Role of the Multiple T Cell Receptor (TCR)-zeta Chain Signaling Motifs in Selection of the T Cell Repertoire

By Elizabeth W. Shores,* Tom Tran,* Alexander Grinberg,Dagger Connie L. Sommers,Dagger Howard Shen,Dagger and Paul E. LoveDagger

From the * Division of Hematologic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892; and the Dagger  Laboratory of Mammalian Genes and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892

Summary
Materials and Methods
Results
Discussion
Footnotes
Acknowledgements
References


Summary

Immature thymocytes undergo a selection process within the thymus based on their T cell antigen receptor (TCR) specificity that results either in their maturation into functionally competent, self-MHC-restricted T cells (positive selection) or their deletion (negative selection). The outcome of thymocyte selection is thought to be controlled by signals transduced by the TCR that vary in relation to the avidity of the TCR-ligand interaction. The TCR is composed of four distinct signal transducing subunits (CD3-gamma , -delta , -epsilon , and zeta ) that contain either one (CD3-gamma , -delta , -epsilon ) or three (-zeta ) signaling motifs (ITAMs) within their intracytoplasmic domains. A possible function for multiple TCR ITAMs could be to amplify signals generated by the TCR during selection. To determine the importance of the multiple TCR-zeta chain ITAMs in thymocyte selection, transgenes encoding alpha /beta TCRs with known specificity were bred into mice in which zeta  chains lacking one or more ITAMs had been genetically substituted for endogenous zeta . A direct relationship was observed between the number of zeta  chain ITAMs within the TCR complex and the efficiency of both positive and negative selection. These results reveal a role for multiple TCR ITAMs in thymocyte selection and identify a function for TCR signal amplification in formation of the T cell repertoire.


During their development, thymocytes are subjected to a selection process that results in the survival of functionally competent, self-MHC-restricted cells (positive selection), and the deletion of potentially autoreactive cells (negative selection; reference 1). The fate of immature thymocytes is ultimately dictated by the specificity of their TCRs (2) and is presumably controlled by TCR-mediated signals that vary with the avidity of TCR-self ligand interactions within the thymus. The TCR complex contains multiple signal transducing subunits (CD3-gamma , -delta , -epsilon , and zeta ) that share a common functional sequence, the immunoreceptor tyrosine-based activation motif (ITAM)1 within their intracytoplasmic domains (3, 4). After TCR engagement, phosphorylation of ITAMs leads to the recruitment of SH2 domain-containing proteins (e.g., tyrosine kinases) to the TCR complex and initiation of the T cell activation cascade (4). The CD3 subunits each contain a single ITAM, whereas zeta contains three ITAMs within its longer cytoplasmic tail. ITAM sequences are conserved but nonidentical, and it remains unclear whether individual motifs perform unique or similar functions (8). It has been suggested that due to its singular configuration, zeta  chain may function as the predominant TCR signaling structure and that its triplicated ITAMs may serve primarily to facilitate TCR signal amplification (9, 10).

zeta chain is required for normal T cell development as zeta -deficient (zeta -/-) mice have markedly reduced numbers of both CD4+CD8+ (double positive, DP) and CD4+CD8- and CD4- CD8+ (single positive, SP) thymocytes (11). However, the major developmental defects in zeta -/- mice were subsequently shown to be the result of impaired TCR surface expression rather than lack of zeta -mediated signals per se, as transgene-encoded zeta  chain variants that lacked ITAM sequences but could still facilitate TCR surface expression (zeta -0 ITAM), restored T cell development in zeta -/- mice (15). These results demonstrated that zeta  chain signals were not specifically required for T cell development as signal transduction by TCR complexes that contain the CD3 subunit ITAMs, but not zeta  chain ITAMs, was sufficient for the generation of mature T cells.

A specific role for the multiple ITAM TCR structure could be in selection of the T cell repertoire during thymocyte development. Recent data support the idea that relatively low affinity TCR-ligand interactions can promote the selection of immature thymocytes, whereas higher affinity interactions are required for mature T cell activation (2, 16). Thus, the multiple zeta  chain ITAMs could be required to amplify TCR signals originating from the relatively weak interactions that mediate thymocyte selection. Since this question could not be easily addressed in mice with a heterogeneous population of T cells with diverse TCR specificities, we fixed the specificity of the TCRs expressed on developing thymocytes by introducing transgenes that encode productively rearranged TCR-alpha and -beta chains (alpha /beta -TCR Tg) into zeta -/- mice (21, 22). We then bred into the alpha /beta -TCR Tg/zeta -/- background transgenes encoding either full-length zeta  chain (3 ITAM), zeta  chains containing a single ITAM (1 ITAM), or a zeta  chain lacking all three ITAMs (zeta - 0 ITAM). The results of these studies reveal that zeta  chain signals influence both positive and negative thymocyte selection and identify a role for the multiple ITAM TCR structure in selection of the T cell repertoire.


