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Functional Similarity and Differences Between Selection-Independent CD4^-CD8^- T Cells and Positively Selected CD8 T Cells Expressing the Same TCR and the Induction of Anergy in CD4^-CD8^- T Cells in Antigen-Expressing Mice¹

Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada

	Abstract

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In TCR- transgenic mice, CD4^-CD8^- TCR-⁺ ( DN) cells arise in the absence of positively selecting MHC molecules and are resistant to clonal deletion in Ag-expressing mice. In this study the activation requirements and functional properties of double-negative (DN) cells were compared with those of positively selected CD8⁺ cells expressing equivalent levels of the same MHC class I-restricted transgenic TCR. We found that positively selected CD8⁺ cells required a lower density of the antigenic ligand for optimal proliferative responses compared with DN cells derived from nonpositively selecting mice. However, when the CD8 coreceptor on CD8⁺ cells was blocked with an anti-CD8 mAb, both DN and CD8⁺ cells exhibited the same dose-response curve to the antigenic ligand and the same dependence on CD28/B7 costimulation. Positively selected CD8⁺ cells also differed from DN cells in that they differentiated into more efficient killers and IL-2 producers after Ag stimulation, even after CD8 blockade. However, Ag-activated DN and CD8⁺ cells were equally efficient in producing IFN-, suggesting that this functional property is independent of positive selection. We also found that DN cells recovered from the lymph nodes of Ag-expressing mice were functionally anergic. This anergic state was associated with defective proliferation and IL-2 production in response to Ag stimulation. These observations indicate that DN cells can be anergized in vivo by physiological levels of the antigenic ligand.

	Introduction

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CD4⁺ and CD8⁺ T cells are the products of positive selection of immature CD4⁺CD8⁺ thymocytes by MHC class II and class I molecules, respectively (1). They constitute the majority of peripheral TCR-⁺ T cells in normal mice. Although much is known regarding the mechanisms by which CD4⁺ and CD8⁺ T cells are positively selected, relatively little is known regarding the mechanisms by which CD4⁺ and CD8⁺ T cells acquire distinct functional characteristics. It is also unclear whether the positive selection process imparts signals that lead to the commitment of CD4⁺ or CD8⁺ T cells to the helper or cytotoxic lineage, respectively (2).

In addition to the major CD4⁺ and CD8⁺ subsets of T cells, a minor population of functionally mature CD4^-CD8^- TCR-⁺ cells (herein referred to as DN⁴ cells) exists in the thymus and peripheral lymphoid organs of normal mice (3, 4). This subset first appears late in thymic development (5). The TCR- on DN thymocytes is capable of transducing signals that lead to proliferation, lymphokine production, and cytolysis in vitro (6, 7). Studies of the methylation status of the CD8 gene in DN thymocytes indicate that they bear a demethylated CD8 gene, reflecting previous expression of CD8 (5). The number of DN thymocytes is also reduced by about 90% in ₂m^-/- mice (8). These data suggest that the development of the DN thymocytes requires positive selection by MHC class I or class I-like molecules and the participation of the CD8 coreceptor in this selection process. The requirement for selection is further supported by the expression of the activation markers CD44 and CD69 by these cells (4). Positive selection by MHC class I molecules has also been postulated to be the basis for the development of an NK1.1⁺ subset of DN thymocytes (8, 9). Some DN cells are autoreactive, as illustrated by the observation that cultured DN thymocytes of normal mice are cytolytic toward syngeneic target cells, a property not manifested by other thymic subsets (10).

The development of DN cells in TCR- transgenic mice is thymus dependent (11). However, their development is independent of positively selecting MHC molecules (11, 12), and they are resistant to clonal deletion in Ag-expressing mice (12, 13). These observations support the idea that DN cells in TCR transgenic mice are intrinsically indifferent to the positive and negative selection processes that affect CD4⁺ and CD8⁺ T cells. Further analysis of DN cells in TCR transgenic mice showed that these cells do not express endogenous TCR genes, have maintained the TCR locus on both chromosomes, and can coexpress TCR and TCR on the cell surface (14). This finding led to the suggestion that DN cells in TCR transgenic mice may result from the premature expression of the and TCR transgenes in the lineage. Another similarity between DN cells from TCR transgenic mice and T cells in normal mice is that the development of T cells is not dependent on the expression of MHC class I molecules (15, 16).

