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The Journal of Immunology, 2000, 164: 4601-4606.
Copyright © 2000 by The American Association of Immunologists

A Physiological Ligand of Positive Selection Is Seen with High Specificity1

Stefan Irion*,{dagger}, Rance E. Berg*,{ddagger} and Uwe D. Staerz2,*,{ddagger}

* Department of Medicine, National Jewish Medical and Research Center, Denver, CO 80206; {dagger} Abteilung fuer Immunologie, Interdisziplinaeres Institut fuer Zellbiologie, Eberhard-Karls-Universitaet, Tuebingen, Germany; and {ddagger} Cancer Center and Department of Immunology, University of Colorado Health Sciences Center, Denver, CO 80262


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Positive selection is a process that ensures that peripheral T cells express TCR that are restricted to self-MHC molecules. This process requires both self-MHC and self-peptides. We have recently established a TCR transgenic mouse model (C10.4 TCRtrans+) in which the transgenic TCR was selected on the nonclassical MHC class Ib molecule H2-M3 in conjunction with a physiologically occurring peptide derived from the mitochondrial NADH-dehydrogenase subunit 1 gene (9-mer peptide). Here, the specificity of positive selection of C10.4 TCRtrans+ T cells was examined using a fetal thymic organ culture system. We demonstrated that at low peptide concentrations, shortening the NADH-dehydrogenase subunit 1 gene 9-mer peptide or mutating its surface-exposed side chains severely impaired its ability to induce positive selection. We concluded that under physiological conditions positive selection of C10.4 TCRtrans+ T cells was highly specific and occurred at low epitope densities.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In the thymus, immature T cells generated in the bone marrow undergo two education processes. The first one, termed positive selection, ensures that T cell recognition is MHC restricted (1, 2, 3). During positive selection, immature T cells interact with self-peptides presented in conjunction with self-MHC molecules (4, 5, 6, 7). The second process, known as negative selection, either eliminates (8, 9, 10) or anergizes (11, 12, 13) potentially autoreactive T cells.

Earlier studies used cognate peptides or their variants to probe T cell development (14, 15, 16), because physiological ligands of positive selection had not been defined. Different experimental systems led to different hypotheses of positive selection. The "antagonism theory" postulated that T cells were positively selected on peptides antagonistic to the cognate Ag (15, 17). The "differential avidity" hypothesis suggested that positive selection depended on low-avidity interactions between the TCR and the MHC/peptide complex (14). If one assumed that TCR-MHC/peptide interactions by themselves were of low affinity, this theory could further explain how positive selection was promiscuous (18) or even independent of the recognition of a specific peptide (19). The "differential avidity" hypothesis also predicted that a low TCR affinity could be compensated for by large numbers of MHC/peptide epitopes. Thus, positive selection of immature T cells could be induced by a "gemisch" of self-peptides rather than a specific Ag (20, 21). However, recent experiments did not support this last conclusion. They suggested that positive selection of T cells was a specific interaction that occurred on low-abundance self-peptides (22).

The question of specificity of positive selection can be best addressed in a model system in which the physiological ligand of positive selection is known. We recently produced a TCR transgenic (TCRtrans+)3 mouse that expressed a TCR specific for a Listeria monocytogenes-derived formylated Methionine (fM) peptide in conjunction with the MHC class Ib molecule H2-M3 (23, 24). We had chosen this system because H2-M3 molecules preferentially bind peptides that carry a fM in the N-terminal position (25, 26). In the mouse, only 13 mitochondrial genes give rise to fM peptides (27). This limited number of fM peptides enabled us to define a NADH dehydrogenase subunit 1 (ND1)-derived peptide as the physiological ligand of positive selection for the C10.4 TCRtrans (23). In this report, we studied how alterations of the ND1 9-mer self-peptide (ND1/9-mer) affected positive selection using a fetal thymic organ culture (FTOC) system. We shortened the ND1 self-peptide and mutated the exposed amino acid side chains as determined by the crystal structure of the H2-M3/ND1 complex (28). Our experiments demonstrated that a natural ligand of positive selection, such as the ND1/9-mer self-peptide, was recognized with exquisite specificity when offered at physiological epitope densities.


