On the role of high- and low-abundance class II MHCpeptide complexes in the thymic positive selection of CD4+ T cells
Bartosz Chmielowski,
Pawel Muranski,
Pawel Kisielow1 and
Leszek Ignatowicz
Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912, USA
1 Basel Insitute for Immunology, CH-4005 Basel, Switzerland
Correspondence to:
L. Ignatowicz
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Abstract
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The role of self-peptides bound to MHC molecules in the selection of T cells in the thymus remains controversial. Here, we have tested whether a high-abundance single class II MHCpeptide complex has a dominant effect on the repertoire of CD4+ T cells selected by low-abundance class II MHCpeptide complexes. For these studies, we have used H-2b mice that lack an invariant chain (Ii) (AbIi) and their transgenic variant (AbAbEpIi) that co-expresses Ab molecules covalently bound with a single peptide Ep(5268). In these latter mice, close to 50% of all Ab molecules are occupied by Ep(5268) peptide. Although the AbEp complex was abundantly expressed in the thymus under conditions excluding negative selection on bone marrow-derived cells, no striking quantitative difference between repertoires of TCR expressed on CD4+ T cells in AbIi and AbAbEpIi mice was noticed. Our results are consistent with the view that diverse, low-abundance self-peptides play an important role in thymic positive selection and do not support the notion that dominant, high-abundance peptides may be critical for shaping the TCR repertoire.
Keywords: MHCpeptide complex, positive selection, T cell repertoire
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Introduction
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Selection of antigen-specific repertoires of the majority of
ß T cells occurs in the thymus and involves interaction of the
ß TCR with MHC molecules presenting peptides derived from the degradation of self-proteins. Whereas the role of self-peptides in determining the specificity of the negative selection of self-antigen reactive T cells has never been disputed, the role of self-peptides in the positive selection of T cells responding to foreign antigens remains controversial (1). Three alternative possibilities have been considered. First, positive selection is highly peptide specific (2,3), i.e. only one or a few related peptides are responsible for the selection of each particular TCR. Second, positive selection is promiscuous (4), i.e. a single peptide is able to select many different TCR. Third, self-peptides play no role in positive selection other than stabilizing the surface expression of MHC molecules at appropriate levels, allowing interactions of MHC epitopes with TCR (5). The last possibility seems to be excluded by in vivo experiments showing that reduction of self-peptide diversity on class II MHC molecules without lowering the expression level of MHC molecules results in reduced numbers of selected CD4+ T cells with a semidiverse TCR repertoire (6,7). This leaves two other possibilities open because conclusive experiments discriminating between the first and the second have not yet been done. In fact, both possibilities could be true because it has not been excluded that some MHCpeptide complexes select significantly less or significantly more diverse TCR than others. The distribution of self-peptides bound by MHC molecules is not equal, i.e. different MHCpeptide complexes may be present at vastly different frequencies (8,9), and it was suggested that a few dominant self-peptides bound to class II MHC molecules may be primarily responsible for T cell selection in the thymus (10). The finding that many different TCR were selected in the mouse expressing a single MHC class IIpeptide complex [AbEp(5268)] that is naturally expressed as a high-abundance complex (11) seemed consistent with this notion, but uncertainty exists whether other peptides present at undetectable levels are not contributing to positive selection in these mice. In fact, a recent paper (12) published while the present work was in progress suggested that the positive selection of CD4+ T cells in mice expressing Ep(5268) peptide on 95% of class II MHC molecules resulted exclusively from the interaction of TCR with a diverse set of self-peptides bound by the remaining 5% of class II MHC molecules. However, since it has been shown by others that the high expression level of AbEp(5268) complex reported in that paper (12) has a dominant negative effect on the outcome of thymic selection (13), its results are inconclusive. In our study, to test the role of dominant peptide in selection of the TCR repertoire and to assess the contribution of low-abundance peptides to positive selection, we compared the repertoire of CD4+ T cells selected in invariant chain (Ii)-deficient mice (AbIi) that express a low number of class II MHC molecules occupied with a few, Ii-independent self-peptides (14,15) with the repertoire in their transgenic variant (AbAbEpIi) that co-expresses Ep(5268) peptide on ~50% of its class II MHC molecules at the level shown to be permissive for positive selection (4,13).
