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The Journal of Immunology, 1998, 160: 718-727.
Copyright © 1998 by The American Association of Immunologists

Positive Selection of Thymocytes Expressing the Same TCR by Different MHC Ligands Results in the Production of Functionally Distinct Thymocytes Distinguished by Differential Expression of the Heat Stable Antigen1

Hung-Sia Teh2, Bruce Motyka and Soo-Jeet Teh

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The issue of whether the signaling process during positive selection can affect the efficiency by which the positively selected T cells respond to antigenic stimulation has not been addressed. We approached this question by determining the consequences of positive selection of a particular transgenic TCR (2C TCR) in the H-2b and the H-2k thymus. The H-2b thymus provides a strong positive-selecting environment for the 2C TCR, whereas the H-2k thymus selects weakly for the 2C TCR. Although the positively selected CD8 thymocytes from the H-2b or H-2k thymus expressed similar levels of the CD8 coreceptor molecule, those for the H-2k thymus expressed a slightly lower level of the 2C TCR. This lower level of 2C TCR expression by H-2k CD8 thymocytes was not a result of coexpression of endogenous TCRs. Interestingly, CD8 thymocytes from H-2k mice were hyporesponsive to Ag stimulation compared with those from the H-2b mice. The functional maturity of positively selected CD8 thymocytes from the H-2b or H-2k thymus was inversely correlated with the level of heat stable Ag expressed by these cells. Furthermore, TCR-derived signals appear to be more efficiently coupled to downstream pathways leading to proliferation and cytokine production in CD8 thymocytes from H-2b 2C mice than those derived from H-2k 2C mice. These results provide the first demonstration that the intensity of the signaling process during positive selection affects the efficiency by which TCR-derived signals in positively selected thymocytes are coupled to downstream effector pathways.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Positive selection refers to the development of mature, functional single-positive (SP)3 (CD4+CD8- and CD4-CD8+) thymocytes from immature double-positive (DP) (CD4+CD8+) thymocytes. This process is dependent on specific signaling events involving TCR and CD4/CD8 recognition of MHC molecules on thymic stromal cells (1). During the selection process, the selecting MHC molecules coordinately recognize the TCR and CD4 or CD8 coreceptor molecules (1, 2, 3). The result of such a selection process is the mutually exclusive expression of CD4 and CD8 coreceptor molecules on mature T cells. Those bearing class I-restricted TCRs express only CD8, whereas those bearing class II-restricted TCRs express only CD4.

Early studies examining the effect of mutations in the peptide-binding groove of MHC class I molecules implicated a role for MHC-bound peptides in the positive selection process (4, 5, 6). More recently, the function of specific peptides in the positive selection of T cells expressing MHC class I-restricted TCRs was determined in fetal thymic organ cultures of mice with a null mutation in genes encoding either the ß2-microglobulin (7, 8, 9, 10) or the TAP-1 molecule (11, 12). The addition of specific peptides to these fetal thymic organ cultures facilitated the determination of the structural requirements for MHC class I-bound peptides in the positive selection process. An adenovirus-mediated delivery of invariant chain and a given peptide was used to define the peptide requirements for the selection of MHC class II-restricted TCRs (13). These studies led to the conclusion that peptides that are structurally related to the antigenic peptide (8, 9, 10, 12, 14, 15), or even unrelated ones (13, 16), can cause the positive selection of TCRs with defined Ag specificity. In these studies, the read-out for positive selection was the generation of phenotypically mature SP thymocytes (10, 12) and, in some instances, the demonstration that the selected thymocytes or T cells were responsive to Ag stimulation (8, 9, 13, 14, 15, 17).

The above-mentioned studies do not address the issue of whether positive selection results in the production of functionally homogeneous mature SP thymocytes or whether positive selection can lead to the production of SP thymocytes at different stages of functional maturity. Other earlier studies have indicated that in normal mice the majority of CD4 SP thymocytes are functionally immature (18, 19). These functionally immature CD4 SP thymocytes expressed high levels of the heat stable Ag (HSA), whereas functionally mature SP thymocytes expressed low levels of HSA (18, 19).

The 2C TCR was derived from a CD4-CD8+ cytotoxic T cell clone of H-2b origin. This TCR is specific for the p2Ca peptide (derived from a mitochondrial protein) presented by Ld class I molecules and has a high affinity for this ligand (KA = 2 x 106 M-1) (20, 21, 22). The 2C TCR is positively selected strongly by Kb and weakly by Kbm8 (5) and is not positively selected by Kbm10, Kbm1 (5), and H-2s (23). In this study, we have followed the positive selection of the 2C TCR in the H-2b and H-2k mice. We found that positive selection of CD4-CD8+ 2C TCR+ thymocytes occurs with different efficiency in H-2b and H-2k mice. More interestingly, on a per cell basis, the positively selected CD8 thymocytes from H-2k 2C TCR transgenic mice (k2C) were less responsive to Ag stimulation than those from H-2b 2C TCR transgenic mice (b2C), and the functional maturity of the CD8 thymocytes and T cells correlated inversely with HSA expression. These results provide the first demonstration that the signaling process during positive selection can affect the efficiency by which TCR-derived signals are coupled to downstream effector pathways in the positively selected thymocytes.


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

Breeders for the H-2b 2C TCR transgenic mice (23, 24) were kindly provided by Dr. Dennis Loh (Nippon Roche Research Center, Kamakura, Kanagawa, Japan). The H-2b 2C mice were in the seventh to eight generation of backcross to C57BL/6 mice. The H-2b 2C mice were also backcrossed to B10.BR (H-2k) mice. The H-2k 2C mice used in this study were in the fourth generation of backcross to B10.BR mice. Breeders for the C57BL/6, B10.BR, and DBA/2 mice were obtained from The Jackson Laboratory, Bar Harbor, ME. BDF1 mice were F1 mice from matings of C57BL/6 mice with DBA/2 mice.

