complex. Fifty-six of these were sequenced. As shown in Table IGo, 39 CDR3 sequences differed at the protein level and 44 at the nucleotide level (data not shown), implying that TCR{alpha}-chain repertoires are diverse in all mice. In both single, but not double, Ab-peptide chimeras, >30% of the V{alpha}3+ segments were found rearranged to the dominant J{alpha}24 segment. Additionally, 90% of the V{alpha}3+ TCRs selected by AbEp complex used one of three J{alpha} segments (J{alpha}18, J{alpha}24, and J{alpha}40), whereas V{alpha}3+ TCRs selected in chimeras expressing AbEp58K or AbEpAbEp58K complexes used 9 or 12 different J{alpha} regions, respectively. The lower number of the repetitive CDR3 sequences and the greater number of J{alpha} segments found in "double peptide" mice suggested that the diversity of the TCR repertoire may be increased due to an additive effect of selection on both Ab-peptide ligands (also visible in the CDR3 profiles of V{alpha}1+, V{alpha}2+, and V{alpha}4+ TCR). Four CDR3 sequences (10, 17, 19, 21) were found in more than one type of chimera, and one CDR3 (17) was present in all three chimeras. These CDR3s may represent TCRs selected on Ab with little peptide contribution. The CDR3s of clones 17, 19, 20, and 37 were identical at the protein level but different at the nucleotide level. These clones probably represent TCRs subject to selective pressure for a particular CDR3 amino acid sequence (24).


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  		Table I. Sequences of V{alpha}3-J{alpha} rearrangements contributing to a CDR3 length of 9 and 10 aa

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recent studies using mice with MHC molecules bound with one peptide, or a dominant peptide, have provided important information on the role of peptides in thymic selection, peripheral survival, and allorecognition (24, 25, 26, 27, 28). We compared repertoires of TCRs positively selected in vivo on two covalent peptides bound to Ab via the same anchor residues, to determine whether these TCRs repertoires would be similar or different. Furthermore, to investigate whether the diversity of {alpha}{beta}TCRs would change due to incremental increase in the number of selecting MHC-peptide ligands, we characterized the repertoire of TCRs positively selected on thymic epithelium that coexpressed both covalent Ab-peptide complexes. All analyzed TCRs expressed one TCR{beta}-chain, were tolerant to wt peptides bound to Ab, and used different endogenously rearranged TCR{alpha}-chains.

First, we found that two covalent Ab-peptide complexes select different number of CD4+ T cells in vivo. This finding is not surprising in the context of the earlier report where different individual peptides added to FTOC positively selected various number of thymocytes (29). The number of CD4+ T cells (both TCR transgenic or nontransgenic) selected by AbEp58K complex exceeded the number of CD4+ T cells selected on Abwt in the absence of Ii (R. Pacholczyk, unpublished observations). Moreover, two other transgenic mouse lines expressing Ab covalently bound with Ep analogue with three residues (58, 61, and 63) substituted with lysine or exogenous peptide pigeon cytochrome c 43–58 had the same number of selected CD4+ T cells as mice expressing AbEp complex (R. Pacholczyk, manuscript in preparation). These two former peptides bind Ab with low affinity and could be efficiently replaced with exogenously added peptides, implying that more efficient selection of CD4+ T cells in AbEp58KIi- mice is not simply a result of a "leak" of endogenously derived peptides (also see below). Conceivably, a higher level of expression of the AbEp58K complex might result in increased positive selection of CD4+ T cells. However, this interpretation is at odds with the outcome of positive selection in transgenic mice expressing various amounts of the AbEp complexes (30). In these studies the transgenic animals expressing the highest level of the AbEp complex had the lowest number of selected CD4+ T cells.

It is also possible that replacement of the glycine in position 58 by lysine might reduce overall the affinity of interactions between the covalent Ab-peptide complex and multiple, endogenous TCR{alpha}-chains. Therefore results of our experiments do not support the hypothesis that class II MHC-peptide complexes, with the flat surface exposed toward the TCR, are predisposed to positively select the highest number of CD4+ T cells (31). Instead, one may imagine that the large side chain projecting toward the TCR{alpha}-chain may bind more TCRs with low affinity, which is sufficient to induce positive selection of considerably more CD4+ T cells.

Second, we found that TCR repertoires selected on each of the Ab-peptide complexes are different. Although our experimental setup with fixed TCR{beta}-chain and TCR{alpha}-chain positioned over the substituted residue in the covalently bound peptides might enhance the TCR{alpha}-chain diversity, the lack of more significant overlap between the two repertoires selected on each of the single Ab-peptide complexes was unexpected. The differences between the selected TCR repertories were noticed either by TCR{alpha} CDR3 spectratyping or DNA sequencing of the limited sets of the TCR{alpha}-chains. Of 39 different CDR3 sequences cloned from two "single peptide" mice, only one CDR3 sequence was the same at the amino acid level.