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Methods
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Animals
AbIi mice were kindly provided to us by E. Bikkoff and R. Germain (15). AbAbEpIi mice were produced by crossing AbIi and AbEpIi mice. All mice were housed under specific pathogen-free conditions in the Animal Care Facility at the Medical College of Georgia (Augusta, GA). C57BL/6 (Ab) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). To generate chimeric mice, 6- to 12-week-old AbIi and AbAbEpIi animals were lethally irradiated (950 rad) and reconstituted on the same day with 5 x 106 fetal liver cells from Ab fetuses (day 15, gestational age). Chimeras were used for experiments no earlier than 8 weeks after reconstitution.
Flow cytometry analysis
mAb specific for CD4, CD8 and different V
/Vß segments of TCR conjugated with FITC and phycoerythrin were prepared in our laboratory or purchased from PharMingen (San Diego, CA). Standard staining procedure was used as previously described (4). Cells were analyzed on a FACSCalibur instrument (Becton Dickinson, San Jose, CA) and using the CellQuest software. Viable cells were identified by gating on forward and side scatter. Data is shown as logarithmic dot-plots.
T cell hybridomas
The Ab
AbIi and Ab
AbAbEpIi chimeras were immunized with synthetic peptides Ep58K (ASFEAQKALANIAVDKA) and Ep63K (ASFEAQGALANKAVDKA) in complete Freund's adjuvant (2.5 mg/ml). Cells from the draining lymph nodes were converted into T cell hybridomas as described (16). Hybridomas were analyzed using flow cytometry for expression of CD4 and TCR. Double-positive hybridomas were tested for peptide-specific response using a standard IL-2-release assay. Briefly, T cell hybridomas (1x105) were stimulated in the presence or absence of a specific peptide using 5x105 Ab splenocytes. After 24 h, supernatants were tested for the presence of IL-2 by measuring proliferation of IL-2-dependent cells (HT-2) using an MTT assay (Sigma, St Louis, MO) (17).
Microscopy
Thymi from 2-month-old Ab and AbAbEpIi mice were embedded in OCT compound (Sakura Finetek, Torrance, CA) and frozen in 2-methylbutane (Fisher Scientific, Pittsburgh, PA) in dry ice. Frozen sections cut with a CM3000 cryostat (Leica, Deerfield, IL) were mounted on gelatin-coated slides and allowed to air-dry before fixation in cold acetone (5 min, 4°C). Sections were then incubated for 60 min at room temperature in a solution of normal mouse serum and 10% culture supernatant of anti-Fc receptor antibody 2.4G2 to block non-specific binding, incubated for 60 min with biotinylated CDR1 antibody (anti-mouse thymic epithelium), and washed twice with PBS. Finally, sections were exposed to streptavidin-conjugated indodicarbocyanine (Jackson ImmunoResearch, West Grove, PA) and either FITC-conjugated Y3P (anti I-Ab) or YAe (anti-I-AbEp) antibody. After staining, sections were mounted using ProLong Antifade kit (Molecular Probes, Eugene, OR). Slides were analyzed with a Diaphot 200 Nikon microscope and pictures were processed with Adobe Photoshop software (Adobe Systems, Mountain View, CA).
Analysis of the DNA sequences of the V
and Vß segments of TCR
To analyze the DNA sequences of the V
and the Vß segments of T cell hybridomas, RNA was isolated using the Ultraspec RNA isolation kit (Biotecx Laboratories, Houston, TX) and converted to cDNA using the Reverse Transcription System (Promega, Madison, WI). After amplification of TCR
and ß chains using constant region and V
/ß-specific primers, PCR products were sequenced by automated fluorescent sequencing on an ABI Prism 377 DNA sequencer (PE Applied Biosystems, Foster City, CA).
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Results
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In AbAbEpIi mice, the AbEp complex is dominantly expressed on class II MHC-positive thymic epithelial cells
Recently, we have shown that, in AbAbEp Ii mice, a transgenic AbEp complex is dominantly expressed on antigen-presenting spleen cells, where close to 50% of all class II MHC molecules are occupied by this single peptide (18). To prove that the introduced transgenic AbEp complex is also dominantly expressed on thymic epithelial cells that positively select T cells, we have now examined the expression of class II MHC molecules on sections of thymus from AbIi and AbAbEpIi mice. Thymic epithelial cells, identified by mAb CDR-1, were stained with mAb specific for all Ab complexes (Y3P) or AbEp complex only (YAe) (Fig. 1
). Thymic epithelium from AbAbEpIi mice, but not from AbIi mice, stained strongly with mAb specific for the AbEp complex. This clearly showed that the transgenic AbEp complex is dominantly expressed on thymic epithelial cells.