In this study we have compared the activation requirements and functional properties of nonpositively selected DN cells with those of positively selected CD8⁺ cells. These cells were isolated from 2C TCR transgenic mice with different MHC backgrounds. The 2C TCR is specific for the p2Ca peptide (derived from a mitochondrial protein) presented by L^d MHC class I molecules (17, 18) and is positively selected by K^b MHC class I molecules (19). The 2C TCR has a relatively high affinity (1 x 10⁶ M^-1) for the p2Ca/L^d ligand (20). We compared the activation requirements and functional properties of DN 2C TCR⁺ T cells isolated from MHC class I^-/- 2C mice with that of positively selected CD8⁺ 2C TCR⁺ T cells from H-2^b 2C mice. In this way we were able to determine which unique properties of CD8⁺ T cells were conferred by the positive selection process. We also found that DN 2C TCR⁺ cells isolated from Ag (H-2^d)-expressing mice were functionally anergic compared with those isolated from transgenic mice that did not express the H-2^d Ag. This observation indicates that physiological levels of the p2Ca/L^d ligand are sufficient to anergize DN cells.

	Materials and Methods

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Mice

2C ₂m^-/- mice were produced by back-crossing H-2^b 2C TCR transgenic mice (21) (provided by Dr. Dennis Loh) to H-2^b ₂m^-/- mice (22) (provided by Dr. Oliver Smithies). H-2^b TAP-1^-/- mice (23) were obtained from The Jackson Laboratory (Bar Harbor, ME). H-2^b/d 2C mice were produced by mating H-2^b 2C TCR transgenic mice with DBA/2 (H-2^d) mice. H-2^b H-Y TCR transgenic mice were produced as previously described (24, 25). All animals were maintained and bred in the animal facility of the Department of Microbiology and Immunology, University of British Columbia.

Cells and cell culture conditions

CD4^-CD8⁺ cells were purified from the lymph nodes of 6- to 12-wk-old mice as follows. The cells were incubated with biotinylated anti-CD8 mAb 53.58 followed by positive selection using a MACS MS⁺ separation column and MiniMACS magnet following the procedure described by the manufacturer (Miltenyi Biotech, Auburn, CA). This procedure yielded a population of cells of which >95% were CD4^-8⁺ as determined by FACS. In this case, CD8 expression was detected by the anti-CD8 mAb 53.67. CD4^-CD8^- (DN) cells were purified from the lymph nodes of 6- to 12-wk-old mice by first incubating the lymph node cells with a mixture of anti-CD4 (GK1.5) and anti-CD8 (53.67) mAbs and depletion of CD4⁺, CD8⁺, and Ig⁺ cells with anti-mouse Ig-coated Dynabeads (Dynal, Oslo, Norway). The purified DN cells were >95% CD4^-CD8^-Ig^-. The TAP-deficient cell line, T2-L^d (derived from a human T x B hybridoma transfected with murine L^d) (26), was used as APCs for the p2Ca peptide. Spleen cells from TAP-1^-/- mice were used for presentation of the H-Y peptide (27). All cells were cultured in I medium (Iscove’s DMEM (Life Technologies, Burlington, Canada) supplemented with 10% (v/v) heat-inactivated FBS (Life Technologies), 100 U of penicillin G/ml, 100 µg of streptomycin/ml, and 5 x 10^-5 M 2-ME).

The proliferative potential of the purified cells was determined by culturing 1 x 10⁴ purified cells with 5 x 10⁴ mitomycin-treated T2-L^d cells or 5 x 10⁵ irradiated TAP-1^-/- spleen cells as APCs and the indicated concentration of the antigenic peptide. The cells were incubated in 96-well round-bottom plates in 200 µl of I medium. Where indicated, a saturating concentration of anti-CD8 mAb (10 µg/ml) was used to block signaling through the CD8 coreceptor. In some cultures the CD28/B7 costimulatory pathway was blocked with 5 µg/ml of CTLA-4 Ig fusion protein (28). All cultures were set up in triplicate. Where indicated, the cultures were supplemented with 20 U/ml of rIL-2. The rIL-2 was provided in the form of spent culture medium of IL-2 gene-transfected X63/0 cells (29), which typically contained 3000 U of IL-2/ml. One microcurie of [³H]thymidine was added to the cultures in the last 6 h of a 72-h culture period.