Abs and flow cytometry

The following Abs were used: anti-CD4 (GK1.5), anti-CD8 (53-6.7), anti-2C TCR Id (1B2), anti-CD3{epsilon} (2C11), anti-CD2 (RM2-5), anti-CD5 (53-7.3), anti-CD69 (H1.2F3), anti-heat stable Ag (M1/69), and anti-CD25 (PC61). Biotinylated Abs specific for CD2, CD5, CD8ß (53–5.8), CD69, and the TCR ß-chain (H57-597) were obtained from PharMingen (supplied by Cedarlane, Hornby, Ontario, Canada). The streptavidin-Tricolor reagent used to detect biotinylated Abs was obtained from Cedarlane. The hybridoma lines producing mAbs specific for HSA, CD4, CD8, and CD25 were obtained from the American Type Culture Collection (ATCC), Rockville, MD. The 2C11 hybridoma line (25) was provided by Dr. Jeffrey Bluestone, University of Chicago, IL, and the 1B2 hybridoma line (26) was provided by Dr. Herman Eisen, Massachusetts Institute of Technology, Cambridge, MA. Cell staining and flow cytometry were performed according to standard procedures. The LYSYS II software program (Becton Dickinson, Mountain View, CA) was used for data acquisition and analysis. For three-color analysis, a total of 30,000 events were acquired.

Isolation of CD8 SP thymocytes and lymph node cells

Thymocytes from b2C or k2C mice were first depleted of CD4+ T cells by incubating with anti-CD4 (GK1.5) mAb followed by depletion of CD4+ cells by anti-mouse Ig coated Dynabeads (Dynal, Oslo, Norway). The nonadherent cells after this treatment contained CD4-CD8- and CD4-CD8+ thymocytes. These nonadherent cells were then incubated with biotinylated anti-CD8ß mAb and the CD8+ cells positively selected with streptavidin-conjugated MicroBeads (Miltenyi Biotec Inc., Auburn, CA). The purity of the positively selected cells was determined by staining the positively selected cells with fluoresceinated goat F(ab')2 anti-mouse Ig Abs (Southern Biotechnology Associates, Inc., Birmingham, AL), which reacted with the anti-CD8ß mAb, and phycoerythrin-conjugated anti-CD4 mAb. Thymocytes purified in this manner are >99% CD4-CD8+. Highly purified CD4-CD8+ lymph node cells from b2C and k2C mice were isolated by the same method.

Proliferation assays

Purified CD4-CD8+ thymocytes or lymph node cells (1 x 104) were stimulated with 5 x 105 irradiated (20 Gy) spleen cells or with 10 µg/ml of anti-CD3{epsilon} mAb in a volume of 0.20 ml of Iscove’s modified Dulbecco’s medium supplemented with 5 x 10-5 M 2-ME and antibiotics (I-media). All cultures were set up in triplicates. 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 (27), which typically contained ~3000 U IL-2/ml. One microcurie of [3H]thymidine was added to the cultures in the last 6 h of a 72-h culture period.

Cytokine assays

Thymocytes and lymph node cells from b2C or k2C mice were first depleted of CD4+ cells by incubating with anti-CD4 mAb followed by depletion of CD4+ cells with anti-mouse Ig coated Dynabeads. After this step, the nonadherent cells were either of the CD4-CD8-1B2+ or CD4-CD8+1B2+ phenotype. For cytokine production, the equivalent of 1 x 105 CD4-CD8+1B2+ cells were stimulated with 3 x 104 dendritic cells from either BDF1 or C57BL/6 mice in 0.20 ml of I-medium. Dendritic cells were prepared as previously described (28). Supernatants from these cultures were harvested after 36 h. The amount of IL-2 and IFN-{gamma} in the culture supernatants was 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-{gamma}. The R4-6A2 hybridoma cell line were obtained from ATCC, and the XMG1.2 cell line (29) was obtained from Dr. Tim Mossman, University of Alberta, Edmonton, Canada. The JES6-1A12 and JES6-5H4 mAbs were obtained from PharMingen.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Phenotypic analysis of thymocytes and lymph node cells from b2C and k2C mice

In the course of our studies on the role of CD2 as a regulator of positive selection, we found that the H-2k thymus provides a weakly selecting environment for the 2C TCR when compared with the H-2b thymus (30). k2C and b2C thymi differed in the yield of thymocytes. An average of ~1 x 108 thymocytes was recovered from the k2C thymus as opposed to ~2 x 107 thymocytes recovered from the b2C thymus. This marked reduction in thymocyte yield correlated with the low percentage of DP thymocytes recovered from b2C mice (Fig. 1GoA).



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  		FIGURE 1. Expression pattern of various differentiation markers by thymocyte subsets from B10.BR, H-2k 2C, and H-2b 2C mice. Thymocytes from these three lines of mice were stained with phycoerythrin-labeled anti-CD4, fluorescein-labeled anti-CD8, and except for the anti-CD3{epsilon} mAb, the indicated biotinylated mAb followed by streptavidin-Tricolor. For determination of the cell surface expression of CD3{epsilon} by the indicated thymocyte subset, the thymocytes were stained with phycoerythrin-labeled anti-CD4, fluorescein labeled anti-CD3{epsilon}, and biotinylated anti-CD8 mAb followed by streptavidin-Tricolor. A total of 30,000 events were acquired using the FACScan flow cytometer and analyzed with the LYSYS II software program. A, CD4/CD8 phenotypes of thymocytes from these three lines of mice. The numbers in each quadrant represent the percentages of cells in these quadrants. R2 and R3 refer to the gates for CD4+CD8+ and CD4-CD8+ thymocytes for the expression of differentiation markers by CD4+CD8+ (B) and CD4-CD8+ (C) thymocytes. The R2 and R3 gates used for the analysis of CD3{epsilon} expression by these thymocyte subsets are not shown.