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Fig. 1. The transgenic AbEp complex is expressed on thymic epithelial cells in AbAbEpIi mice, but not in AbIi mice. Frozen sections of thymi from AbIi (A and C) and AbAbEpIi animals (B and D) were stained with biotinylated CDR1 antibody (anti-mouse thymic epithelium) and streptavidin-conjugated indodicarbocyanine as a secondary reagent to identify thymic epithelial cells (red). Additionally, sections were stained with FITC-conjugated Y3P (anti-I-Ab) (A and B) or YAe (anti-I-AbEp) (C and D).
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Dominant expression of the AbEp complex does not quantitatively enhance positive selection of CD4+ T cells
To test the effect of dominant AbEp complex on selection of CD4+ T cells, we first compared the proportions of CD4+ T cells in the thymus and periphery of AbIi and AbAbEpIi mice. As shown in Fig. 2
, the proportions of CD4+ T cells in AbIi and AbAbEpIi mice were very similar. Next, we compared the Vß repertoire of TCR expressed on CD4+ T cells in both types of mice. As shown in Fig. 3
, the only difference was that Vß4-bearing CD4+ T cells appeared to be slightly more frequent in AbAbEpIi mice than in AbIi mice. From this set of experiments, we concluded that dominant expression of the AbEp complex does not result in a proportionally strong skewing of the TCR repertoire selected by MHCpeptide complexes expressed at low frequency. However, although the expression level of AbEp complex in our AbEp transgenic Ii mice was shown to be permissive for positive selection of CD4+ T cells (4), we had to consider the possibility that the lack of a noticeable, quantitative contribution of AbEp to positive selection was a result of negative selection (13). Furthermore, the possibility remained that the presence of the AbEp complex in AbAbEpIi mice could exert a more subtle effect by skewing the TCR repertoire on CD4+ T cells toward Ep-like antigens.

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Fig. 2. The percentage and total number of CD4+ T cells in the thymus and periphery are similar in the presence and absence of AbEp complex. Thymocytes and lymph node cells from AbIi and AbAbEpIi were stained with anti-CD4 and anti-CD8 mAb. The percentage of CD4+ cells in the total population of T cells is indicated. The total numbers of CD4+ T cells in AbIi mice and in AbAbEpIi mice were 9.56 (± 1.6) x 106 and 7.72 (± 2.77) x 106 respectively. The number of CD4+ cells was estimated from the number of cells recovered from the spleen and the pool of inguinal, axillary and mesenteric lymph nodes using immunofluorescence analysis. The total number of CD4+ T cells was obtained by adding the number of CD4+ T cells from spleen and twice the number from lymph nodes to account for the number of circulating cells as described (25).
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Fig. 3. Comparison of the repertoire of Vß segments of TCR on CD4+ cells in AbIi and AbAbEpIi mice. Lymph node cells were stained with antibodies against CD4 and TCRVß segments. Only live cells were analyzed. The percentage of usage of a particular Vß segment was calculated from the number of CD4+ T cells.
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Negative selection by bone marrow-derived cells expressing AbEp complex is not responsible for the lack of dominant influence of this complex on positive selection
To minimize the possible contribution of AbEp complex to negative selection, we have made radiation chimeras. The AbAbEpIi and AbIi mice were lethally irradiated and reconstituted with fetal liver cells from Ab wild-type mice. These two types of radiation chimeras differ only by the expression of the AbEp complex on thymic epithelium, with negative selection occurring on the wild-type set of self-peptides. As shown in Fig. 4
, in the absence of negative selection by AbEp on bone marrow-derived cells, there was only a subtle increase of CD4+ T cells in Ab
AbAbEpIi chimeras. Moreover, distribution of various TCR Vß segments on CD4+ T cells in Ab
AbIi and Ab
AbAbEpIi chimeras were also very similar (Fig. 5
). These results indicate that negative selection on AbEp complex expressed on the bone marrow-derived cells does not explain the lack of dominant effect of this complex on positive selection occurring on MHCpeptide complexes expressed at a lower frequency.
The repertoire of CD4+ T cells selected in AbIi mice is not deficient in AbEp-specific TCR and dominant expression of the AbEp complex changes it only qualitatively.