Abs and flow cytometry

Abs and their specificities were as follows: 1B2, 2C TCR Id (30); 53.67, CD8 (American Type Culture Collection (ATCC), Manassas, VA); 53.58, CD8 (ATCC); GK1.5, CD4 (ATCC); PC61, CD25 (ATCC); Pgp-1, CD44 (ATCC); Mel-14, CD62L (ATCC); I3/2, pan-specific CD45;and 16A, CD45RB (PharMingen, San Diego, CA). All FITC and biotin conjugations of mAb, with the exception of FITC-goat anti-mouse Ig (obtained from Southern Biotechnology Associates, Birmingham, AL), and CD45RB (PharMingen) were performed in our laboratory. CD4-PE was obtained from Becton Dickinson (Mountain View, CA). Streptavidin-Tricolor (PharMingen) was used to detect biotinylated mAb. Cell staining and flow cytometric analysis were performed according to standard procedures. A FACScan equipped with LYSYS II software (Becton Dickinson) was used to acquire and analyze data. For three-color analysis, a total of 30,000 events were acquired.

Peptides

The following peptides were synthesized at the University of British Columbia: p2Ca (LSPFPFDL) (17), pMCMV (YPHFMPTNL) (20), and H-Yp (KCSRNRQYL) (27).

Cytokine assays

For cytokine production 1 x 10⁵ purified CD4^-CD8⁺1B2⁺ or CD4^-CD8^-1B2⁺ cells were stimulated with 5 x 10⁴ mitomycin-treated T2-L^d cells and the indicated peptide in a volume of 0.20 ml in I medium. Supernatants from these cultures were harvested after 40 h. The amounts of IL-2 and IFN- in the culture supernatants were determined by ELISA. The capture and biotinylated mAbs were as follows: JES6-1A12 and JES6-5H4 for IL-2 and R4-6A2 and XMG1.2 for IFN-. The R4-6A2 hybridoma cell line was obtained from the ATCC, and the XMG1.2 cell line (31) was obtained from Dr. Tim Mossman, University of Alberta (Edmonton, Canada). The JES6-1A12 and JES6-5H4 mAbs were obtained from PharMingen.

Cytotoxic assay

Effector cells were produced by incubating 1 x 10⁵ purified CD4^-CD8⁺ 1B2⁺ cells or CD4^-CD8^-1B2⁺ cells with 5 x 10⁵ mitomycin-treated T2-L^d cells and 1 µM p2Ca in a volume of 2.0 ml of I medium in 24-well plates. The activated cells were split as necessary with I medium containing 20 U/ml of IL-2. After 6 days of culture, 5 x 10⁴ activated cells were assayed for their ability to lyse ⁵¹Cr-labeled T2-L^d target cells (1 x 10⁴/well) with the indicated concentration of p2Ca peptide. In some experiments, the H-2^d mastocytoma, P815 (ATCC), was used as the target cells. Where indicated, a saturating concentration of anti-CD8 mAb (10 µg/ml) was used to block signaling through the CD8 coreceptor. The incubation time for the ⁵¹Cr release assay was 3 h. Spontaneous release in all assays ranged from 10 to 15% of the maximum releasable counts.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Development of DN 2C TCR⁺ in non-Ag-expressing and Ag-expressing mice

The 2C TCR is positively selected by K^b (19), negatively selected by L^d (21), and not selected in H-2^b ₂m^-/- mice (32), which lack MHC class I expression (22, 33). The CD4/CD8 phenotypes of thymocytes derived from 6- to 8-wk-old H-2^b, H-2^b/d, and H-2^b ₂m^-/- 2C transgenic mice are shown in Fig. 1. These patterns are typical for thymocytes that expressed the transgenic 2C TCR in positively selecting, negatively selecting, and nonselecting MHC backgrounds, respectively (21, 32). The yields of thymocytes from H-2^b, H-2^b/d, and H-2^b ₂m^-/- 2C TCR transgenic mice were 2 x 10⁷, 1 x 10⁷, and 15 x 10⁷, respectively. The relatively low yield of thymocytes from H-2^b 2C mice is attributed to deletion caused by the relatively high affinity/avidity of the 2C TCR for the positively selecting ligand(s) in H-2^b mice (34). The total numbers of lymph node cells from H-2^b, H-2^b/d, and H-2^b ₂m^-/- 2C mice were 3.3 x 10⁷, 3.3 x 10⁷, and 2.0 x 10⁷, respectively. As expected, only lymph node cells from H-2^b 2C mice have a well-defined CD4^-CD8⁺ population. DN cells from the lymph nodes of H-2^b, H-2^b/d, and H-2^b ₂m^-/- 2C mice contained 24.4, 42.7, and 21.3% 2C TCR⁺ cells, respectively (Fig. 1). The 2C TCR is detected by the anti-idiotypic mAb, 1B2 (30). The total numbers of DN 1B2⁺ cells that could be recovered from the lymph nodes of H-2^b, H-2^b/d, and H-2^b ₂m^-/- 2C mice were about 8 x 10⁶, 14 x 10⁶, and 4 x 10⁶, respectively. These results indicate that DN 2C TCR⁺ T cells are produced in significant numbers in all three lines of 2C transgenic mice and are the most abundant in H-2^b/d 2C mice.

