Plasticity in the positive selection of T cells: affinity of the selecting antigen and IL-7 affect T cell responsiveness
Robert L. Rubin and
Tracee M. Hermanson
Department of Molecular Genetics and Microbiology, MSC08 4660, 1 University of New Mexico Medical School, Albuquerque, NM 87131, USA
Correspondence to: R. L. Rubin; Email: rlrubin{at}salud.unm.edu
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Abstract
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The current study examines how responsiveness of T cells is affected by the avidity of the peptide/MHC engaged during positive selection of their thymocyte precursors. We used a thymus reaggregate culture system in which CD4+CD8+ thymocytes from AND TCR transgenic mice were induced to undergo positive selection by pigeon cytochrome c (PCC) peptide or its analogs presented by I-Ek class II MHC on a thymic epithelial cell line. When low-affinity peptide analogs drove positive selection, up to 100 µM was needed to produce >50% CD4+ T cells, and these cells were highly responsive to PCC. In contrast, <0.2 µM high-affinity peptides was required to achieve similar selection efficiency, but the resultant cells failed to respond to PCC. However, these cells were not dead based on dye exclusion and capacity to respond to phorbal ester and to agonist if IL-2 was also present, supporting the view that non-responsiveness of cells selected on high-affinity peptides is a form of central T cell tolerance distinct from deletion. Cells selected on intermediate-affinity peptides showed variable responsiveness which was suppressed 5- to 10-fold by addition during reaggregate culture of antibody to the IL-7R. Similarly, supplementary IL-7 in the reaggregate culture produced CD4+ T cells that were promiscuously responsive. Overall, this study demonstrates that the responsiveness of T cells is not rigidly controlled and that the presence of IL-7 during T cell development has the potential to negate central T cell tolerance and produce autoreactive T cells.
Keywords: autoimmunity, clonal anergy, lymphocyte activation, self-tolerance, thymus gland/immunology
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Introduction
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While an agonist with high affinity for the TCR can stimulate peripheral T cells, the same peptide can trigger cell death if present during T cell development in the thymus (1, 2). At lower concentrations of peptide that induce this process of negative selection, an agonist peptide can rescue a pre-T cell from programed cell death in a process termed positive selection (36). Lower affinity analogs that behave as partial agonists or antagonists to peripheral T cells can also support positive selection if present at sufficient concentration (5, 79). The differential avidity model has been proposed to describe these phenomena in which the fate of a T cell is determined by the number of TCRs engaged during development, which in turn is determined by the intrinsic affinity of the peptide/MHC for the TCR and the density of these molecules on the interacting cell surfaces (3). This model accounts for the correlation between the efficiency of positive selection and the concentration and affinity of the peptide for the TCR (10) and for the requirement of higher ligand concentration to induce negative selection (1, 2, 5, 11).
Positive selection is accompanied by numerous changes in transcription and in intracellular signaling molecules that are affected by and affect expression of cell-surface molecules, including up-regulation of the TCR, CD2, CD5, CD69 [reviewed in (12, 13)] and sialylation level (14) and down-modulation of CD4 or CD8. Most studies on the role of peptides in positive selection have focused on these convenient surface markers of development and maturation. In contrast, there are limited data on the importance of selecting peptide on the subsequent functionality of the mature T cell as measured by proliferative response to agonists. In systems in which negative selection is partially inhibited or cannot occur, T cells selected on high-affinity agonists are usually non-responsive to subsequent challenge (4, 1520). In contrast, CD4 or CD8 thymocytes capable of responding to a high-affinity antigen were typically selected on a much lower affinity analog or on an antigen of no obvious structural relationship to the challenge antigen (5, 15, 2130). However, disagreement exists as to how degenerate the selection process is (3133) and whether cells selected on high-affinity agonists are always non-functional (11, 3436). In addition, there has been no systematic study of how responsiveness of T cells is affected by the avidity of the peptide/MHC engaged during positive selection of precursor thymocytes. This issue is important not only for understanding the physical basis of positive selection but has substantial ramifications for disease because aberrations in this process may be the basis for failure of T cell tolerance in autoimmunity or insufficient T cell functionality in infection.
The current study was undertaken to test the hypothesis that responsiveness of T cells is affected by the affinity of the peptide engaged during positive selection of its precursor thymocyte. The idea that characteristics of T cells are affected by the strength of signaling experienced during positive selection in the thymus has been variously described as tuning (3740) or adaptation (41). The simplest prediction is that T cells would be educated during positive selection to have the capacity to respond only to antigens of higher affinity for the TCR than the selecting antigens as a result of the proportionate accumulation of negative regulators of T cell activation. However, it is unclear whether there is a threshold or a quantitative inverse relationship between selecting peptide affinity and subsequent T cell responsiveness or whether T cell function depends on features of the selecting peptide structure not necessarily manifested as binding or functional avidity.