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

C10.4 TCRtrans+ mice expressing Vß8.1 and V{alpha}4.2 TCR chains specific for a L. monocytogenes fM peptide (called AttM) in the context of H2-M3wt have recently been described (23). C57BL/6 and TAP1-/- mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Recombination activating gene 2 deficient (RAG2-/-) mice were kindly provided by Fred Alt (Harvard Medical School, Boston, MA). All breeding was done at the Biological Resource Center at the National Jewish Medical and Research Center (Denver, CO). All animal studies had been approved by the Institutional Animal Care and Use Committee of the National Jewish Medical and Research Center.

Cells, cell lines, and cell culture media

EL4 is an H-2b C57BL/6 thymoma obtained from American Type Culture Collection (TIB-39; Manassas, VA). The 13S2 fibroblast line obtained from Robert Rich (Emory University, Atlanta, GA) and John Rodgers (Washington State University, Pullman, WA) had been transfected with a chimeric H2-M3wt/Ld molecule (29). C10.4 TCRtranshigh T cell lines were generated from the spleens of C10.4 TCRtrans+ mice bred onto an RAG2-/- background. C10.4 TCRtranshigh T cell lines were maintained by weekly restimulation with irradiated C57BL/6 spleen cells that had been incubated with the cognate AttM fM peptide (fMIVTLF). Cells were cultured at 37°C in 7% CO2 in IMDM (Sigma, St. Louis, MO) supplemented with 5 mM HEPES, 2 mM glutamine, 1 mM hydroxypyruvate, 50 mM 2-ME, nonessential amino acids, 100 IU/ml penicillin, 100 mg/ml streptomycin, 50 mg/ml gentamicin (all supplements from Sigma), and 10% FCS (HyClone, Logan, UT) (complete IMDM). FTOC media was supplemented with 10% FCS (Intergen, Purchase, NY), 1% Nutridoma-SP (Boehringer Mannheim, Mannheim, Germany), 5 µg/ml human ß2-microglobulin (Vital Products, St. Louis, MO), potato carboxypeptidase inhibitor (10-4 nM final concentration), captopril (10-7 mM final concentration), and E-64 (10-2 mM final concentration) (all from Sigma). Media for the H2-M3 up-regulation assay was complete IMDM containing potato carboxypeptidase inhibitor (10-4 nM final concentration), captopril (10-7 mM final concentration), and E-64 (10-2 mM final concentration) (all from Sigma). The C10.4 TCRtranshigh T cell line had 4% rat Con A supernatant added to the media to provide cytokines and growth factors.

Peptides

fM peptides were synthesized by the Molecular Research Center at the National Jewish Medical and Research Center as previously described (24). Crude peptides were purified using a reverse-phase HPLC system (Rainin Instruments, Emeryville, CA). Integrity was confirmed using a reverse-phase matrix-assisted laser desorption ionization time-of-flight mass spectrometer (Applied Biosystems, Foster City, CA). Peptide stock solutions were prepared at 10 mM in 100% DMSO and stored at -20°C.

Abs and flow cytometric analyses

Monoclonal Abs specific for CD4 (RM4-5) and CD8 (53-6.7) directly conjugated to either PE or FITC, respectively, were purchased from PharMingen (San Diego, CA). Streptavidin Cy-Chrome and streptavidin PE for revealing biotinylated mAbs were purchased from PharMingen. The anti-Cß mAb, H57-597 (30), the anti-Vß8.1–8.3 mAb, F23.1 (31), and the anti-{alpha}3 Ld mAb, 28-14-8S (32) were purified from culture supernatants using protein G Sepharose beads (Pharmacia, Piscataway, NJ) and then biotinylated, if indicated, following standard protocols. Surface immunofluorescence staining was performed as previously described (23). The extent of fluorescence was analyzed on a FACScan (Becton Dickinson, San Diego, CA) using CellQuest software (Becton Dickinson).