 
Phenotypic analyses of DP thymocytes from b2C, k2C, and B10.BR mice revealed differences in the expression of CD2, CD5, TCR/CD3, and CD8 by these populations (Fig. 1GoB). DP thymocytes from b2C mice expressed uniformly high levels of CD2, CD5, CD3{epsilon}, and the 2C TCR Id (detected by the 1B2 mAb). By contrast, those from k2C mice expressed slightly lower levels of CD2, CD5, CD3{epsilon}, and the 2C TCR Id. High levels of expression of these markers have been used as indicators of positive selection (30, 31, 32). DP thymocytes from B10.BR mice expressed the lowest level of CD5, and the expression of CD2 was biphasic, with a negative and an intermediate peak. As expected, B10.BR thymocytes did not stain with the 1B2 mAb, and staining with the anti-CD3{epsilon} mAb revealed that the majority of DP thymocytes from B10.BR mice expressed very low levels of CD3{epsilon}, but few of these cells expressed as high a level of CD3{epsilon} as those from b2C mice. The DP thymocytes from k2C and B10.BR mice expressed high levels of CD8, whereas those from b2C mice were deficient in cells that expressed high levels of CD8. All three DP populations expressed high levels of HSA.

Similar phenotypic analyses were performed on the CD8 SP thymocytes from b2C, k2C, and B10.BR mice (Fig. 1GoC). All three populations expressed uniformly high levels of CD2 and CD5. The level of CD8 expressed by CD8 SP thymocytes from b2C and k2C mice was also similar. However, CD8 SP thymocytes from b2C mice expressed a higher level of the 2C TCR Id compared with those from k2C mice. One potential explanation for the lower expression of the 2C TCR Id is that the CD8 SP thymocytes from k2C mice could have been positively selected by TCRs that expressed endogenous TCR {alpha}-chains, but cells selected by these TCRs continued to coexpress the 2C TCR. However, this explanation is unlikely, since staining of CD8 SP thymocytes with the anti-CD3{epsilon} mAb, which detects {alpha}ß TCRs of both transgenic and endogenous origin, revealed a similar decrease in the expression of CD3{epsilon} in these cells (Fig. 1GoC). The most significant phenotypic difference between CD8 SP thymocytes from b2C and k2C mice is in the level of HSA expressed. Whereas those from k2C mice expressed mainly high levels of HSA, those from b2C mice were either negative or expressed lower levels of HSA. By contrast, the pattern of HSA expression by CD8 SP thymocytes from B10.BR mice was very heterogeneous, ranging from negative to intermediate and high. In summary, positive selection of the 2C TCR in k2C mice resulted in the production of CD8 SP thymocytes that expressed a uniformly high level of HSA, normal levels of CD8, but slightly lower levels of the 2C TCR; this lower level of 2C TCR was not due to the coexpression of endogenous {alpha}ß TCRs on these cells.

The CD4/CD8 phenotypes of lymph node cells from b2C, k2C, and B10.BR mice are shown in Figure 2Go. b2C lymph node cells contained a high proportion of CD8 SP cells (36.6%) but were deficient in CD4 SP cells (4.9%). By contrast, k2C lymph node cells contained a higher proportion of CD4 SP cells (11.0%) but a smaller proportion of CD8 SP cells (13.3%). However, the proportion of CD4 and CD8 SP thymocytes in k2C lymph nodes was smaller than those observed in normal B10.BR mice. Thus, k2C mice were inefficient in accumulating CD4 and CD8 SP cells in their lymph nodes. Three-color analyses of lymph node cells indicated that the vast majority of CD8 SP cells from k2C mice expressed low levels of HSA (Fig. 2Go). This level of HSA was similar to that expressed by the vast majority of CD8 SP cells from b2C and B10.BR mice. However, CD8 SP lymph node cells from k2C mice differed from those of b2C mice in their expression of the CD8 coreceptor and the 2C TCR. Those from b2C mice expressed a uniformly high level of CD8 and 2C TCR molecules. However, those from k2C mice expressed either a high or a lower level of the 2C TCR. Subgating of these cells on the basis of CD8 expression indicated that the CD8low cells were those that expressed high levels of the 2C TCR, whereas CD8high cells expressed lower levels of the 2C TCR (data not shown). These results suggest that the CD8 SP lymph node cells from k2C mice comprised two populations, a CD8low population, that expressed the same level of the 2C TCR as those from b2C mice, and a population of cells that expressed a high level of CD8 but a much lower level of the 2C TCR than cells from b2C mice. The existence of CD4-CD8low TCR{alpha}ß+ T cells has also been observed in another line of transgenic mice expressing a MHC class I-restricted TCR, i.e., the H-Y TCR (33).These cells differ from conventional TCR-{alpha}ß+ cells in many aspects, and Bruno et al. (34) have provided evidence that they may in fact be {gamma}{delta} lineage cells. Another characteristic of the H-Y TCR transgenic mice is that the composition of the {alpha}ß TCRs of CD8 SP thymocytes and peripheral T cells is different in these mice (35, 36). It has been suggested that while the vast majority of CD8 SP thymocytes in H-Y TCR transgenic mice express the transgenic TCR, a minority of these cells are selected by cells that express endogenous TCR {alpha}-chains. Although this population of cells constitutes a minority of positively selected CD8 SP thymocytes, they have a selective advantage in peripheral lymphoid organs because these are the T cells that will respond to environmental Ags and pathogens (36). It is possible, therefore, that the lower expression of the 2C TCR by peripheral CD8 T cells in k2C mice may reflect the preferential expansion of cells that were selected by endogenous TCRs.