The current work employs the well-characterized thymus reaggregate culture system (42, 43). In these organ-like cultures, CD4+CD8+ pre-selection thymocytes from the AND TCR transgenic mouse which develop into the CD4 lineage were subjected to pigeon cytochrome c (PCC) peptide analogs of graded affinities for the TCR presented by a thymic epithelial cell line expressing a permissive allele of the class II MHC as previously described (10, 44). After incubation as a cell aggregate, the capacity of developed CD4+ cells to respond to agonist challenge was tested in a second stage culture. Because the CD4+CD8+ pre-T cells are derived from a mouse that has a non-permissive MHC class II allele, positive selection is synchronously driven in vitro predominately by the experimental peptide. Since the thymic epithelial cell line employed does not express the co-stimulatory or adhesion molecules B7-1, B7-2, ICAM-1 or CD40, its antigen-presenting function is limited to positive selection. Also, because the antigen-presenting cells (APCs) cannot support T cell proliferative or negative selection (44), confounding effects such as activation during reaggregate culture of possible contaminating post-selection thymocytes are largely eliminated. Since classical negative selection does not occur in this in vitro culture system (45), the viability and functionality of cells selected on high- as well as low-avidity antigens could be compared.
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Methods
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Mice
Mice transgenic for the
- and ß-chain of the TCR specific for PCC peptide 88104 (AND) originally developed by Kaye et al. (46) were obtained from Jackson Laboratory, Bar Harbor, ME, USA, for those on the C57BL/6 background (stock no. 2408) and from Stephen Hedrick (University of California, San Diego, CA, USA) through Howard M. Grey (La Jolla Institute for Allergy and Immunology, San Diego, CA, USA) for those on the B10.A(4R) background. B10.BR and B10.A(4R) mice were obtained from Jackson Laboratory and bred at the University of New Mexico (UNM) vivarium. A colony of homozygous AND B10.A(4R) mice was maintained at UNM, and the males were bred with the B10.A(4R) females to produce F1[B10.A(4R) x AND B10.A(4R)] neonatal mice.
Reagants
Reagants for FACS® analysis were FITC-labeled anti-CD4 and PE-labeled anti-CD8 (Caltag, Inc., Burlingame, CA, USA) and 7-aminoactinomycin D (7-AAD) was obtained from Molecular Probes, Inc. (Portland, OR, USA). IL-2 and IL-7 were obtained from R&D Systems, Minneapolis, MN, USA. Peptides were chemically synthesized on a Rainin Symphony synthesizer (Peptide Technologies, Inc., Washington, DC, USA). They were purified to >95% purity by reverse-phase HPLC and their identity is shown in Table 2, verified by amino acid analysis or mass spectrometry. Standard tissue culture medium was RPMI 1640 medium base (GIBCO Invitrogen, Grand Island, NY, USA) with 10% fetal bovine serum (GIBCO), 20 mM HEPES, 0.1 mM non-essential amino acids, 1 mM pyruvate, 2 mM glutamine, 50 µM mercaptoethanol, 0.5 µg ml1 fungizone and 50 µg ml1 gentamycin. Anti-mouse IL-7 antibody (polyclonal; Cell Science, Inc., Canton, MA, USA) and anti-mouse IL-7R
-chain antibody (clone SB/14; BD PharMingen, San Diego, CA, USA) were added to some reaggregate cultures.
Reaggregate cultures
An organ culture system in which CD4+CD8+ double positive (DP) AND thymocytes were driven to CD4+ (single positive, SP) by peptide presented by ANV/I-Ek cells was adapted from Wang et al. (10). ANV cells are a cortical epithelial cell line described by Nelson et al. (44), transfected with the I-Ek
- and ß-chain genes (10) and maintained at sub-confluency in a short-term culture in the presence of 200 µg ml1 G418 (GIBCO) in RPMI 1640 medium. A single-cell suspension of thymocytes was made from a litter of newborn (3- to 6-day old) [B10.A(4R) x AND B10.A(4R)]F1 mice, and DP thymocytes were further purified by magnetic separation with anti-CD8a (Ly-2) microbeads from Miltenyi Biotec (Auburn, CA, USA) according to the manufacturer's recommendations. Washed DP cells were suspended at 20 million ml1 in RPMI 1640 medium and mixed with ANV/I-EK cells at a ratio of 7: 1 (thymocytes: ANV). The cell mixture was centrifuged at 2000 r.p.m. for 7 min without use of the brake, and the supernatant was removed. Aliquots of the pellet containing one million thymocytes were transferred in a volume of 2 µl to a 13-mm diameter, 0.8-µM nucleopore track-edge membrane (Whatman, Clifton, NJ, USA) placed on a Gelfoam gelatin sponge (2 cm2 x 7 mm thick) (Pharmacia & Upjohn, Kalamazoo, MI, USA) in a 15 x 60-mm Falcon 3004 tissue culture dish (Becton Dickinson, Franklin Lakes, NJ, USA) with a total volume of 3 ml RPMI 1640 medium. Up to nine aliquots were distributed onto a single membrane. Test peptide with or without IL-7 or anti-IL-7R antibody at the indicated concentrations was added to the medium as noted. Reaggregates were harvested after 4 days culture in a 5% CO2 incubator by pooling replicates into a 1.5-ml microfuge tube, briefly centrifuging and dispersing the pooled pellets with the aid of a polypropylene pestle (Kimble/Kontes, Vineland, NJ, USA). A uniform percentage of each preparation (usually 20%) was stained with anti-CD4, anti-CD8 and 7-AAD and examined with a FACScalibur or FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA); the remainder was washed twice and suspended in a standard volume for measurement of the proliferative capacity. In some experiments CD4+ thymocytes or CD4+ peripheral T cells were purified from harvested reaggregates or spleens from AND C57BL/6 mice, respectively, using anti-CD4 magnetic beads (Miltenyi).