CTL assays

C10.4 TCRtranshigh CTL lines were grown from C10.4 TCRtrans+/+ RAG2-/- spleens (23). Standard CTL assays were performed as previously described (24). Briefly, targets were pulsed with 51Cr (Na2-51CrO4; ICN Pharmaceuticals, Costa Mesa, CA) and sensitized with the different peptides. Effector CTLs were then added at the E:T ratios as indicated in the figure legends. After a 4-h incubation, supernatant was harvested and 51Cr release was measured on an automatic gamma counter (Micromedic Systems, Huntsville, AL). Percent specific lysis was calculated as [(experimental release - spontaneous release)/(maximum release - spontaneous release)] x 100. Results are expressed as the mean of triplicates.

FTOC

Day 16 thymic lobes from C10.4+/+ TAP1-/- fetuses were cultured at the air-liquid interphase in 6-well plates (Falcon; Becton Dickinson Labware, Franklin Lake, NJ) on nitrocellulose membrane carriers (0.45 µM pore size; Gelman Sciences, Ann Arbor, MI) supported by gelatin sponges (Pharmacia and Upjohn, Kalamazoo, MI) (18). The different peptides were added to the cultures at decreasing concentrations. After 6 days of culture, thymic lobes were harvested and single-cell thymocyte suspensions were stained for CD4, CD8, and TCR surface expression levels. Positive control cultures contained 39 nM ND1/9-mer peptide (23), while 0.1% DMSO was used as a negative control.

H2-M3 up-regulation assay

The relative binding affinities of the different peptides were measured using an MHC up-regulation assay as previously described (29). 13S2 fibroblast cells expressing the H2-M3wt/Ld chimeric molecule were plated in 48-well plates (Costar, Cambridge, MA), and 100 U/ml of IFN-{gamma} (Genzyme, Cambridge, MA) was added to up-regulate MHC expression. After overnight incubation at 37°C, peptides were added at various concentrations. The cells were then incubated overnight at 27°C at 7% CO2, harvested, and stained for expression of the chimeric H2-M3wt/Ld molecule using the biotinylated 28-14-8S mAb and streptavidin PE as a secondary reagent. Cultures to which the OVA-derived SIINFEKL-peptide or no peptide had been added were used as a negative control (data not shown). Peptide concentration for half-maximal binding was determined as a mean of two experiments.

Expansion of FTOC thymocytes and their use in CTL assays

After FTOCs were cultured for 6 days, a single-cell suspension of thymocytes was added to 24-well plates (Costar) that had been coated with 5 µg/ml of the anti-Cß mAb H57-597. Culture media was complete IMDM with 4% rat Con A supernatant. After 3 days, the cells were harvested, counted, and used as effectors in a CTL assay at an E:T ratio of 10:1. EL4 cells that had been coated with the cognate AttM fM peptide fMIVTLF or the control peptide fMIGWII (known to bind H2-M3 without sensitizing C10.4 T cells) were used as targets (24, 33).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Design of ND1 peptide mutants

Mitochondrial fM peptides are presented on the cell surface by H2-M3 MHC class Ib molecules. The exact lengths of these naturally occurring peptides have not been determined. On normal cells, surface expression of H2-M3 is barely detectable due to the paucity of natural fM peptides (27) and the low level of H2-M3 gene expression (34). We had chosen the 9-aa length of the ND1 self-peptide for our original studies of positive selection for the following reasons. Most peptides extracted from surface MHC class Ia molecules were 9-aa long (35). Although fM peptides as short as 2 aa bound to H2-M3 (36), and Listeria-derived 5-mer fM peptides in context of H2-M3 could be recognized by T cells (24), CTLs specific for naturally occurring mitochondrial fM peptides recognized longer peptides. Several CTLs had been described that were specific for ND1 fM peptides of 9-aa lengths (37). Finally, structural analysis of an H2-M3/ND1 complex had been performed with the ND1/9-mer peptide (28).