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  		FIGURE 2. Phenotypic analyses of lymph node cells from B10.BR, H-2k 2C, and H-2b 2C mice. Lymph node cells from these three lines of mice were stained with phycoerythrin-labeled anti-CD4, fluorescein-labeled anti-CD8, and the indicated biotinylated mAb followed by streptavidin-Tricolor. The labeled cells were analyzed as described in Figure 1Go. A, CD4/CD8 phenotype of lymph node cells. The numbers in the quadrants indicate the percentages of cells in these quadrants. The expression of level of the HSA, CD8, and 1B2 by CD4-CD8+ cells (R1 gate) are shown in B.

 
CD8 SP thymocytes and lymph node T cells from k2C mice are hyporesponsive to Ag stimulation

We next determined the responsiveness of CD8 SP thymocytes from b2C and k2C mice to Ag stimulation. Thymocytes from b2C and k2C mice contained CD8 SP and CD4-CD8- double-negative (DN) cells that expressed high levels of the 2C TCR Id. It was therefore necessary to obtain pure populations of CD8 SP thymocytes from b2C and k2C mice so that the responses of these cells to Ag stimulation could be compared on a per cell basis. Thymocytes were first depleted of CD4+CD8- and CD4+CD8+ cells by treating them with the anti-CD4 (GK1.5) mAb, followed by removal of CD4+ cells with anti-mouse Ig-coated magnetic beads. The nonadherent cells recovered from this step were either of the CD4-CD8- or the CD4-CD8+ phenotype. The CD4-CD8+ population was then recovered from this mixture by first incubating it with a biotinylated anti-CD8ß mAb followed by positive selection with streptavidin-conjugated MicroBeads. The levels of CD8, 1B2, and TCR ß-chain expressed by the purified thymocytes (>99% CD8+) are shown in Figure 3Go. It is clear from these data that the CD8 SP thymocytes from either b2C or k2C mice expressed an equivalent level of CD8. However, as noted in Figure 1GoC, CD8 SP thymocytes from k2C mice expressed a slightly lower level of the 2C TCR. The lower expression of the 2C TCR on these cells was not a result of the coexpression of endogenous TCRs, since the lower expression of TCR was also evident when these cells were stained with an anti-TCR ß-chain mAb, which detects all {alpha}ß TCRs (Fig. 3Go).



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  		FIGURE 3. Phenotypic analysis of highly purified CD8 SP thymocytes and lymph node cells. Thymocytes and lymph node cells from either b2C or k2C mice were first depleted of CD4+ and Ig+ cells by incubation with anti-CD4 mAb and anti-mouse Ig-coated magnetic beads. CD8 SP cells were then recovered from the nonadherent population by first incubating these cells with biotinylated anti-CD8ß mAb followed by positive selection with streptavidin-conjugated MicroBeads as described in Materials and Methods. The level of CD8 expression was determined by staining the purified cells with fluorescein-labeled anti-mouse Ig Abs. The level of 2C TCR Id (1B2) and TCRß (H57-597) chain expression was determined by staining these cells with either of the biotinylated mAbs followed by streptavidin-Tricolor. The cells were analyzed in the FACScan; a total of 10,000 events were analyzed.

 
CD8 SP cells from the lymph nodes of b2C and k2C mice were purified by the same method as described above. While CD8 SP lymph node cells from b2C mice expressed fairly similar levels of CD8, 1B2, and TCR ß-chain when compared with their thymic counterpart, this was not the case for CD8 SP cells from k2C lymph nodes (Fig. 3Go). As noted in Figure 2Go, these cells expressed either a high or a lower level of CD8. The high level of CD8 expressed by these cells was identical to that expressed by CD8 SP lymph node cells from B10.BR mice (overlays not shown) and was slightly higher than that expressed by CD8 SP lymph node cells from b2C mice. The level of 1B2 expressed by CD8 SP lymph node cells from k2C mice was heterogeneous. The high level was similar to that of CD8 SP lymph node cells from b2C mice; the lower levels of 1B2 expressed by these cells were either similar to those expressed by CD8 SP thymocytes from k2C mice or at lower levels than in k2C thymocytes. Interestingly, these cells expressed a level of the TCR ß-chain that was identical to that expressed by peripheral CD8 SP cells from b2C mice (Fig. 3Go). These observations are consistent with the conclusion that CD8 SP cells from k2C lymph nodes contained two populations. The first population is the CD8low population, which expressed the same level of 2C TCR as did CD8 SP cells from b2C mice. The second is the CD8high population, which coexpressed both the 2C TCR and endogenous TCRs. The total level of TCR-{alpha}ß expressed by CD8 cells from k2C mice was the same as CD8 SP T cells from b2C mice, since both populations expressed the same level of TCR ß-chain.