T cell proliferation assay
The standard method was to add the T cell source and the test peptide in triplicate to wells of a flat-bottom microtiter plate containing 500 000 B10.BR splenocytes irradiated at 3000 rad as the source of APCs and to incubate for 3 days, with the last 8 h in the presence of 1 µCi [3H]thymidine ([3H]TdR). Cells were harvested on glass fiber filters and radioactivity was determined by liquid scintillation spectrometry. In comparing the proliferative response of thymocytes obtained from reaggregates grown under different conditions within the same experiment, cells from pooled, replicate reaggregates were normalized by bringing up to the same volume of the medium. The positive control for each preparation was either phorbol myristate acetate (PMA) at 200 ng ml1 + ionomycin at 20 µg ml1 (both from Sigma Chemical Co., St Louis, MO, USA) or IL-2 at 200 U ml1 + agonist as indicated. In testing the antigenicity of various peptides, AND splenocytes from C57BL/6 mice were typically used at 50 000100 000 cells per well. In some experiments CD4+ splenocytes were first purified using anti-CD4 magnetic beads (Miltenyi). Results are expressed either as raw average counts per minute (c.p.m.) or as the stimulation index (SI), which is the ratio of the average response to the test peptide divided by the c.p.m. in the absence of agonist.
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Results
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Peptide-mediated differentiation of CD4+CD8+ thymocytes into CD4+ T cells
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2% of total thymocytes in AND transgenic B10.A(4R) mice can proceed in development because the AND TCR does not recognize I-Ak, the only MHC class II complex expressed in these mice (Fig. 1A). About two-thirds of the thymocytes are CD4+CD8+ DP, and these cells were purified by magnetic anti-CD8 microbeads (Fig. 1A) to produce a starting population that was 95% DP and <1% CD4+ cells. For each reaggregate, one million AND DP thymocytes were centrifuged together with ANV cells to form a pellet. ANV cells were derived by Nelson et al. (44) from thymus cortical epithelium transformed in vivo by human papilloma virus E6 and E7 oncogenes linked to the keratin 14 promoter; they displayed a cell-surface phenotype and response to thymic cytokines very similar to that of thymus cortical epithelium in situ. The derivative line used in these studies could not normally express the invariant chain needed to present endogenously processed protein-derived peptides (10, 47) and was made to express the I-Ek MHC at a level comparable to that of normal thymic epithelial cells by transfection with the I-Ek
- and ß-chain genes (10). Thus, ANV cells can only present exogenous peptides on this class II molecule.

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Fig. 1. FACS analysis of AND T cells before and after selection on various peptides. Part A displays the CD4 and CD8 staining of total AND B10.A(4R) thymocytes and of thymocytes subjected to anti-CD8 magnetic beads. The CD4+CD8+ DP thymocytes were the starting preparation in thymus reaggregate cultures. Part B shows typical FACS analysis after 4 days of reaggregate culture with ANV cells and the indicated peptide. Displayed events are gated for viable cells based on exclusion of the dye 7-AAD, and the percentage of cells in each quadrant is shown.
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Various concentrations of peptides structurally related to PCC, the high-affinity index peptide of peripheral AND T cells, were added to DP thymocyte/ANV cell pellets and incubated over 4 days in a thymus reaggregate culture environment. After preparation of a single-cell suspension, a standardized fraction of the cells was labeled with FITC-conjugated anti-CD4 and PE-conjugated anti-CD8, mixed with the vital dye 7-AAD and examined by three-color FACS®. We compared the efficacy of various peptides to support positive selection in this system by production of viable CD4+ T cells. Shown in Fig. 1(B) are typical FACS analysis results at concentrations of each peptide that produced 6080% CD4+ viable cells, and the average yield of viable CD4 SP cells is summarized in Table 1. Without peptide addition few viable CD4+ cells were detected, representing 2% of the average CD4 SP cells recovered with the addition of selecting peptides (Table 1) and consistent with previous studies that invariant chain-deficient I-Ek-positive thymic epithelial cells cannot support positive selection of AND T cells (10, 47). As detailed later the highest affinity peptides [PCC and moth cytochrome c (MCC)] required the lowest concentration to achieve robust production of CD4+ viable cells, whereas the lowest affinity peptides used in this study required up to a 1000-fold higher concentration to produce good selection efficiency. With some reaggregate cultures there was considerable heterogeneity in the CD4 staining; CD4 dulling resulted in the appearance of CD8 SP or double negative cells as previously reported with AND cells in the reaggregate culture (44), the significance of which is unclear.