Because we already knew that positive selection of C10.4 TCRtrans+ T cells depended on the N-terminal fM group, we looked for crucial residues at the other terminus of the ND1/9-mer peptide by removing C-terminal amino acids (ND1/8-mer and ND1/7-mer). In the crystal structure of the H2-M3/ND1 complex, amino acid side chains at position P2, P5, P8, and P9 were surface exposed (28). In addition, functional studies demonstrated that some specific CTLs recognized amino acid side chains in position P6 (24, 26). Therefore, we synthesized ND1/9-mer peptide variations in which the amino acids in position P2, P5, P6, P8, and P9 were exchanged for the amino acid alanine or the amino acids in positions P9 or P8 and P9 were removed (Table IGo).


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  		Table I. Nomenclature, sequence, and binding of the peptides used in this study1

 
We first studied how changes in the ND1/9-mer peptide sequence affected binding to H2-M3. We measured the relative binding abilities of the different ND1 peptide mutants using a very sensitive H2-M3 up-regulation assay. 13S2 fibroblasts transfected with the chimeric H2-M3wt/Ld molecule, which is expressed at high numbers, were incubated overnight with the different ND1 peptide mutants. H2-M3 binding peptides stabilized the MHC/peptide complex on the surface. Indeed, addition of exogenous peptides resulted in numbers of surface MHC molecules that far exceeded those seen on normal cells (29). These high expression levels allowed us to measure the binding capabilities of the peptide variants. Surface expression of the chimeric H2-M3wt/Ld molecule, as a measure of MHC/peptide affinity, was determined using the anti-Ld-specific mAb, 28-14-8S (Table IGo). Binding of a non-fM peptide, the OVA-derived SIINFEKL, to H2-M3 was not detectable above background levels (data not shown).

Removing the amino acid in position P9 (ND1/8-mer) did not alter affinity to H2-M3 (Table IGo). The ND1/7-mer peptide, in which the two C-terminal amino acids had been removed, required 60-fold higher peptide concentrations for half-maximal H2-M3 stabilization compared with the concentration of ND1/9-mer peptide required for this stabilization. Changing position 9 (ND1/P9) to an alanine did not decrease the stability of the MHC/peptide complex. However, individual changes at position 2 (ND1/P2), 5 (ND1/P5), 6 (ND1/P6), or 8 (ND1/P8), as well as multiple changes at positions 8 and 9 (ND1/P89) or 2, 5, 8, and 9 (ND1/P2589) reduced peptide binding by 6- to 36-fold.

Recognition of ND1 mutant peptides

Positive selection induces shifts in the surface phenotype of immature thymocytes and leads to the functional maturation of T cells. In the case of positive selection of MHC class I-restricted T cells, CD4+CD8+ double-positive thymocytes mature into CD8+ single-positive thymocytes (20). We had previously used a FTOC system to establish that a ND1-derived fM peptide represented the physiological ligand of positive selection for C10.4 TCRtrans+ T cells (23). We had further determined that the ND1/9-mer peptide is not susceptible to peptide degredation by serum peptidases in the experimental system we established (data not shown). In addition to that we used a peptidase inhibitor cocktail as described in Materials and Methods. We now used this system to study how mutations within the ND1/9-mer sequence affected this T cell differentiation process. C10.4+/+ TAP1-/- FTOCs were supplemented with decreasing concentrations of the different peptide variants. After 6 days of culture, the thymic lobes were harvested and the developing thymocytes were analyzed for surface expression of CD4, CD8, and TCR (using the F23.1 and H57-597 mAbs). The ND1/9-mer induced physiological levels of CD8+ Vß8+ single-positive thymocytes (20–30%) when added at 39 nM (23) (Fig. 1Go). As previously reported, the overall number of thymocytes did not increase, and the newly appearing mature-type thymocytes were not blast-like in nature (data not shown). None of the peptide variants was as effective as the ND1/9-mer peptide in inducing development of C10.4 TCRtrans+ T cells (Fig. 2Go). Therefore, staining patterns for these peptides are only presented for the highest concentration (Fig. 1Go). Neither the ND1/8-mer nor the ND1/7-mer peptide induced thymocyte maturation at the highest peptide concentrations of 10 µM even though the ND1/8-mer peptide bound to H2-M3 with high affinity. One of the peptide mutants, namely the ND1/P2, induced phenotypic shifts toward mature thymocytes at the two highest peptide concentrations. However, even this most potent peptide mutant was at least 100-fold less effective than the ND1/9-mer peptide in inducing positive selection in this assay. The ND1/P5 and the ND1/P2589 peptides showed activity only at the highest peptide concentration of 10 µM.