The responsiveness of the highly purified CD8 SP thymocytes from b2C and k2C mice to Ag stimulation was determined by stimulating them with irradiated BDF1 (H-2b/d) or B6 (H-2b) spleen cells in the absence or presence of exogenous IL-2 (Fig. 4Go). The TCR signaling capacity of these cells was also determined by stimulating them with anti-CD3{epsilon} mAb in the absence or presence of exogenous IL-2 (Fig. 4Go). In the absence of exogenous IL-2, CD8 SP thymocytes from b2C mice gave a significant proliferative response when stimulated with BDF1 spleen cells. The response of b2C to BDF1 was Ag specific, since these cells did not respond to B6. In the presence of exogenous IL-2, b2C CD8 SP thymocytes responded much more vigorously to stimulation by BDF1 spleen cells. By contrast, in the absence of exogenous IL-2, CD8 SP thymocytes from k2C mice were unable to respond to BDF1 or B6 stimulation. In the presence of IL-2, CD8 SP thymocytes from k2C mice mounted an Ag-specific response to BDF1 spleen cells. However, this response (4,906 ± 250; mean ± SEM) was much weaker than similarly stimulated CD8 SP thymocytes from b2C mice (36,257 ± 2,032). CD8 SP thymocytes from k2C mice were also hyporesponsive to stimulation by anti-CD3{epsilon} mAb and IL-2 when compared with those from b2C mice (8,385 ± 717 vs 33,079 ± 1,013). These results indicate that CD8 SP thymocytes from k2C mice differed from those from b2C mice in the requirement for exogenous IL-2 for an Ag-specific response and in the magnitude of the response. Signaling via the TCR is also less efficient in CD8 SP thymocytes from k2C mice, since these cells were less responsive to stimulation with anti-CD3{epsilon} mAb and IL-2 when compared with those from b2C mice. The hyporesponsiveness of k2C thymocytes to Ag or anti-TCR stimulation correlated with a high level of HSA expression by these cells (Fig. 1GoC).



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  		FIGURE 4. CD4-CD8+ thymocytes from H-2k 2C mice are hyporesponsive to stimulation by BDF1 spleen cells. CD8 SP cells were prepared as described in Figure 3Go. 1 x 104 CD4-CD8+1B2+ cells were stimulated with 5 x 105 irradiated (20 Gy) spleen cells from the indicated mice or with 10 µg/ml of 2C11 (anti-CD3{epsilon}) in the absence or presence of 20 U/ml of rIL-2. The cultures were set up in triplicates and assayed for thymidine incorporation after a 3-day culture period.

 
We next compared the responsiveness of highly purified CD8 SP cells from the lymph nodes of b2C and k2C mice to stimulation by BDF1 spleen cells. It is clear from the results shown in Figure 5Go that CD8 SP lymph node cells from k2C mice proliferated specifically to stimulation by BDF1 spleen cells even in the absence of exogenous IL-2 (1,622 ± 275 for BDF1 vs 114 ± 5 for B6). However the k2C response was considerably weaker than that observed for CD8 SP cells from b2C lymph nodes (43,615 ± 1,960). Whereas the addition of exogenous IL-2 increased the response of CD8 SP cells from b2C mice by less than 2-fold, the addition of exogenous IL-2 increased the response of CD8 SP cells from k2C mice to BDF1 stimulation by about 20-fold. In the presence of IL-2, the response of k2C cells to BDF1 stimulation was about half of that observed for CD8 SP cells from b2C mice. These data indicate that despite the lower expression of the 2C TCR Id (about half that of the k2C CD8 SP lymph node cells), they were much more responsive to stimulation by BDF1 than the k2C CD8 SP thymocytes, which expressed a uniformly higher level of the 2C TCR Id. Signal transduction via the TCR occurred very efficiently in CD8 SP cells from k2C lymph nodes, since these cells were stimulated to almost the same extent as those from b2C mice by anti-CD3{epsilon} mAb and IL-2 (Fig. 5Go). The greater sensitivity of k2C CD8 SP lymph node cells to Ag and anti-CD3{epsilon} stimulation correlated with the low level of HSA expressed by these cells.



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  		FIGURE 5. H-2k 2C lymph node cells can respond to BDF1 stimulation in the absence of exogenously added IL-2. Culture and assay conditions were the same as those described in Figure 4Go.

 
On a per cell basis, CD8 SP lymph node cells from b2C mice were also more responsive to BDF1 stimulation than CD8 SP thymocytes from these mice (43,615 ± 1,960 vs 9,477 ± 360). This result indicates that the CD8 SP lymph node cells from b2C mice were functionally more mature than CD8 SP thymocytes. This functional maturity also correlated with lower HSA expression.

Further evidence that CD8 SP thymocytes from k2C mice are less functionally mature than those from b2C mice

Ag-activated T cells express the early activation marker CD69 (37) and the IL-2R {alpha}-chain (CD25) (38). We determined whether CD8 SP cells from the thymus and lymph nodes of k2C mice differed in their ability to up-regulate CD69 and CD25 expression in response to Ag stimulation. Thymocytes and lymph node cells from b2C and k2C mice were depleted of CD4+ cells by incubation with anti-CD4 mAb and removal of CD4+ cells and Ig+ cells with anti-mouse Ig-coated magnetic beads. The remaining cells were either of the CD4-CD8- or CD4-CD8+ phenotype. Cells of the CD4-CD8-1B2+ phenotype were unable to respond to stimulation with H-2d spleen cells in the absence of exogenous IL-2 (Ref. 39 and data not shown). To optimize the response to the H-2d alloantigen, these cells were stimulated with dendritic cells from BDF1 or B6 mice. The expression of CD25 and CD69 by Ag-activated CD8 SP cells was determined by three-color flow cytometric analysis; the data are shown in Figure 6Go. These data indicate that almost all of the CD8 SP thymocytes from b2C mice up-regulated CD69 and CD25 when stimulated with BDF1 dendritic cells. This response was Ag-specific, since stimulation of these cells with B6 dendritic cells did not induce any detectable CD69 or CD25 up-regulation (data not shown). In contrast, these markers were very poorly induced in k2C CD8 SP thymocytes when they were stimulated with BDF1 dendritic cells. Only a very small proportion of BDF1-activated cells expressed detectable levels of these markers.