Responsiveness of thymocytes selected on various peptides
Cells from a 4-day reaggregate culture were distributed into microtiter plates along with irradiated APCs from mice expressing the H-2k class II MHC and challenged with excess PCC. Eight-hour proliferative responses were measured on the third day, and typical results from several experiments are shown in Fig. 2. The relatively low yield of cells after reaggregate culture (Table 1) and lack of [3H]TdR incorporation in the absence of challenge peptide indicate that cell division did not take place during reaggregate culture as previously reported (48). Cells selected on PCC, MCC, MCC96N and PCC99R were essentially non-responsive to PCC as compared with the negative control in which the challenge antigen was omitted, although MCC96N produced weakly responsive cells in one of the four experiments. In contrast, cells selected on MCC100N, MCC99Q, MCC102Q, MCC102L and MCC97I were highly responsive to stimulation with PCC. There were similar numbers of viable CD4+ cells regardless of the selecting peptide based on exclusion of the vital dye 7-AAD (Fig. 1 and Table 1), although MCC102Q tended to produce 2- to 3-fold higher CD4 SP cell recovery. Regardless of the selecting peptide, cells were responsive to PMA + ionomycin (Fig. 2), indicating that failure of cells to respond to PCC was for the most part not due to loss of viability.

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Fig. 2. Responsiveness of AND T cells selected on various peptides. After 4 days reaggregate culture with the indicated peptides, cells were harvested and tested for responsiveness to 1.0 µM PCC (filled bar). Controls for each cell preparation include no challenge peptide (diagonally stripped bar) or PMA + ionomycin (positive control, open bar). These are typical results of the raw c.p.m. pooled from several experiments. Variances are standard deviations.
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Figure 3 shows the reproducibility of the responsiveness of AND T cells selected on various peptides. Cells selected on PCC, MCC and MCC96N were always non-responsive, and cells selected on MCC99Q, MCC102Q, MCC102L and MCC97I were consistently responsive, although the SI displayed substantial variation between experiments (Fig. 3). However, the functional properties of cells selected on MCC100N and PCC99R were inconsistent, two extremes of which are shown in Fig. 3. PCC99R-selected cells had an SI of 2 or less in two experiments and an SI = 90 and 280 in two other experiments. Cells selected on MCC100N had an SI of 3 or less in three experiments and 22 and 185 in the other two. MCC100N- and PCC99R-selected cells responded to PMA + ionomycin whether or not they responded to PCC (data not shown), although the response to this positive control tended to be lower if the cells failed to respond to PCC.

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Fig. 3. Reproducibility of responsiveness after selection on various peptides. Proliferative response expressed as SI of cells selected on different peptides. Responses of cells selected on the same peptide in different experiments are grouped together. The values for off-scale SIs are imbedded in bars.
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Overall, these results demonstrate that qualitative features of the peptide driving positive selection have a profound effect on the responsiveness of the selected cells. In addition, while peptides can be generally classified as producing either responsive or non-responsive T cells, there seemed to be considerable inter-experimental variability in the magnitude of this response, and two of the peptides showed sporadic capacity to produce responsive T cells, suggesting that other factor(s) in this system can affect the functional properties of cells undergoing selection.
Despite the criteria of vital dye exclusion and capacity to respond to PMA, an agonist that bypasses the TCR, the viability of cells that failed to respond to PCC was a nagging concern in these experiments. The inability of viable CD4+ thymocytes selected on certain peptides to respond to the index peptide is reminiscent of peripheral T cells exposed to altered peptide ligands (49) or made anergic by exposure to strong agonists in the absence of co-stimulation (50). Typically, such cells respond to peptide in the presence of exogenous IL-2 (51), and this was tested in the current system. As shown in Fig. 4, cells that failed to respond to PCC after selection on PCC or MCC100N displayed robust response to PCC when IL-2 was present during the challenge stage. IL-2 also substantially enhanced the responsiveness of cells selected on MCC96N, MCC102L and MCC99Q. In the absence of challenge peptide, IL-2 had no effect on proliferation (data not shown). Response to the index peptide in the presence of IL-2 supports the view that otherwise non-responsive cells are not dead, and suggests that such cells have functional features similar to those of anergic peripheral T cells.

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Fig. 4. Viability of cells selected on various peptides. After selection, cells were challenged as indicated. The selecting peptides were 0.1 µM PCC (open bar), 0.5 µM MCC100N (left diagonally stripped bar), 50 µM MCC96N (vertically stripped bar), 100 µM MCC102L (right diagonally stripped bar) and 50 µM MCC99Q (horizontally stripped bar).