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  		FIGURE 1. FTOCs using different ND1 variations. The peptide variants were added at the indicated concentration to C10.4+/+ TAP1-/- FTOCs. After 6 days in the FTOC, thymocytes were teased into a single-cell suspension and analyzed by flow cytometry using the mAbs shown. A, The staining patterns for cells selected on 39 nM ND1/9-mer peptide is shown. B–J, Results for the different peptide mutants (all at 10 µM) are shown. K, A FTOC pulsed with no peptide as a negative control is shown. Each quadrant label shows the percentage of total live gated cells (as determined by forward/side scatter) within the quadrant. The marker labels show percentage CD8+Vß8+ or CD8+TCR+ T cells of all live gated cells.

 


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  		FIGURE 2. Dependence of positive selection on the concentrations of the peptide mutants. An overview of the percentage of CD8+Vß8+ positively selected T cells is provided covering the titration range of each peptide mutant used in FTOCs (as indicated in the figure). Percentages are expressed as the mean of two thymic lobes.

 
Functionality of the resulting thymocytes

Positive selection not only induces phenotypic T cell maturation, but also generates functional T cells. Indeed, we had demonstrated earlier that positively selected C10.4 TCRtrans+ thymocytes (defined as FTOCs resulting in about 20–30% CD8+ Vß8+ thymoctes), when expanded in vitro by TCR cross-linkage, developed into specific CTLs (23). This level of mature-type T cells was reached in FTOCs to which ND1/9-mer (39 nM), ND1/P2 (2.5 µM), ND1/P5 (10 µM), or ND1/P2589 (10 µM) had been added. Thymocytes were harvested from all FTOCs, expanded, and tested for specific CTL responses (Fig. 3Go). CD8high TCRhigh T cells proliferated from all FTOCs, including those that had not shown signs of positive selection (data not shown). Only the ND1/9-mer, the ND1/P2, and the ND1/P2589 peptides induced the development of specific CTLs. The ND1/P5 peptide, which had been the weakest inducer of the phenotype shift, showed only marginal CTL activity. T cells grown from any of the other cultures did not show any specific lytic activity. Thus, this finding indicated that at physiological levels of positive selection functionally mature thymocytes developed.



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  		FIGURE 3. Functionality of the matured thymocytes from FTOCs. After 6 days of FTOC with the different peptides, thymocytes were expanded for 3 days by TCR cross-linkage and tested for specific killing in CTL assays. EL4 target cells were labeled either with the cognate AttM fM peptide fMIVTLF ({blacksquare}) or with the unrelated H2-M3 binding control peptide fMIGWII ({square}). The E:T ratio was 10:1. Expanded thymocytes were derived from FTOCs supplemented with the ND1/9-mer peptide at 39 nM, the ND1/P2 peptide at 2.5 µM, and all other peptides at 10 µM.

 
TCR sensitization in the periphery induced by the peptide variants

We next examined whether any of these peptides was seen by mature C10.4 TCRtrans+ CTLs (Fig. 4Go). The ND1/9-mer peptide sensitized targets at peptide concentrations that were higher than those required for positive selection in FTOCs. When we incubated target cells with either the ND1/P2 or the ND1/P2589 peptide at concentrations at which they had induced T cell maturation, the targets were not lysed by C10.4 TCRtrans+ CTLs. In Table IGo, we had determined that these peptides bound to H2-M3 molecules. However, these peptide variants could not be recognized by C10.4 TCRtrans+ T cells.