Finally, we found that coexpression of both covalent Ab-peptide complexes results in selection of TCRs that are different from the ones found in mice expressing individual Ab-peptide complexes. For example, the V{alpha}4+ CDR3 profiles selected on AbEpAbEp58K complexes had the highest number of different lengths. These findings implied that the individual Ab-peptide complex has an additive effect on the selected TCR repertoire. The sequencing of a narrow set of V{alpha}3+ CDR3s that are only 9–10 aa long and were selected on tested Ab-peptide complexes revealed that these sequences are different between the analyzed repertoires. It is also possible that the different TCRs found in these mice appeared as a result of the changed density of each covalent Ab-peptide complex. Alternatively, these TCRs could use a "gemish" of both covalent Ab-peptide complexes (7). Because more CD4+ T cells were found in mice expressing the AbEp58K, rather than the AbEp complex, conceivably, the former Ab-peptide complex likely contributed more to positive selection in "double peptide" mice. One may doubt that the V{alpha}3+ CDR3 sequences presented here do not sufficiently represent TCRs selected on the covalent Ab-peptide complexes. Although we may have not sequenced all relevant CDR3s, the analyzed TCR{alpha}-chain sequences were restrained not only by pairing with a fixed TCR{beta}-chain and by positive selection on covalent Ab-peptide complex(es), but also by extensive negative selection and the narrowed length of CDR3s, which was not executed in the previously published studies (24).

One may interpret that the observed difference in selected TCRs in AbEpIi- vs AbEp58KIi- mice comes only from different contributions of low-abundance peptides (21). The following argues against this interpretation. The blocking experiments in FTOCs showed that at least the vast majority of CD4+ T cells is selected on covalently bound peptides. The number of the CD4+ T cells in double peptide mice is not a simple sum of the number seen in single peptide mice, regardless of the "doubled" vulnerability for leakage of endogenous peptides. Additionally, sequencing of the relatively small number of CDR3 sequences reveals selection at the protein level, which we would not see if these TCRs were selected on many low-abundant MHC-peptide complexes (21).

In retrospect, similar studies have examined TCRs on CD4+ thymocytes selected in wt or H-2M-deficient mice, where the latter expressed Ab molecules predominantly bound with Ii-derived class II-associated Ii peptide (24). The TCRs selected in H-2M-deficient mice had limited sequence diversity in the CDR3s of a particular TCR{alpha}-chain, prompting the conclusion that positive selection primarily shapes the repertoire of TCRs expressed on peripheral T cells. However, the spectra of self-peptides bound to class II MHC in wt and H-2M- mice differ significantly (32, 33) and therefore those experiments could not assess the contribution of the individual peptide to the positive selection of {alpha}{beta}TCR expressed on CD4+ T cells. In this study, by comparing {alpha}{beta}TCRs positively selected on thymic epithelium expressing two covalent Ab-peptide complexes we show that each of these selects a number of different TCRs. Thymic cortical epithelium express 10-fold less class II MHC molecules than peripheral APCs, and are likely to be bound by 103–104 endogenous peptides (9, 34). The lowest limit of different {alpha}{beta}TCRs expressed on naive T cells in blood has been estimated at 2.5 x 107, implicating that an impact of the individual MHC-peptide complex on positive selection of T cells may indeed be detectable (14, 35). Consequently, we found that as the diversity of positively selecting Ab-peptide complexes increases incrementally, the repertoire of TCRs expressed on CD4+ T cells changes, demonstrating how a restrained set of self-MHC-peptide complexes manages to select a large repertoire of {alpha}{beta}TCRs.


   Acknowledgments
 
We thank Dr. R. Markowitz for editing the manuscript. We also thank H. Ignatowicz and G. Pacholczyk for their help in producing transgenic mice, J. Nechtman and C. Leithner for help with DNA sequencing, and Drs. D. Mathis, Ch. Benoist, L. van Kaer, and A. Rudensky for their generous gifts of knockout mice, DNA constructs, or Abs. All work involving animals was conducted under protocols approved by the Animal Care and Use Committee at Medical College of Georgia.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Leszek Ignatowicz, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912-2600. Back

2 Abbreviations used in this paper: wt, wild type; Ab, MHC class II molecule; Ep, E{alpha}52–68 peptide; {beta}2m, {beta}2-microglobulin; FTOC, fetal thymic organ culture; CDR3, complementarity-determining region. Back

Received for publication September 7, 2000. Accepted for publication November 30, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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