View larger version (53K): [in this window] [in a new window]   FIGURE 1. DN 1B2+ cells are not positively selected and are not deleted in Ag-expressing mice. Thymocytes and lymph node cells from H-2b, H-2b/d, and H-2b 2m-/- 2C mice were incubated with anti-CD8-FITC, anti-CD4-PE, and biotinylated 1B2 mAb. The cells were then washed and stained with streptavidin-Tricolor. The labeled cells were analyzed using the FACScan flow cytometer. A total of 30,000 events were acquired, and the data were analyzed with the LYSYS II software program. The dot plots indicate the CD4/CD8 phenotypes of thymocytes and lymph node cells from the 2C mice with the indicated MHC background. The numbers in each quadrant indicate the percentages of cells in that quadrant. The expression of 1B2 by gated CD4-CD8- and CD4-CD8+ lymph node cells from the indicated mice are shown as histograms.

DN 1B2⁺ cells from H-2^b ₂m^-/- 2C mice are independent of MHC class I molecules for their development, whereas the development of CD8⁺ 1B2⁺ T cells is dependent on positive selection by K^b MHC class I molecules (19). Because these two populations of T cells express equivalent levels of the 2C TCR (Fig. 1), it was of interest to determine whether positively selected CD8⁺ 1B2⁺ cells differ from DN 1B2⁺ cells with regard to their activation requirements and function. The proliferative response of these two population of T cells to Ag stimulation was determined by stimulating them with varying doses of the p2Ca peptide presented by the L^d-transfectant of the peptide transporter-deficient cell line, T2 (T2-L^d) (26). It was found that CD8⁺ 1B2⁺ cells proliferated more efficiently at low, but not at high, p2Ca/L^d ligand concentrations (Fig. 2). This proliferative response is specific for the p2Ca/L^d ligand, because the addition of another L^d-binding peptide, pMCMV (20), did not lead to any proliferative response above background levels (data not shown). Therefore, the expression of CD8 on positively selected cells led to a lowering of the activation threshold on positively selected cells. However, when the CD8 molecules on the CD8⁺ T cells were blocked with an anti-CD8 mAb, the dose-response curve of CD8⁺ cells to the p2Ca/L^d ligand was similar to that of DN cells.

View larger version (29K): [in this window] [in a new window]   FIGURE 2. DN 1B2+ cells have the same requirement for the p2Ca/Ld ligand as CD4-CD8+ 1B2+ cells when the CD8 coreceptor is blocked. The responder cells (1 x 104/well) were either purified DN 1B2+ lymph node cells from H-2b 2C 2m-/- mice or purified CD8+ 1B2+ lymph node cells from H-2b 2C mice. They were incubated with mitomycin-C treated T2-Ld cells (5 x 104/well) and the indicated amount of p2Ca peptide. Anti-CD8 mAb was added to the indicated cultures at 10 µg/ml. The cultures were assayed for [3H]thymidine incorporation in the last 8 h of a 72-h culture period. The error bars denote SDs.

View this table: [in this window] [in a new window]   Table I. Similar proliferative response of DN cells from H-2b 2C and H-2b 2m-/- 2C mice to the p2Ca/Ld liganda

View larger version (27K): [in this window] [in a new window]   FIGURE 3. Ag-stimulated DN 1B2+ cells are dependent on CD28/B7 costimulation for optimal proliferation. Culture and assay conditions are described in Fig. 2. CTLA-4 Ig was added at 5 µg/ml to the indicated cultures.