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Relationship between functional affinity of the selecting peptide and responsiveness
The collection of peptides used in this study was characterized for functional affinity by measuring their capacity to stimulated peripheral AND CD4+ T cells or splenocytes from AND C57BL/6 mice in the presence of H-2k APCs. Doseresponse curves were used to estimate the concentration of the peptide that produces 20% of the maximum response of AND cells, based on response to 1.0 µM PCC as maximum. Table 2 compares these values to previously published data (10) with the same set of peptides. In general, there was good agreement between the two studies. Two exceptions for unknown reasons were PCC99R which had a 60-fold higher functional affinity and MCC97I which had a 14-fold lower affinity in the current study compared with that of Wang et al. (10).
Cells selected on various peptides were tested for their capacity to respond to high concentrations of various peptides and to increasing concentrations of PCC. As shown in Fig. 5, cells selected on PCC, MCC or MCC96N showed negligible response to all peptides as expected. In this experiment, cells selected on MCC100N did not respond to any peptide, but PCC99R-selected cells responded well to even low concentrations of PCC as well as to 10 µM PCC99R but not to peptides of lower affinity. Cells selected on the low-affinity peptides MCC99Q, MCC102Q or MCC102L showed strong, dose-dependent responses to PCC and to 10 µM PCC99R. These cells also showed substantial response to MCC and MCC96N and even had detectable response to the very low-affinity peptides, MCC102Q and MCC102L. Therefore, positive selection on low-affinity peptides produced cells that showed the same differential responsiveness to PCC analogs as displayed by mature, peripheral T cells. A minor discrepancy was the higher response of positively selected thymocytes to MCC102L and MCC102Q than to MCC99Q as compared with peripheral T cells; however, for both cell types responses to these very low-affinity agonists were very weak.
A summary of these data is shown in Fig. 6. The antigenicity (current data, Table 2) of each of the nine tested peptides is plotted against the concentration of the same peptide needed to achieve 4080% production of CD4+ cells in thymus reaggregate culture (Fig. 1). Three categories of peptide are indicated based on the functional activity of the selected thymocytes. The lowest affinity peptides, MCC99Q, MCC102Q, MCC102L and MCC97I, produced the most functionally responsive cells, and two of the three highest affinity peptides, PCC and MCC, produced cells that did not respond to any agonist. A peptide of intermediate affinity, MCC96N, also fell into the category of producing largely non-antigen-responsive T cells. Two peptides, PCC99R and MCC100N, had sporadic capacity to generate functional T cells. While there was a general inverse relationship between selecting peptide affinity and subsequent cell responsiveness, the several exceptions suggest that special features of peptide structure are important in determining the responsiveness of the selected cells. In addition, the inter-experimental variability in the responsiveness of cells selected on certain peptides suggests that other factors influence in a remarkably sensitive way the properties of positively selected thymocytes.

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Fig. 6. Relationship between peptide antigenicity, positive selection efficiency and the capacity to produce responsive T cells in thymus reaggregate culture. Antigenicity is the concentration of peptide resulting in 20% of the maximum proliferative capacity of peripheral AND T cells. Positive selection efficiency is the concentration of peptide required to convert 4080% of DP thymocytes to the CD4+ phenotype in thymus reaggregate culture. Reaction of selected thymocytes to challenge with PCC was consistently non-responsive (filled circles), consistently highly responsive (open triangles) or demonstrated inter-experimental variability in response behavior (open circles).
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Effect of IL-7 during thymus reaggregate culture
To further address the inability of thymocytes selected on high-affinity peptides to respond to signaling through the TCR, we introduced IL-7 into the thymus reaggregate culture system. IL-7 is known to be the survival cytokine for murine T and B cells and is produced in the thymus (52, 53). However, unexpected effects occurred when IL-7 was added to reaggregate cultures at 10 ng ml1. Thymocytes selected on PCC or MCC100N at this concentration of IL-7 showed a strong proliferative response to as little as 0.001 µM PCC, and response to PCC reached an SI of 592 for MCC100N-selected cells (Fig. 7). These cells also responded to MCC100N and even to the low-affinity antigen MCC99Q, with SIs >200. In contrast, cells selected on these peptides in the absence of exogenous IL-7 did not respond to challenge with any peptide (Fig. 7), while these cells showed good proliferative responses when IL-2 was added as shown previously. By FACS analysis, there was a 6.7 ± 1.0-fold increase (mean ± SD) in the total number of viable CD4 SP cells recovered in the presence of IL-7 (Fig. 7), but there was no difference in the percent of CD4+ viable cells. This suggests that 10 ng ml1 IL-7 enhanced the recovery of viable thymocytes regardless of their phenotype, although it remained possible that a component of the enhanced responsiveness of cells selected in the presence of IL-7 was due to non-specific effects on survival. In addition, while the bulk of the viable cells used in the challenge assay after thymus reaggregate culture were CD4+, there were significant numbers of DP, CD8+ and double negative cells as well, and it is possible that the increased proliferative response was the result of IL-7 enhancing the survival of one of these sub-populations.