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  		FIGURE 4. Sensitization of EL4 cells for lysis by the C10.4 TCRtrans+ CTLs. 51Cr-labeled EL4 target cells were incubated with decreasing concentrations of the different peptide mutants and exposed to C10.4 TCRtrans+ CTLs at an E:T ratio of 5:1. After 4 h, supernatant was collected and percent specific lysis was determined. All data was collected in triplicates.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
One extension of the "differential avidity" hypothesis was that recognition during positive selection could occur on a "gemisch" of peptides and therefore it was promiscuous. Indeed, we had earlier seen that at high peptide concentrations several unrelated peptides were able to induce positive selection (18). Changing to the present experimental system, we found that at low epitope density, positive selection of the C10.4 TCRtrans+ mouse occurred only with a single mitochondrial fM peptide (23). Thus, these studies suggested that positive selection was specific rather than promiscuous. Recent experimental findings by others lead to a similar conclusion (22). This animal model further provided an opportunity directly to determine whether this ligand is recognized with high or low specificity. We studied how mutations of this peptide affected its ability to induce positive selection.

T cell recognition depends on the interaction of a TCR with an MHC/peptide complex. Because stability of the MHC/peptide complex is a crucial component of T cell recognition, we determined how variants of the ND1/9-mer peptide bound to H2-M3. Although H2-M3 preferentially binds fM peptides, a peptide motif able to predict binding to H2-M3 has not been defined yet (38). Therefore, we used the most sensitive assay available to study MHC/peptide binding (29). As seen in Table IGo, some, but not all, of the different ND1 variants tested showed reduced binding to H2-M3.

We already knew that naturally occurring fM peptides extracted from surface H2-M3 molecules, as well as the ND1/9-mer peptide, reconstituted physiological levels of positive selection. They had to be added to FTOCs at low nanomolar concentrations (23). We had found also that at very high peptide concentrations, positive selection of the C10.4 TCRtrans+ lost specificity. Others had shown that positive selection occurs within a narrow window of affinities (39). Therefore, we titrated each of the peptide mutants over a broad range of concentrations to include the physiological ranges of avidities. Removing the lysine in P9 abolished the ability of the ND1/8-mer peptide to induce positive selection at any peptide concentration even though binding ability to H2-M3 was not affected. Therefore, at low epitope density, immature C10.4 TCRtrans+ thymocytes depended on the leucine in P9. In addition, mutations of surface side chains profoundly impaired the ability of any of these peptide variants positively to select. There was no apparent correlation between the binding affinity of these peptides to H2-M3 and their ability to induce positive selection in our system. For example, the ND1/8-mer peptide only bound 2-fold less, yet showed no signs of positive selection above background. However the ND1/P2 showed a 35-fold decreased binding capability, yet scored as one of the best positive selectors at high peptide concentrations. Furthermore, we had earlier shown that the unrelated LemA peptide binds H2-M3 more efficiently, yet required high peptide concentrations to show signs of induction of positive selection (23). Therefore, recognition during positive selection seemed to be specific.

Recent experiments further revealed that the cognate AttM/9-mer peptide was able to comparably bind to the H2-M3/Ld molecule and induce positive selection at a significant lower peptide concentration.4 The physiological ligand of positive selection, the ND1 peptide (fMFFINILTL), and the cognate AttM peptide (fMIVTLFYSA) are nearly unrelated in their sequences. Thus, we have now found that C10.4 TCRtrans+ T cells recognized two unrelated peptides with specificity, albeit at different maturation stages. An even broader search of peptide variants or a search of random peptide libraries might have identified additional peptides seen by the C10.4 TCRtrans.

A phenotypic shift from double-positive to single-positive thymocytes is not the only sign of positive selection. Fully mature CD8+ thymocytes can also be induced to become functional CTLs. When we expanded thymocytes from these FTOCs by TCR cross-linkage, we found that they gave rise to CTLs able to lyse target cells coated with the cognate AttM peptide. Although thymocytes harvested from all FTOCs expanded after TCR cross-linkage, only FTOCs that had shown signs of positive selection gave rise to functional CTLs. The ND1/P2, ND1/P2589, and ND1/P5 peptides only induced phenotypic shifts at high peptide concentrations (Fig. 2Go). From these FTOCs, we could expand CTLs albeit at low (ND1/P2 and ND1/P2589) to borderline levels (ND1/P5) (Fig. 3Go). Expanded effectors from nonpositively selected FTOCs could not lyse target cells. Thus, the efficacy to induce phenotypic shifts in FTOCs correlated with the CTL activity after in vitro expansion. We could exclude the possibility that positive selection on ND1 variants depended on the expression of endogenous TCR chains. We had run control experiments in FTOCs established from C10.4 TCRtrans+ TAP1-/- RAG-/- mice and received similar results (data not shown). Therefore, we concluded that thymocytes selected on high levels of one of the peptide variants were functionally similar to thymocytes selected on low levels of the ND1/9-mer self-peptide.