Ag-activated CD8⁺ 1B2⁺ cells differentiate into cytotoxic T cells specific for the p2Ca/L^d ligand. We compared the efficacy by which Ag-activated DN 1B2⁺ and CD8⁺ 1B2⁺ T cells kill target cells expressing varying densities of the p2Ca/L^d ligand. The results in Fig. 4 showed that in the absence of anti-CD8 mAb, Ag-activated CD8⁺ 1B2⁺ cells killed target cells expressing the p2Ca/L^d ligand with a higher efficiency than Ag-activated DN 1B2⁺ cells. When the CD8 coreceptor on CD8⁺ cells was blocked with anti-CD8 mAb, this higher efficiency was only observed at higher density of the p2Ca/L^d ligand. The higher efficacy of Ag-activated CD8⁺ 1B2⁺ cells in killing target cells was also evident when an H-2^d mastocytoma cell line, P815, which expressed physiological levels of the p2Ca/L^d ligand, was used as the target cell (Fig. 5). This observation suggests that although both DN 1B2⁺ and CD8⁺ 1B2⁺ T cells have the potential to differentiate into cytotoxic effector cells, the positive selection process may have facilitated the differentiation of CD8⁺ 1B2⁺ cells into more effective killers.

View larger version (27K): [in this window] [in a new window]   FIGURE 4. Ag-activated DN 1B2+ cells differentiate into cytotoxic effector cells. Purified DN 1B2+ lymph node cells from H-2b 2m-/- 2C mice or purified CD8+ 1B2+ lymph node cells from H-2b 2C mice were cultured with T2-Ld and p2Ca as described in Materials and Methods. After 6 days of culture the Ag-activated cells (5 x 104/well) were assayed for their ability to lyse 51Cr-labeled T2-Ld target cells (1 x 104/well) in the presence of the indicated concentration of exogenous p2Ca peptide. Where indicated, anti-CD8 mAb was added at 10 µg/ml during the cytotoxic assay.

View larger version (23K): [in this window] [in a new window]   FIGURE 5. Less efficient killing of P815 by p2Ca/Ld-activated cells from H-2b 2m-/- 2C mice. p2Ca/Ld-activated CD8+ 1B2+ and DN 1B2+ cells were generated as described in Fig. 4. The target cells in this experiment were 51Cr-labeled P815 (H-2d) mastocytoma cells (1 x 104/well). The cytotoxic assays were performed with the indicated E:T cell ratios in the presence or the absence of anti-CD8 mAb (10 µg/ml).

CD8⁺ T cells produce IFN- and IL-2 in response to Ag stimulation. We compared the amounts of IFN- and IL-2 produced by these two cell types in response to Ag stimulation. It was found that DN 1B2⁺ cells were as efficient as CD8⁺ 1B2⁺ cells in producing IFN- in response to stimulation by the p2Ca/L^d ligand (Fig. 6A). By contrast, CD8⁺ 1B2⁺ cells were much more efficient than DN 1B2⁺ cells in producing IL-2 in response to p2Ca/L^d stimulation (Fig. 6B). The anti-CD8 mAb inhibited IL-2 production by CD8⁺ 1B2⁺ cells only at low, but not high, p2Ca/L^d concentrations. This result suggests that the positive selection process may have contributed to the more efficient production of IL-2 by Ag-stimulated CD8⁺ 1B2⁺ cells.

View larger version (19K): [in this window] [in a new window]   FIGURE 6. Ag-activated DN 1B2+ cells produced normal levels of IFN-, but reduced levels of IL-2. For cytokine production 1 x 105 purified CD4-CD8+1B2+ or DN 1B2+ cells were stimulated with 5 x 104 mitomycin-treated T2-Ld cells and the p2Ca peptide in a volume of 0.20 ml in I medium. Where indicated, anti-CD8 mAb (10 µg/ml) was included at the beginning of the culture period. Supernatants from these cultures were harvested after 40 h. The amounts of IFN- (A) and IL-2 (B) in the culture supernatants were determined by ELISA as described in Materials and Methods. In these assays, 10 U of IFN- = 1 ng of IFN- and 16 U of IL-2 = 1 ng of IL-2.