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  		FIGURE 6. H-2k 2C thymocytes do not up-regulate CD69 and CD25 when stimulated with BDF1 dendritic cells. Thymocytes and lymph node cells from b2C and k2C mice were depleted of CD4+ and Ig+ cells as described in Materials and Methods. The remaining cells were of the CD4-CD8+1B2+ or CD4-CD8-1B2+ phenotype. The number of responding cells in each well was adjusted to contain 1 x 105 CD4-CD8+1B2+ cells, and these cells were stimulated with 3 x 104 dendritic cells from BDF1 or B6 mice. After 36 h of culture, the cells were harvested and stained with phycoerythrin-labeled anti-CD4, fluorescein-labeled anti-CD8, and biotinylated mAbs specific for either CD69 or CD25 followed by streptavidin-Tricolor. The expression level of CD69 and CD25 by BDF1-stimulated CD4-CD8+ cells are depicted. The background expression of these markers was determined by staining these cells with phycoerythrin-labeled anti-CD4 and fluorescein-labeled anti-CD8 mAbs followed by streptavidin-Tricolor. The FL3 expression level of gated CD4-CD8+ cells was used to depict the level of these markers in unstained controls.

 
Lymph node cells from b2C and k2C mice were purified and stimulated in a similar manner. All BDF1-stimulated CD8 SP lymph node cells from b2C mice expressed a very high level of CD69 and CD25. No detectable up-regulation of these markers occurred in these cells when they were stimulated with B6 dendritic cells (data not shown). Stimulation of CD8 SP lymph node cells from k2C mice with BDF1 dendritic cells yielded a more heterogeneous pattern of CD69 and CD25 expression. The BDF1-activated cells expressed either a high or background level of CD69 expression. CD25 expression in these activated cells was either at a background, intermediate, or high level. These results indicated that a subpopulation of CD8 SP lymph node cells was unresponsive to activation by BDF1 dendritic cells.

Ag-activated CD8 cells produce IFN-{gamma} and IL-2. We therefore determined whether Ag-activated CD8 SP thymocytes and lymph node cells from b2C and k2C mice differed in their ability to produce these cytokines. The results shown in Table IGo indicate that CD8 SP thymocytes from k2C mice did not produce detectable amounts of IFN-{gamma} or IL-2 when they were stimulated with BDF1 dendritic cells. The corresponding cells from the b2C thymus produced small amounts of IFN-{gamma} when stimulated with BDF1 dendritic cells. The CD8 SP cells from k2C lymph nodes behaved like CD8 SP thymocytes from b2C mice in that they produced small amounts of IFN-{gamma} when stimulated with BDF1 dendritic cells. CD8 SP lymph node cells from b2C mice were most responsive to BDF1 stimulation and produced four times the amount of IFN-{gamma} compared with CD8 cells from k2C lymph nodes and readily detectable amounts of IL-2. These data provide additional evidence that CD8 SP thymocytes from k2C mice are functionally less mature than those from b2C mice.


View this table:
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  		Table I. Cytokine production by BDF1-stimulated thymocytes and lymph node cellsa

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We determined whether the signaling process during positive selection can affect the functional maturity of the positively selected thymocytes. This was done by comparing the phenotype and functional maturity of CD8 SP thymocytes that expressed the 2C TCR in H-2b and H-2k mice. The data indicate that CD8 SP thymocytes from b2C and k2C mice differ qualitatively and quantitatively in response to Ag stimulation. These observations indicate that the signaling process during positive selection process can affect the functional maturity of the positively selected thymocytes.

The following observations support the conclusion that the 2C TCR is strongly positively selected in b2C mice. First, DP thymocytes from b2C mice expressed very high levels of TCR CD2, and CD5, which are markers of positive selection. Second, DP thymocytes from b2C mice are deficient in cells that expressed high levels of CD8. Furthermore, the transgenic expression of the CD8 coreceptor molecule led to virtually a complete deletion of DP thymocytes in b2C mice, although this level of CD8 expression did not cause the deletion of DP thymocytes expressing another transgenic TCR, i.e., the H-Y TCR, in a positively selecting environment (40, 41). Third, b2C mice have a very small thymus, low percentages of DP thymocytes, and deficient development of CD4 SP thymocytes and T cells. Previous studies have shown that deletion of DP thymocytes expressing a MHC class I-restricted TCR resulted in the poor development of CD4 SP thymocytes and T cells (42). These observations suggest that deletion of DP CD8high thymocytes occurred in b2C mice. One likely explanation for this deletion is that the 2C TCR is selected by high affinity/avidity interactions in the H-2b mice such that DP thymocytes expressing high levels of CD8 are deleted by these high affinity/avidity ligands. By contrast, k2C mice have a normal size thymus, and the majority of DP thymocytes expressed normal levels of the CD8 molecule. However, k2C DP thymocytes differ from those in B10.BR mice in that they expressed higher levels of TCR, CD2, and CD5. Furthermore, although the k2C thymus contained a higher proportion of CD8 SP thymocytes (4.0%) when compared with the B10.BR thymus (1.7%), this proportion of CD8 SP thymocytes was considerably lower than those found in the b2C thymus (11.1%). Collectively, these observations suggest that CD8 SP thymocytes in k2C mice are selected by ligand(s) with lower affinity/avidity than those present in b2C mice. However, the 2C TCR likely interfered with the selection of CD4 SP thymocytes in k2C mice, since k2C thymi contained only a low percentage of CD4 SP cells (2.7%) compared with the higher percentage observed in B10.BR mice (7.6%) or in a line of transgenic mice expressing another MHC class I-restricted TCR (i.e., the H-Y TCR) (35). This inefficient selection of CD4 SP thymocytes could be due to the highly efficient pairing of the 2C TCR heterodimer, resulting in a more restricted usage of endogenous TCR {alpha}-chains in k2C mice.