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Fig. 7. Effect of IL-7 at 10 ng ml1 on thymocytes undergoing positive selection. AND DP thymocytes were selected on 0.1 µM PCC or 0.5 µM MCC100N in the presence or absence of 10 ng ml1 IL-7. After extensive washing, cells were challenged with the indicated agonist. The yield of CD4 SP cells in the no peptide control + IL-7 was 28 912 viable cells. The average yield of CD4 SP viable cells with selecting peptide was 223 637 cells in the absence of IL-7 and 1 427 614 cells in the presence of 10 ng ml1 IL-7.
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In order to minimize effects of IL-7 on non-specific enhancement of survival, reaggregate cultures were performed at lower IL-7 concentrations, and the resultant cells were purified. By FACS analysis, the presence of IL-7 at 1.5 ng ml1 increased the yield of viable CD4+ selected cells by 2540%, but this increase was similar for both DP and CD4+ cells (data not shown). CD4+ cells purified after positive selection on MCC in the presence of 1.5 ng ml1 IL-7 retained and therefore accounted for the robust proliferative response to PCC + IL-2 displayed by the unfractionated cell population (Table 3). While IL-7 enhanced the survival of AND DP thymocytes cultured in single-cell suspension for 4 days, such cells were capable on a per cell basis of only 23% of the proliferative response to PCC + IL-2 displayed by AND DP thymocytes after reaggregate culture. Therefore, thymocytes had to develop and differentiate in thymus reaggregate culture to become activatable in the challenge assay whether or not IL-7 was present. This result as well as the no peptide selecting antigen negative control as shown in Figs 2, 3 and 7 makes it unlikely that the robust proliferative response demonstrated after reaggregate culture was due in part to possible contamination with thymocytes pre-selected in vivo on self-antigen or that they may have been preserved in vitro due to the presence of IL-7. Table 3 also shows that while fresh CD4+ peripheral AND T cells had a proliferative response comparable to that of CD4+ SP thymocytes selected on MCC + IL-7, peripheral splenocytes showed negligible responsiveness after 4 days culture in the presence or absence of IL-7. This result makes it very unlikely that IL-7 was supporting the growth or survival of post-selection CD4+ SP AND transgenic thymocytes possibly contaminating the starting population, indicating that IL-7 was acting during positive selection to prevent acquisition of tolerance to the selecting antigen.
We wondered whether endogenous IL-7 in the reaggregate cultures was affecting the responsiveness of cells undergoing positive selection, especially on cells selected on peptides of intermediate affinity that displayed variable response characteristics (Fig. 3). Addition of anti-IL-7 antibody at 1 µg ml1 during the selection stage had no effect (data not shown). In the experiment shown in Table 4, AND cells selected on the intermediate-affinity peptides PCC99R or MCC100N were highly responsive to the tested agonists, consistent with the results of several previous experiments. However, if positive selection was done in the presence of anti-IL-7R, there was a 5- to 10-fold reduction in proliferative response to MCC and PCC99R and an almost 100-fold lower response to MCC100N (Table 4). In contrast, the presence of IL-7R antibody had no effect on the responsiveness of cells selected on the low-affinity peptide MCC97I which continued to show robust response to all the challenge agonists. The lower responsiveness of cells selected in the presence of IL-7R antibody could not generally be explained by the loss of viability because cells selected on PCC99R or MCC97I showed no reduction in responsiveness if IL-2 was present during challenge. By FACS analysis, the presence of anti-IL-7R antibody reduced the number of CD4+ viable cells recovered after selection on PCC99R and MCC100N by 30 and 66%, respectively, but there was no substantial difference in the percent of CD4+ cells (Fig. 8), indicating that the presence of anti-IL-7R could account for a maximum of only a 2-fold decrease in yield. These results suggest that IL-7 was endogenously produced during these reaggregate cultures, resulting in enhanced responsiveness of cells selected on certain peptides such as PCC99R and MCC100N.
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Table 4. Effect of anti-IL-7R antibody during reaggregate culture on subsequent responsiveness of cells selected on various peptidesa
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Fig. 8. FACS analysis after positive selection in the presence or absence of anti-IL-7R antibody. Displayed events are gated for viable cells based on exclusion of the dye 7-AAD. The percentage of cells in each quadrant and the number of recovered viable CD4 SP cells are shown.