The above studies indicate that DN 1B2⁺ cells from H-2^b ₂m^-/- 2C mice are readily activated by the p2Ca/L^d ligand. DN 1B2⁺ are also present in large numbers in L^d-expressing mice (Fig. 1). We have shown that even relatively low concentrations of the p2Ca/L^d ligand can activate DN 1B2⁺ cells (Fig. 2). It is therefore not surprising to find that DN 1B2⁺ cells from H-2^b/d mice, which express the p2Ca/L^d ligand, have a cell surface phenotype that reflects past interaction with Ag. This is illustrated by the expression of high levels of CD44 on DN 1B2⁺ cells from H-2^b/d 2C mice (Fig. 7). However, DN 1B2⁺ cells from H-2^b/d 2C do not have the classical memory phenotype, in that they expressed very high levels of CD45RB (Fig. 7). The expressions of CD62L on DN 1B2⁺ cells from H-2^b/d and H-2^b ₂m^-/- 2C mice also differed. Those from H-2^b ₂m^-/- 2C mice expressed either an intermediate or a high level of the CD62L, whereas those from H-2^b/d 2C mice expressed a uniformly high level of the CD62L. The data in Fig. 7 also indicate that DN cells from H-2^b/d and H-2^b ₂m^-/- 2C mice expressed equivalent levels of the 2C TCR, CD25, and total levels of CD45 (detected by the I3/2 mAb).

View larger version (22K): [in this window] [in a new window]   FIGURE 7. Up-regulation of CD44 and CD45RB in DN 1B2+ cells from H-2b/d 2C mice. DN cells from the lymph nodes of H-2b 2C 2m-/- and H-2b/d 2C mice were purified as described in Materials and Methods. The purified DN cells were incubated with anti-CD4-PE, anti-CD8-FITC and biotinylated mAb specific for the 2C TCR (1B2), CD62L, CD44, CD45RB, or CD25. The cells were then washed and incubated with streptavidin-Tricolor and analyzed using FACScan. For analysis of total CD45 expression by these cells, the cells were incubated with anti-CD4-PE, anti-I3/2-FITC, and biotinylated 1B2 mAb. The cells were washed and then stained with streptavidin-Tricolor. The levels of these cell surface markers expressed by gated DN cells are indicated in the histograms. The two different methods for staining CD45RB (TriColor-labeled) and total CD45 (FITC-labeled) led to differences in the level of fluorescence for these two cell surface Ags and do not reflect higher expression of CD45RB relative to total CD45 levels. DN cells from the lymph nodes of H-2b 2C mice expressed the same levels of these cell surface molecules as those from H-2b 2m-/- 2C mice (data not shown).

The DN 1B2⁺ cells that are present in H-2^b/d mice are a potential source of autoreactive cells. The high expression level of CD44 on these cells also suggests that they have interacted with Ag in vivo. However, H-2^b/d 2C mice do not exhibit any overt signs of autoimmune disease. It is therefore possible that the exposure of these cells to self Ag have rendered them anergic to further Ag stimulation. We tested this possibility by determining the proliferative response of DN 1B2⁺ cells from H-2^b/d 2C mice to the p2Ca/L^d ligand. The results in Fig. 8 indicate that these cells are hyporesponsive to the p2Ca/L^d ligand relative to DN 1B2⁺ cells from H-2^b ₂m^-/- 2C mice. This result indicates that these cells are functionally anergic.

View larger version (19K): [in this window] [in a new window]   FIGURE 8. DN 1B2+ cells from H-2b/d 2C mice are hyporesponsive to the p2Ca/Ld ligand. DN from H-2b 2m-/- 2C (b2C) mice and DN cells from H-2b/d 2C (bd2C) mice were purified as described in Materials and Methods. The proliferative response of purified DN cells to the p2Ca/Ld ligand was determined as described in Fig. 2.

View larger version (18K): [in this window] [in a new window]   FIGURE 9. Ag-activated DN 1B2+ cells from H-2b/d 2C mice are defective in IL-2 production, but produced normal levels of IFN-. DN 1B2+ cells purified from the lymph nodes of H-2b/d 2C (bd2C) or H-2b 2m-/- 2C (b2C) were stimulated as described in Fig. 6. Culture supernatants were collected from the stimulated cells after a 40-h incubation period and were assayed for IL-2 (A) and IFN- (B) as described in Fig. 6.