Materials and Methods

Transgenic Mice. Transgenes encoding full-length zeta  chain (zeta -3 ITAM Tg) and zeta  chain variants with intact extracellular and transmembrane domains, but containing only a single ITAM (either the membrane proximal ITAM or the membrane distal ITAM; [zeta -1 ITAM Tg]), or containing no ITAMs (zeta -0 ITAM Tg) were generated using the identical (human CD2) promoter/ enhancer cassette. The generation of zeta -/- mice, zeta  transgenes, and genetic reconstitution of zeta -/- mice with zeta  transgenes has been described previously (11, 15). The alpha /beta -TCR transgenes used in these experiments encoded either an MHC class I-restricted TCR specific for male antigen (H-Y; 21) or an MHC class II-restricted TCR specific for pigeon cytochrome C (AND; 22). alpha /beta TCR transgenes were introduced into the zeta -/- background by breeding and identified by Southern blotting or PCR analysis of tail DNA. All mice used for these studies were bred and maintained in a barrier (specific pathogen-free) facility.

Proliferation Assays. Single cell suspensions were prepared from LNs from normal (zeta +/+) or transgene-reconstituted zeta -/- mice. T cells were purified by incubating cells on rabbit anti-mouse Igcoated plates. Accessory cells and APCs (either for FcR-mediated antibody cross-linking or allogeneic stimulator cells) were prepared from spleen cell suspensions of C57BL/6 or BALB/c mice. APCs were depleted of T cells with anti-Thy1.2 + C' and irradiated with 3,000 rads. 105 responder T cells were combined with 5 × 105 accessory cells in flat-bottom 96-well plates in the presence or absence of the indicated stimulants. After the specified time, cells were pulsed for 8 h with 1 µCi [3H]thymidine and harvested. For mitogen stimulation, Con A at 2 µg/ml was added to the culture. For anti-CD3epsilon stimulation, 25% tissue culture supernatant from the 145-2C11 hybridoma was added to the culture. Cells were cultured in presence of syngeneic (C57BL/6) APCs and harvested after 48 h. For the mixed lymphocyte reaction, responder T cells were combined with either syngeneic (C57BL/6) or allogeneic (BALB/c) APCs. Cells were harvested after a total of 96 h.

Flow Cytometry. For multicolor flow cytometry (FCM), thymocytes or LN cells were first incubated with antibody to the Fc receptor (mAb 2.4G2) to prevent FcR binding. For two- and threecolor FCM, cells were incubated with FITC-conjugated, PEconjugated, and biotinylated antibodies, followed by the addition of red 670 streptavidin (GIBCO BRL, Gaithersburg, MD). The FCM was performed on a Becton Dickinson Immunocytometry Systems FACScan® using standard Cell Quest software. Data were collected on 10-20 × 104 viable cells as determined by forward and side light scatter. The majority of monoclonal antibodies used for FCM analysis were purchased from PharMingen (San Diego, CA) and included biotinylated anti-CD4 (RM4.5) and PE-anti-CD8 (6.7), FITC and PE-anti-CD3 (145-2C11), FITC-anti-TCRVbeta 14 (14-2), and FITC-anti-Valpha 11 (RR8-1). Anti-TCRVbeta 8 (F23.1) and anti-H-Y clonotypic receptor (T3.70) were purified from cell culture supernatants and labeled with FITC.

Superantigen-induced Thymocyte Deletion. 4-8-wk-old mice were injected intraperitoneally with PBS alone, 10 µg staphylococcus enterotoxin B (SEB; Sigma Chemical Co., St. Louis, MO) in PBS, or 20 µg SEB in PBS, every other day for 1 wk (three doses). 2 d after the final injection, mice were killed and thymocytes were harvested. Thymocytes were stained with anti-Vbeta 8-FITC or anti-Vbeta 14-FITC, followed by anti-CD3epsilon -PE. Deletion was assessed by software gating on CD3high (SP cells) and determining the percentage of cells expressing individual TCRVbeta chains. Percent deletion was determined by dividing the percentage of CD3high, TCRVbeta 8+ thymocytes remaining after SEB injection by the percentage of CD3high, TCRVbeta 8+ cells present in mice injected with PBS alone.