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Discussion
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While there have been numerous studies comparing the capacity of peptide analogs to promote positive or negative selection as measured by production of CD4+ or CD8+ T cells, this is the first study to systematically evaluate the responsiveness of cells selected on a set of antigens of graded affinity for the TCR. Previous work generally showed that as the affinity of the selecting peptide for the TCR increased, the concentration needed for efficient positive selection decreased (10, 44); negative selection showed a similar relationship but required higher concentrations or a higher affinity ligand (1, 2, 5, 11). The current work is largely in agreement with these studies as assessed by FACS analysis, supporting the differential avidity model of T cell selection in the thymus in which there is no upper limit to peptide affinity in driving positive selection. However, the functionality of post-selection thymocytes displayed a more complex relationship to the affinity of the selecting peptide, suggesting that the functional T cell repertoire is dependent upon the nature of the selecting peptide(s). Low-affinity peptides produced highly responsive T cells, and cells selected on high-affinity peptides were essentially non-responsive to peptide agonists, but certain intermediate-affinity peptides produced cells with variable response as discussed below. Failure of cells selected on high-affinity peptide to respond was not due to killing or initiation of cell death since these cells were viable based on dye exclusion and the ability to proliferate in response to peptide + IL-2 or to PMA + ionomycin. The culture system did not support negative selection because of the lack of B7-1 or B7-2 expression by the APCs used in the reaggregate culture (44, 54, 55) or of any medullary epithelial cells or APCs of hematopoietic origin that mediate either CD28-dependent or -independent negative selection characteristic of a normal thymic environment (56). The viability of cells selected on the high-affinity peptides PCC or MCC was especially robust if IL-7 was present during reaggregate culture (see below). We suggest that non-responsiveness of T cells selected on high-affinity peptides is a form of central T cell tolerance independent of negative selection.
Non-deletional tolerance has been previously reported (16, 17), and these and other studies invariably showed that T cells fail to respond to the selecting antigen [reviewed in (57)]. Although failure to respond is commonly considered a manifestation of negative selection, the present results revealed considerable more subtlety and complexity to this process. We show that even moderately high-affinity antigens such as PCC99R or MCC100N sometimes selected T cells that responded to the index, high-affinity peptide or even to the selecting peptide in the case of PCC99R. Variability in responsiveness was also a feature of cells selected on low-affinity peptides such as MCC102Q and MCC102L, producing cells in different experiments that displayed as much as 10-fold differences in SI. Thymocytes used in this work retained the DNA recombination machinery, and non-allelic exclusion of the
-chain gene could give rise to cells with non-transgeneic TCRs (12). While it is possible that such receptors could drive positive selection in this system, it is unlikely that inter-experimental variability was related to a non-transgenic population of thymocytes because there was no consistent direction in the bias among the various peptides that were used in each experiment, and inter-experimental variability was not a feature of cells selected on the highest affinity peptides (PCC and MCC). Also, it is unlikely that, without the V
11 transgene, thymocytes could recognize PCC or its analogs, although it is possible that such cells could produce idiosyncratic reactions when peptides of intermediate affinity were present. Association between DP TCR transgenic thymocytes and thymic epithelial cells has recently been characterized as either dynamic with a mean contact duration of
30 min or relatively stable for 612 h as the cells underwent positive selection (58). Presumably, productive signaling associated with positive selection (43, 59) requires stable cellcell contact, and uncontrolled variable(s) affecting the intercellular dynamics within the microenvironment of the reaggregate cultures may account for the variability in responsiveness of T cells especially when selected on antigens of low or intermediate affinity for the TCR. Regardless of the cause, these results are consistent with the view proposed by Ohashi (37) that the functionality of T cells undergoing positive selection is not tightly controlled.
Plasticity in the properties of post-selection thymocytes was clearly demonstrated by the effect of IL-7, where heterogeneity in the starting cell population or the culture conditions was not a variable. It is well known that the IL-7/IL-7R system plays a dominant role in growth and survival of DP thymocytes (52, 53), although studies in TCR transgenic mice demonstrated that cell proliferation does not accompany positive selection (60). Previous studies demonstrating that IL-7 enhanced survival of post-selection SP thymocytes probably through up-regulation of Bcl-2 (61) (but not of CD4+ peripheral T cells, Table 3) is consistent with our findings that the presence of IL-7 in thymus reaggregate culture increased the yield of all major thymocyte populations by about one-third. However, by the definitive viability criterion of proliferative response in the presence of IL-2, we could show that 1.5 ng ml1 IL-7 during positive selection enhanced survival of cells selected on high-affinity antigens by >10-fold, a level equivalent to the responsiveness of cells selected on low-affinity peptides. Although these cells were largely non-responsive to agonist if IL-2 was omitted from the challenge assay, cells selected on high-affinity peptides in the presence of 10 ng IL-7 ml1 were promiscuously responsive. They displayed SIs to high-affinity peptides of several hundred fold, including the selecting peptide, and even responded to low-affinity peptides such as MCC100N or MCC99Q. With PCC99R as the selecting peptide, as low as 1.5 ng IL-7 ml1 was sufficient to enhance responsiveness several hundred fold. Responsiveness to the selecting peptide was a property of purified CD4+ post-selection thymocytes. These results suggest that the presence of IL-7 during positive selection results in CD4+ T cells with a lower activation threshold, essentially autoreactive, and the capacity of IL-7 to cause such a dramatic effect on T cell properties is consistent with the view (37) that the functional status of cells undergoing positive selection is not stringently controlled. The finding that addition of anti-IL-7R antibody during positive selection greatly inhibited the promiscuous reactivity of cells selected on PCC99R and MCC100N suggests that endogenous IL-7 underlies the inter-experimental variability in responsiveness of cells selected on these peptides.