Previous studies in H-Y TCR transgenic mice suggest that DN cells derived from either female or male H-Y TCR transgenic mice were not male reactive (11, 13). Furthermore, there is no evidence that the DN H-Y TCR⁺ cells were anergized as a result of in vivo exposure to the male Ag (11). The H-Y TCR is specific for the male peptide (H-Yp) presented by D^b. The sequence of the H-Y peptide for this transgenic TCR has been determined, and it was shown to bind poorly to the D^b molecule (27). As a result, the H-Y TCR functions like a low affinity/avidity TCR for the H-Yp/D^b ligand. We proceeded to determine whether DN H-Y TCR⁺ cells from either female or male H-2^b H-Y TCR transgenic mice can be activated by high concentrations of the H-Yp/D^b ligand. The results in Fig. 10 indicate that DN H-Y TCR⁺ cells from either female or male mice mount similar proliferative responses to the H-Yp/D^b ligand. A high concentration of H-Yp was required for a proliferative response in the absence of exogenous IL-2. The proliferative response to the H-Yp/D^b ligand was enhanced by exogenous sources of IL-2. These results provide an independent assessment of the lack of anergy induction in DN cells from male H-2^b H-Y TCR transgenic mice. They also suggest that the in vivo induction of anergy in DN cells is determined by the relative affinity/avidity of the TCR for the activating Ag.

View larger version (27K): [in this window] [in a new window]   FIGURE 10. DN H-Y TCR+ cells from male H-2b H-Y TCR transgenic mice are not anergic. DN cells were purified from the lymph nodes of female or male H-2b H-Y TCR transgenic mice. Both populations of DN cells expressed equivalent levels of H-Y TCR (data not shown). The DN cells (3 x 104/well) were stimulated with irradiated spleen cells (5 x 1010) from H-2b TAP-1-/- mice and the indicated concentration of the H-Y peptide. The proliferative response was determined by [3H]-thymidine uptake after a 3-day culture period. Where indicated, IL-2 was added at 20 U/ml.<.>

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The mechanism by which DN 1B2⁺ cells develop in H-2^b ₂m^-/- 2C mice remains to be determined. The bulk of the evidence suggests that DN TCR-⁺ cells in normal mice have previously express CD4 and CD8. Furthermore, a subset of these DN TCR-⁺ cells appears to be selected by MHC class I molecules. By contrast, the development of DN 1B2⁺ cells is independent of MHC class I selection, as they are found in large numbers in ₂m^-/- 2C mice. DN 1B2⁺ cells are also unlikely to be derived from immature CD4⁺CD8⁺ thymocytes, because these cells are deleted in L^d-expressing mice. We therefore favor the suggestion that these cells belong to a lineage that is distinct from DN cells in normal mice (11). Similar to DN H-Y TCR⁺ cells in H-Y TCR transgenic mice, the DN 1B2⁺ cells may, in fact, be of the lineage as a result of the premature expression of the and TCR transgenes in lineage cells (14). This raises the interesting prospect of using this system for detailed analysis of the signaling mechanisms that are required for T cell activation and the functions of Ag-activated T cells. One conclusion of this study is that these putative T cells, as a result of the lack of coreceptor expression, require relatively high ligand density for their activation. This requirement may render these cells ineffective for surveillance against ligands that are present at low density on cell surfaces. Interestingly, when the CD8 coreceptor is blocked, DN 1B2⁺ cells have the same activation requirement as positively selected CD8⁺ cells. This observation suggests that the coreceptor-independent signaling mechanisms that lead to T cell activation are highly conserved in different T cell lineages. Furthermore, this study also indicates that T cells of distinct lineages possess common functional properties, including IFN- production and cytotoxicity, suggesting that positive selection signals are not essential for the acquisition of these common functions.