Results

Reconstitution of zeta -/- mice with a transgene encoding a zeta  variant chain lacking all three zeta  chain ITAMs (zeta -0 ITAM) restores TCR surface expression and promotes the generation of large numbers of CD4+CD8- and CD4-CD8+ SP thymocytes and peripheral T cells (15). Significantly, the T cells generated in zeta -0 ITAM Tg mice are functionally competent as assessed by their ability to respond to TCR-dependent stimuli (Fig. 1; 23). These results demonstrate that TCR-zeta chain signals are not specifically required for either the generation or activation of mature T cells.



Fig. 1. Role of TCR-zeta chain ITAMs in mature T cell function. (A) Proliferative response of LN T cells to anti-CD3epsilon and Con A stimulation. (B) Proliferative response of LN T cells to allogenic stimulator cells. LN T cells were obtained from 4-8-wk-old nontransgenic (zeta +/+/) mice, or zeta -/- mice that express transgenes encoding either full-length zeta  chain (zeta -3 ITAM Tg) or zeta  chain lacking ITAM sequences (zeta -0 ITAM Tg) and stimulated as described in Materials and Methods.
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To examine the importance of the multiple TCR-zeta ITAMs in the selection of the T cell repertoire, we fixed the specificity of the TCRs expressed on developing thymocytes by introducing a transgene that encodes productively rearranged TCR-alpha and -beta chains specific for the male H-Y antigen in the context of H-2Db (H-Y TCR; 21). This approach enabled us to examine both positive selection (H-2Db female mice) and negative selection (H-2Db male mice) of thymocytes bearing a single alpha /beta -TCR.

Examination of zeta -/- mice that express the H-Y transgene revealed that the developmental impairment observed previously in zeta -/- mice was also observed in both male and female H-Y+/zeta -/- mice (Fig. 2). These results were expected because of the extremely low level of TCR surface expression in the absence of zeta  chain (11) and are consistent with a requirement for the TCR for normal thymocyte development and selection. To address the importance of the zeta  chain ITAMs in thymocyte selection, we next examined H-Y+/zeta -/- mice reconstituted with transgenes encoding either full-length zeta  chains (H-Y+/zeta -3 ITAM Tg mice), zeta  chains containing a single ITAM (H-Y+/zeta -1 ITAM Tg mice), or zeta  chains lacking all three ITAMs (H-Y+/zeta - 0 ITAM Tg mice) (15). Each of the zeta  transgenes was capable of restoring TCR surface expression in mice lacking endogenous zeta  chain (Fig. 3; 15). For these experiments transgenic founder lines were chosen that gave comparable levels of TCR surface expression that were equal to or greater than that found on thymocytes from zeta +/+ mice (Fig. 3; 15).