Our conclusion that the 2C TCR is positively selected in k2C mice differs from that of Pawlowski et al. (16). Their conclusion was based strictly on phenotypic analyses; the authors correlated the small percentage of CD8 SP thymocytes expressing the 2C TCR in k2C mice with a lack of positive selection in these mice. We also observed that CD8 SP thymocytes were inefficiently produced in k2C mice relative to b2C mice. Furthermore, we noted that the CD8 SP thymocytes in k2C mice expressed a lower level of the 2C TCR Id. One explanation for this lower level of 2C TCR Id on CD8 SP thymocytes is that they coexpress endogenous TCRs. It can also be argued that these cells were in fact selected by endogenous TCRs but continued to coexpress the transgenic TCR. However, this is unlikely, since staining of these cells with Abs to either the CD3 {epsilon}- or TCR ß-chain, which will detect TCRs of both transgenic or endogenous origin, indicated that the predominant TCR on CD8 SP thymocytes from k2C mice was in fact the 2C TCR. Another study has shown that although a significant number of immature thymocytes in normal mice express two TCRs, only one these TCRs is involved in positive selection. The nonselected TCR is then down-regulated in the positively selected thymocytes (43). It is therefore unlikely that the CD8 SP thymocytes from k2C mice could continue to express the 2C TCR as the predominant TCR if it was not involved in the positive selection process. We also provided evidence that the highly purified CD8 SP thymocytes from k2C mice responded specifically to stimulation by the H-2d alloantigen. Although this response was considerably weaker than that manifested by CD8 SP thymocytes from b2C mice and occurred only in the presence of exogenous IL-2, it nevertheless indicates that positive selection of functional CD8 SP 2C TCR+ cells occurred in k2C mice. The more important conclusion from these observations is that positive selection of thymocytes expressing the same TCR by distinct selecting ligands can lead to selected thymocytes that differ in their functional maturity. This difference in functional maturity is manifested by differences in the level of TCR expression and in the efficiency by which TCR signals are transmitted to downstream activation events.

The following observations suggest that CD8 SP thymocytes from k2C mice are functionally less mature than those from b2C mice. First, the vast majority of CD8 SP thymocytes from k2C mice have not down-regulated HSA relative to their b2C counterpart. Second, they did not proliferate and failed to up-regulate CD25 and CD69 or make IFN-{gamma} when stimulated with BDF1 dendritic cells. They mounted only a very weak proliferative response when they were stimulated with BDF1 spleen cells in the presence of IL-2. An earlier study also showed that HSAhigh SP thymocytes required exogenously added IL-2 for a proliferative response to stimulation by alloantigens (18). By contrast, a high proportion of CD8 SP thymocytes from b2C mice have down-regulated HSA expression, and they up-regulate CD25 and CD69 and produce IFN-{gamma} when stimulated with BDF1 dendritic cells. They proliferated when they were stimulated with BDF1 spleen cells even without an exogenous supply of IL-2. It is noted that although CD8 SP thymocytes from b2C and k2C mice expressed similar levels of the CD8 coreceptor, those from k2C mice express about half the level of the 2C TCR. It is possible that the hyporesponsiveness of k2C CD8 SP thymocytes is due in part to this lower expression of TCR. However, it is interesting to note that these cells were also inefficiently stimulated by the anti-CD3{epsilon} mAb. Thus, a more likely explanation of these data is that positive selection of CD8 SP 2C TCR+ thymocytes in k2C mice resulted in the production of CD8 thymocytes that are less functionally mature than those selected in b2C mice, and this functional immaturity correlates with inefficient TCR-mediated signal transduction and the inability of these cells to down-regulate HSA.

On a per cell basis, the CD8 SP cells from the lymph nodes of b2C mice were more responsive to Ag stimulation than those derived from the thymus. For instance, although the vast majority of b2C CD8 SP thymocytes up-regulated CD69 and CD25 expression as a result of Ag stimulation, their proliferative response in the absence of exogenous IL-2 was about one-fifth that of the corresponding cells from the lymph nodes. The b2C thymocytes were also less responsive on the basis of IFN-{gamma} and IL-2 production after Ag stimulation. Precedent exists for the observation that peripheral T cells relay TCR-derived signals more effectively than do mature thymocytes (19). The more efficient coupling of TCR-derived signals to downstream effector pathways in peripheral T cells may reflect either a normal developmental step for T cells in the periphery or the preferential export of the most functionally mature cells by the thymus to the periphery. The greater responsiveness to Ag stimulation also correlated with the lower expression of HSA by lymph node cells. This lower expression of HSA by lymph node cells may be due to the selective migration of HSAlow cells from the thymus to peripheral lymphoid organs. Alternatively, the thymus may export HSAhigh cells to the periphery and these cells then rapidly down-regulate HSA expression. In this regard, Kelly and Scollay (44) reported that HSA+ thymocytes can migrate to peripheral lymphoid organs and can maintain HSA expression for at least 24 h. Therefore, although these data indicate that the CD8 SP lymph node cells from b2C mice were functionally more mature than their thymic counterpart, more definitive studies are required to determine whether this further maturation process is an intra- or an extrathymic developmental process.