We have shown that DN 1B2⁺ T cells from Ag-expressing mice are anergic to further Ag stimulation. Furthermore, these anergic T cells are defective in IL-2 production. These observations indicate that physiological densities of the p2Ca/L^d ligand on APC are sufficient to anergize DN 1B2⁺ T cells in Ag (L^d)-expressing mice. This observation contrasts with those previously described in H-Y TCR transgenic mice. In the H-Y system the majority of peripheral T cells in Ag-expressing mice are either of the DN or the CD8^low phenotype (13). The CD8^low population in Ag-expressing H-Y TCR transgenic mice are also -like (14). We and others have found that the CD8^low, but not the DN, population was deleted as a result of the transgenic expression of higher levels of CD8 (36, 37). In male mice the CD8^low population expressed high levels of CD44, and they were shown to be autoreactive in adoptive transfer experiments into male nu/nu mice (11). Both the CD8^low and the DN populations from male H-Y TCR transgenic mice were not activated by male APC even when exogenous IL-2 was provided (11). However, these cells proliferated vigorously when they were stimulated with anti-TCR Abs and IL-2 (11, 38). This was used as an argument to support the idea that these cells were not intrinsically anergic. In this study we used an alternative approach to assess the functionality of DN cells derived from male transgenic mice. We showed that DN cells from either female or male transgenic mice were activated by high concentrations of the H-Yp/D^b ligand (Fig. 10) even in the absence of exogenous sources of IL-2. This provides more direct support for the conclusion that the DN cells from male transgenic mice were not functionally anergic. In our previous study we also showed that the transgenic expression of the CD8 molecule in male H-Y TCR transgenic mice led to impaired calcium mobilization in the DN cells (37). This was used to argue in favor of an inhibitory role for the CD8 coreceptor in DN cells. However, in light of our study with the DN 1B2⁺ cells in Ag-expressing mice, we offer the following reinterpretation of our previously published data. Because the H-Y TCR effectively functions as a low avidity TCR for the H-Yp/D^b ligand (27), physiological densities of this ligand on APC are insufficient to activate and/or anergize DN cells. However, expression of the CD8 coreceptor on CD8^low cells enables them to be activated by physiological densities of the H-Yp/D^b ligand, as illustrated by the high level of CD44 expression on these cells (11). Transgenic expression of higher levels of CD8 coreceptor in male H-Y TCR transgenic mice led to the complete deletion of the CD8^low, but not the DN, population (36, 37). We propose that transgenic expression of CD8 will lead to the induction of anergy in DN cells from male H-Y TCR transgenic mice. This hypothesis is consistent with our observation that calcium mobilization is inhibited in DN cells from male H-Y/CD8 double transgenic mice (37). In the 2C system, the 2C TCR has a high affinity for the p2Ca/L^d ligand (20). Our data suggest that physiological densities of the p2Ca/L^d ligand on APC are sufficient to anergize DN 1B2⁺ T cells in Ag (L^d)-expressing mice, and this process is independent of CD8 coreceptor expression.

In this study we have shown that autoreactive DN 1B2⁺ cells are anergized in Ag-expressing mice. Our ongoing studies revealed that this form of T cell anergy has the following unique feature. The anergic cells behave like Ag-primed cells, because they can be activated by a low affinity ligand to express high affinity IL-2R, and they proliferated vigorously in response to the low affinity ligand and exogenous sources of IL-2. Thus, the contribution of this unique form of anergy to autoimmune responses cannot be ignored. Furthermore, if these DN cells are, in fact, DN cells disguised as DN cells, then this system provides a convenient means of evaluating the role of cells in autoimmunity. This system is also amenable for the biochemical analysis of the signaling defects associated with this form of T cell anergy, because large numbers of anergic DN cells can be recovered from Ag-expressing mice.

	Acknowledgments

 
We thank Simon Ip for excellent technical assistance. We thank Dr. Dennis Loh for permission to use the 2C TCR transgenic mice and Dr. Herman Eisen for providing the 1B2 hybridoma cell line and the T2-L^d cell line that was generated in Dr. Peter Creswell’s laboratory.

	Footnotes

 
¹ This work was supported by grants from the National Cancer Institute of Canada with funds from the Canadian Cancer Society, the Medical Research Council of Canada, and the Arthritis Society of Canada. B.M. was a recipient of a fellowship from the Medical Research Council of Canada.

² Current address: Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.

³ Address correspondence to Dr. Hung-Sia Teh, Department of Microbiology and Immunology, 6174 University Boulevard, Vancouver, British Columbia, Canada V6T 1Z3. E-mail address:

⁴ Abbreviations used in this paper: DN, CD4^-CD8^- TCR⁺; p2Ca, LSPFPFDL; H-Y peptide, KCSRNRQYL; T2-L^d, L^d transfectant of a peptide transporter-deficient cell line; ₂m, ₂-microglobulin.

Received for publication February 10, 1999. Accepted for publication May 18, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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