Systematic substitution of amino acids in PCC and other model systems has been used to infer TCR and MHC contact residues and to show that the response of mature T cells can be qualitatively controlled so that only partial activation occurs as measured by gene transcription, cytokine production, phosphorylation or recruitment of molecules involved in signal transduction (6264). Differential signaling by some of the peptides used in the current study may also underlie their idiosyncratic behavior as measured by the proliferative response of post-selection thymocytes. All three of the selecting peptides substituted in any of the two major TCR contact sites (positions 99 and 102) (2, 65) became weak agonists and directed positive selection into highly responsive T cells, supporting the view that low-affinity interactions with the TCR favor functional T cells. Substitution of conservative amino acids, such as arginine for lysine in position 99 or asparagine for glutamine in position 100, resulted in a relatively small decrease in agonist activity or positive selection efficiency, but the functionality of cells that underwent positive selection with these analogs was variable. The basis for the variability with these particular peptides is unclear but appears to be related to endogenous IL-7 production as previously discussed. Two peptides had radical substitutions at residues considered to be minor TCR contact sites. Substitution of the aliphatic amino acid isoleucine for the aromatic tyrosine residue in position 97 did not significantly affect binding to H-2Ek (2) but caused a decrease of three orders of magnitude in antigenicity and over two orders of magnitude in the efficiency of positive selection; cells selected on this analog (MCC97I) were highly responsive, consistent with the conclusion that low-affinity interactions with the TCR support production of functional T cells (66). MCC96N also behaved like a low-affinity peptide based on its capacity to stimulate peripheral T cells and drive positive selection. In this case the polar asparagine residue replaced the small, aliphatic alanine residue adjacent to a major MHC contact site at position 95, possibly reducing peptide affinity for the MHC. While the affinity of MCC96N for H-2Ek has not been reported, a more conservative replacement of isoleucine for alanine at position 96, both of which have hydrophobic character, resulted in a 14-fold decrease in binding to H-2Ek relative to MCC (2), so it is likely that MCC96N binds poorly to H-2Ek. A lower affinity for MHC would explain the requirement for high concentrations of this peptide to stimulate peripheral AND T cells and to drive positive selection, consistent with previous reports (10). However, cells selected on MCC96N behaved like cells selected on high-affinity peptides, being invariably non-responsive. It is possible that this peptide retains strong signaling through the TCR, suggesting that if sufficient MHC occupancy is forced by high peptide concentration, peptides with high intrinsic affinity for the TCR create non-responsive T cells.
In summary, the current results are consistent with prior studies that functional T cells are selected on low-affinity peptides, but we also show that T cells selected on high-affinity peptides, while non-responsive, remained viable and were capable of responding to agonist if IL-2 was present. Thus, cells selected on high-affinity antigens have a behavior similar to peripheral effector T cells or T cell clones made anergic by strong signaling through the TCR in the absence of co-stimulation (67, 68). It is noteworthy that thymic epithelial cells lack co-stimulatory capacity through B7 molecules (55, 6972), and anergy accompanying positive selection has been proposed to be a major form of central T cell tolerance (57). While it could be argued that in a normal in vivo situation, cells with the potential to be tolerized by positive selection on high-affinity antigens would be subjected to negative selection anyway, once cells are tolerized they may be incapable of undergoing deletion. Regardless, central T cell tolerance diminishes the need for deletion in preventing autoimmunity. Deletion may serve as a disposal machine since thymocytes tolerized by encountering high-affinity ligands may be largely useless, although some thymocytes selected on high-affinity agonists appear to be the source of CD4+CD25+ regulatory T cells (73). However, the variable responsiveness of T cells selected on peptides of intermediate affinity and the capacity of IL-7 during the selection process to induce promiscuously responsive T cells indicate that the tolerance machinery that is triggered during positive selection can be readily modulated. Lacking flawless execution of negative selection in eliminating autoreactive T cells, this finding may have considerable ramifications for the etiology of spontaneous autoimmunity.
 |
Acknowledgements
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We thank Howard M. Grey, La Jolla Institute for Allergy and Immunology, where this work was initiated. We also thank Anke Kretz-Rommel, Alexon Antibody Technologies, and Wen Yang, Roger Williams Medical Center, for critical reading of the manuscript. The excellent technical support of Deborah Rampton and Vanessa Arteaga is also appreciated. This work was supported in part by grants NIH R24 CA88339 for Flow Cytometry and the UNM Cancer Research and Treatment Center and by grant number ES06334 from the National Institute of Environmental Health Sciences, NIH.
 |
Abbreviations
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APC | antigen-presenting cell |
7-AAD | 7-aminoactinomycin D |
c.p.m. | counts per minute |
DP | double positive |
[3H]TdR | [3H]thymidine |
MCC | moth cytochrome c |
PCC | pigeon cytochrome c |
PMA | phorbol myristate acetate |
SI | stimulation index |
SP | single positive |
UNM | University of New Mexico |
 |
Notes
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Transmitting editor: P. Ohashi
Received 29 December 2004,
accepted 29 April 2005.
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