In FTOCs conditioned with nonselecting peptides, an increase in the frequency of CD8+ single-positive thymocytes over background was not observed. Even though these background thymocytes could be expanded in vitro, they were not able to lyse specific targets (Fig. 3Go). Recent studies in our laboratory have shown that these nonselected thymocytes are qualitatively different from positively selected cells. They fail to express CD8 {alpha}ß heterodimers seen on normal CTLs.4

We know from previous experiments that recognition of immature C10.4 TCRtrans+ T cells became promiscuous when the selecting peptide concentration was raised to 10 µM (23). Even mitochondrial peptides with no apparent homology to the ND1/9-mer peptide had been shown to induce positive selection of C10.4 TCRtrans+ T cells. Thus, at high peptide concentration levels, recognition of amino acid side chains became less important. This fact would explain why the "flat" ND1/P2589 peptide induced some positive selection at the highest peptide concentration. Its increased activity compared with the other mutants might be caused by slight conformational changes that it induced in the MHC structure (40, 41, 42).

Others have shown that incubating cells with exogenous peptides at these low nanomolar concentrations did not result in detectable up-regulation of normal MHC class Ib molecules such as H2-M3. H2-M3 surface expression indeed is extremely low due to the scarcity of H2-M3 binding peptides and the low levels of specific mRNA (43). If under physiological conditions the number of surface H2-M3/peptide complexes was low, then positive selection of C10.4 TCRtrans+ T cells was driven by few interactions. The signs of positive selection that we had seen at high peptide concentrations were most likely due to high, nonphysiological numbers of MHC/peptide complexes on the cell surface. Indeed other reports had shown that TCR/MHC interactions became promiscuous at these very high peptide concentrations (18, 23).

Our findings demonstrated that under physiological conditions positive selection of C10.4 TCRtrans+ T cells was highly specific and occurred as a low-abundance interaction between the TCR and MHC/peptide complexes. This exquisite specificity was lost at high epitope densities. In contrast to H2-M3, MHC class Ia molecules are present on the cell surface at high numbers. Therefore, high number of MHC/peptide complexes could compensate for the low affinity of a promiscuous TCR. However, that does not necessarily mean that positive selection is promiscuous. Others had provided evidence that a repertoire of T cells restricted to MHC class II molecules was selected on diverse peptides present at low levels (22). Therefore, our observation that under physiological conditions C10.4 TCRtrans+ T cells were selected by a specific and low-abundance interaction might be representative of all positive selection events.


   Acknowledgments
 
We thank Kevin R. Dahl for technical assistance, Kara Lukin for critically reading this manuscript, Amy Marrs and Randy Anselment for synthesis of the fM peptides, Robert Rich and John Rodgers for the 13S2 cell line, and Bill Townend and Shirley Sobus for assistance with flow cytometry.


   Footnotes
 
1 This work was supported in parts by Grants HD26841C, AI35194A, and AI22295D from the National Institutes of Health and a grant from the Cancer League of Colorado (all to U.D.S.). Back

2 Address correspondence and reprint requests to Dr. Uwe D. Staerz, Department of Medicine, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. Back

3 Abbreviations used in this paper: TCRtrans+, TCR transgenic; fM, formylated Methionine; FTOC, fetal thymic organ culture; ND1, NADH dehyrogenase subunit 1; RAG, recombination activating gene; wt, wild type. Back

4 R. E. Berg, S. Irion, S. J. Kattman, M. F. Princiotta, and U. D. Staerz. Submitted for publication. Back

Received for publication October 6, 1999. Accepted for publication February 24, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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