CD8 SP lymph node cells from k2C mice were also more responsive than thymocytes to stimulation by the H-2d alloantigen. However, in the case of lymph node cells from k2C mice, the situation is complicated by the existence of CD8 cells with more than one phenotype. Phenotypic analysis of CD8 SP lymph node cells from k2C mice suggests that these cells comprised at least two distinct populations: a CD4-CD8low 2C TCRhigh population and a CD4-CD8high 2C TCRlow population. CD4-CD8low, TCR{alpha}ß+ T cells have been observed in another line of TCR transgenic mice (33). These cells have been shown to be unresponsive to stimulation by cognate Ag although they can be activated by anti-TCR mAbs in the presence of exogenous IL-2 (33, 36). The lack of up-regulation of CD69 and CD25 in a subpopulation of H-2d stimulated CD8 SP cells from k2C lymph nodes is consistent with the conclusion that the unresponsive cells were of the CD8low 2C TCRhigh phenotype. More importantly, despite the presence of this nonfunctional population and the generally lower expression of the 2C TCR, these cells were much more responsive than CD8 SP thymocytes from k2C mice to stimulation by the H-2d alloantigen. Thus, these cells responded specifically to H-2d stimulation even in the absence of exogenous IL-2. They also produced detectable levels of IFN-{gamma} when stimulated with the H-2d alloantigen. These results indicate that CD8 SP lymph node cells are more functionally mature than those from the thymi of these mice. However, what is unclear is the origin of these functionally more mature CD8 SP lymph node cells of k2C mice. It is noted from the data in Figure 3Go that the level of CD8 expressed by CD8high lymph node cells in k2C mice is slightly higher than that expressed by CD8 SP thymocytes from these mice. These CD8high cells are also the cells that expressed a high level of TCR ß-chain and lower levels of 2C TCR. The higher level of CD8 expressed by these cells and the higher expression of endogenous TCRs by these cells rendered them unlikely descendants of the CD8 SP thymocytes that were selected by the 2C TCR and that expressed 2C TCR as the predominant TCR. A more likely explanation for the presence of these cells in k2C lymph nodes is that they represent a minor population of CD8 SP thymocytes that was selected by endogenous TCRs with higher affinity for the selecting ligands than the 2C TCR. These positively selected thymocytes would then express high levels of the endogenous TCR but continue to coexpress lower levels of the 2C TCR. Therefore, a likely explanation for the increased responsiveness of k2C lymph node cells to stimulation by H-2d is that these cells were positively selected by endogenous TCRs with putatively higher affinity ligands and had undergone a normal maturation pathway. The efficiency by which the TCR signaling pathway is coupled to downstream signaling pathways leading to proliferation and cytokine production in these cells is expected to be normal, which was observed (Fig. 5Go). The lower response of these cells to stimulation by H-2d Ag can be explained, at least in part, by the much reduced level of 2C TCR expression by these cells. The apparent lack of CD8 SP cells that expressed similar levels of the 2C TCR and the TCR ß-chain in the lymph nodes of k2C mice also implies that the CD8 SP thymocytes that were positively selected by 2C TCR were either unable to colonize peripheral lymphoid organs or unable to compete effectively against those that were selected by endogenous TCRs for expansion in the lymph nodes. Further experimentation is required to distinguish between these two possibilities.

The observations reported here provide a simple explanation for the heterogeneous level of HSA expression by SP thymocytes and the functional immaturity of HSAhigh SP thymocytes in normal mice. The TCR repertoire expressed by DP thymocytes from normal mice is extremely heterogeneous and therefore can be selected by an immense variety of ligands. We propose that DP thymocytes that are positively selected by either high, intermediate, or low affinity/avidity ligands first differentiate into SP TCRhighHSAhigh thymocytes. At this stage of their developmental pathway, they are functionally immature, since the TCR signaling pathway is inefficiently coupled to downstream signaling pathways that lead to cytokine production and proliferation. We propose that SP thymocytes that are selected by high affinity/avidity ligands undergo a fairly rapid transition from the functionally immature HSAhigh stage to the functionally more mature HSAlow stage, as exemplified by the CD8 SP thymocytes from b2C mice. SP TCRhighHSAhigh thymocytes that are selected by very low affinity/avidity ligands undergo the transition from the functionally immature HSAhigh stage to a functionally more mature stage very inefficiently. These SP thymocytes maintain their high level of HSA expression, and the TCR signaling pathway in these cells is inefficiently coupled to downstream effector pathways. This scenario is exemplified by CD8 SP thymocytes from the k2C mice. CD8 thymocytes that are selected by ligands of intermediate affinity/avidity are expected to have a phenotype in between those of b2C and k2C CD8 SP thymocytes. The extremely heterogeneous level of HSA expression by CD8 SP thymocytes from normal mice, as exemplified here by those from B10.BR mice, is consistent with our hypothesis that these thymocytes are selected by TCRs that have either high, intermediate, or low affinity/avidity for the positively selecting ligands.


   Acknowledgments
 
We thank Simon Ip for excellent technical assistance, and Drs. Chris Ong and Jan Dutz for helpful discussions. We thank Dr. Dennis Loh for providing breeders for the 2C TCR transgenic mice and Dr. Herman Eisen for providing the 1B2 hybridoma cell line.


   Footnotes
 
1 This work was supported by the Medical Research Council of Canada and the Arthritis Society of Canada. B.M. is supported by a postdoctoral fellowship from the Medical Research Council of Canada. Back

2 Address correspondence and reprint requests to Dr. Hung-Sia Teh, Department of Microbiology and Immunology, 6174 University Boulevard, Vancouver, B. C., Canada V6T 1Z3. Back

3 Abbreviations used in this paper: SP, single positive (CD4+CD8- or CD4-CD8+); DP, double positive (CD4+CD8+); HSA, heat stable antigen; b2C, H-2b 2C TCR transgenic mice; k2C, H-2k 2C TCR transgenic mice. Back

Received for publication July 8, 1997. Accepted for publication October 2, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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