Next, we tested if the AbEp complex co-expressed with a limited set of Ab/self-peptide complexes on thymic epithelium of AbAbEpIi mice may have a qualitative influence on selection by skewing the repertoire of CD4+ T cells towards Ep-like antigens. The Ab
AbAbEpIi and Ab
AbIi chimeras were primed with two Ep analogs, each with a single amino acid substitution, Ep58K or Ep63K. These two Ep analogs were previously found to be able to trigger specific CD4+ T cells that were selected on a single AbEp complex and that were tolerant to wild-type Ab (19). After 1 week, CD4+ T cells from lymph nodes were expanded in vitro in the presence of a specific peptide and converted into T cell hybridomas as described (20). We were able to generate CD4+ T cell hybridomas specific for both Ep analogs not only from Ab
AbAbEpIi chimeras but also, surprisingly, from Ab
AbIi chimeras. CD4+ T cell hybridomas specific for Ep(5268) were also generated from Ab
AbIi chimeras (data not shown). These results contradicted the recent report (21) that Ep(5268) is not stimulatory for CD4+ T cells selected in Ab
AbIi chimeras and indicated that self-derived peptides binding to Ab molecules in the absence of Ii can positively select CD4+ T cells specific for soluble Ep or Ep analogs. Analysis of the Vß repertoires of TCR specific for both analogs of Ep showed that the spectrum of TCR specific for the Ep58K and Ep63K analogs has similar profiles in both types of chimeras (Fig. 6
), thus failing to provide evidence for the dominant role of the dominantly expressed AbEp complex. However, despite the fact that CD4+ T cells from both radiation chimeras responded to both Ep analogs with a similar magnitude, the responding T cells selected in Ab
AbIi and Ab
AbAbEpIi chimeras could use different TCR. To test this possibility, we further characterized the subpopulation of Vß6-bearing CD4+ T cells that dominated the responses to the Ep58K analog in both Ab
AbIi and Ab
AbAbEpIi radiation chimeras. Six TCR from each chimera were sequenced and were found to represent four different TCR (Fig. 7
). Two of them were derived from Ab
AbIi and two from Ab
AbAbEpIi chimera, indicating that positive selection of CD4+ T cells by the dominant AbEp complex may have a qualitative influence on the repertoire of TCR specific for Ep58K peptide.
This conclusion was further supported by the sequence analysis of Vß14+ TCR specific for the Ep63K analog. Three different TCR were found among nine T cell hybridomas derived from Ab
AbIi chimeras and six different TCR were found among 14 TCR hybridomas derived from Ab
AbAbEpIi chimeras respectively. It should be noted that these radiation chimeras have very few CD4+ T cells (Fig. 4
) and therefore antigen specific TCR analyzed here are probably representative of the Ep58K- and Ep63K-specific-repertoire selected in these animals. The fact that none of the TCR in one type of chimera was similar to the TCR in the other type of chimera implies that expression of the AbEp complex has a qualitative effect on the thymic selection processes and alters the peripheral repertoire of TCR expressed on CD4+ T cells.
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Discussion
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This study demonstrates that the
ßTCR repertoires selected in AbIi mice by a limited set of low-abundance peptides and the repertoire selected in AbAbEpIi mice that express the high-abundance AbEp complex in addition to the low-abundance peptides were similar but not identical. This indicates that positive selection by the dominant AbEp complex does not contribute quantitatively the majority of CD4+ T cells to the repertoire. Analysis of bone marrow chimeras in which the AbEp complex was expressed exclusively on thymic epithelial cells at the level permissive for positive selection allowed us to exclude the possibility that negative selection interfered with the potential of the AbEp complex to dominate positive selection in AbAbEpIi mice. Thus, our results contradict the postulate that a few peptides abundantly expressed in the thymus could be responsible for positive selection of a complete repertoire of TCR specificities (10). Our observations are consistent with the view that a high diversity of peptides is essential for positive selection of a fully diverse T cell repertoire (2224). These conclusions, however, do not allow one to make generalized inferences about the degree of peptide specificity of positive selection (12) because the instances in which the contribution of single MHCpeptide complexes to positive selection has been analyzed may not be representative of all self-peptides expressed in the individual. In fact, only one repertoire of TCR positively selected by a single Abpeptide complex and tolerant to wild-type self-peptide has been analyzed so far and it is limited but capable of responding to several antigenic peptides (19). Theoretically then, positive selection could be both highly specific and highly degenerate, depending on individual TCRMHCpeptide interactions. Some receptors could be selected by only one MHCpeptide complex, while others could be selected by several. Conversely, some MHCpeptide complexes could be able to select a single TCR specificity while others could be able to select many. None of the existing evidence excludes these possibilities. It therefore seems premature to deduce general rules concerning the peptide specificity of positive selection, based on available data.
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Acknowledgments
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This work was supported by National Institutes of Health Grant AI41145. The Basel Institute for Immunology was founded and is supported by Hoffmann-La Roche, Basel, Switzerland.
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Abbreviations
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Notes
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Transmitting editor: T. Sasazuki
Received 18 June 1999,
accepted 5 October 1999.
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