Fig. 2. Phenotype of H-Y-/zeta -/- and H-Y+/zeta -/- mice. Data show immunofluorescence and multicolor FCM analysis of thymocytes and LN T cells from adult (6-12-wk-old) H-2Db mice. Three-color FCM analysis was performed on cells stained with anti-H-Y clonotypic antibody (T3.70) conjugated to FITC, anti-CD8-PE and anti-CD4-biotin, followed by streptavidin-red 670 (16). CD4 versus CD8 two-color profiles are displayed on total thymocytes or total lymph node T cells. Single-color profiles (solid lines) depict T3.70 staining on total thymocytes. Dotted lines reflect staining with negative control antibody.
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Fig. 3. Role of TCRzeta -mediated signals in thymocyte positive and negative selection. (A) Phenotype of thymocytes from H-Y+/zeta -/-; zeta  Tg female mice. (B) Phenotype of thymocytes from H-Y+/zeta -/-; zeta  Tg male mice. H-Y+/zeta -/- mice were reconstituted with transgenes encoding either full-length zeta  chains (zeta -3 ITAM Tg), zeta  chains that contain a single ITAM (either the first or third, zeta -1 ITAM Tg) or zeta  chains that lack ITAMs (zeta -0 ITAM Tg). Data show immunofluorescence and multicolor FCM analysis of thymocytes from adult H-2Db mice. Three-color FCM was performed on cells stained with anti-H-Y clonotypic antibody (T3.70) conjugated to FITC, anti-CD8-PE, and anti-CD4- biotin, followed by streptavidin- red 670 (16). Two-color plots show total thymocytes or software-gated T3.70+ thymocytes. Numbers in the quadrants reflect the percentage of total thymocytes in that quadrant. Singlecolor profiles (solid lines) depict T3.70 staining on either CD4+CD8+ thymocytes (A) or total thymocytes (B). Dotted lines reflect staining with negative control antibody.
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In female H-2Db, H-Y+/zeta +/+ mice, interaction of the H-Y TCR on immature thymocytes with an unidentified ligand results in positive selection and generation of CD8 SP thymocytes that express high surface levels of the H-Y TCR (detected by reactivity with the clonotype-specific antibody, T3.70; reference 24; Fig. 3 A, Table 1 A). Importantly, a phenotype similar to that of H-Y+/zeta +/+ mice was observed in female H-Y+/zeta -/- mice reconstituted with the full-length zeta  transgene (H-Y+/zeta -3 ITAM Tg; Fig. 3 A, Table 1 A). These mice also had large thymi that contained a high percentage of T3.70+, CD8 SP cells indicating that positive selection was effectively restored in zeta -/- mice by expression of a transgene-encoded full-length (3 ITAM) zeta  chain (Fig. 3). However, in female H-Y+/zeta -/- mice reconstituted with a transgene encoding the signaling-deficient zeta  chain (H-Y+/zeta -0 ITAM Tg), positive selection of T3.70+ thymocytes was markedly impaired as evidenced by the extremely low percentage of T3.70+, CD8 SP thymocytes (Fig. 3 A, Table 1 A). Interestingly, H-Y+/zeta -/- females reconstituted with transgenic zeta  chains that contain a single ITAM (either the first [membrane proximal] or third [membrane distal]) exhibited an intermediate phenotype (Fig. 3 A). Although there were subtle differences in the two H-Y+/zeta -1 ITAM Tg lines, the percentage of T3.70+, CD8 SP thymocytes in both lines consistently fell between those observed in mice reconstituted with either the zeta -3 ITAM or zeta -0 ITAM transgenes. The direct relationship between the number of zeta  chain ITAMs and the generation of clonotypic (T3.70+) CD8 SP thymocytes was even more evident when absolute numbers of T3.70+, CD8+ thymocytes in the various transgenic lines were compared (Table 1 A). Examination of LNs from H-Y+/zeta -0 ITAM Tg females also revealed a lower percentage of T3.70+, CD8+ T cells relative to H-Y+/zeta -3 ITAM Tg females (Fig. 4). A similar relationship between the number of TCR-zeta ITAMs and the efficiency of positive selection was also observed with a class II-restricted TCR-alpha beta Tg (AND; reference 22; Fig. 5). Together, these results demonstrate that TCR ligand interactions that can generate signals that promote positive selection in the presence of full-length zeta  chains are unable to generate these signals in the absence of zeta  chain ITAMs.

Table 1. Numbers of Thymocytes in H-Y+/zeta +/+ Mice and H-Y+/zeta -/- Mice Reconstituted with Transgenes Encoding the Full-length zeta  Chain (zeta -3 ITAM Tg), a zeta  Chain Containing the Third ITAM (zeta -1ITAM Tg) or a zeta  Chain lacking ITAMs (zeta -0 ITAM Tg)


Females  zeta +/+ n = 3  zeta -3 ITAM Tg n = 3  zeta -1 ITAM Tg n = 6  zeta -0 ITAM Tg n = 6

Total
Thymocytes        97 ± 42 166 ± 74 122 ± 45        56 ± 22
Total T3.70+        53 ± 25 117 ± 33        91 ± 51        47 ± 18
T3.70+ DN*     8.2 ± 3     6.2 ± 3     3.3 ± 2     1.9 ± 2
T3.70+ DP*        28 ± 10        89 ± 29        74 ± 26        42 ± 16
T3.70+ CD8 SP* 15.5 ± 12 17 ± 6     9.8 ± 5     0.6 ± 0.2
Males n = 4 n = 5 n = 6 n = 7
Total
Thymocytes     6.0 ± 1.0     5.6 ± 2.7          7.6 ± 5.2        17 ± 10
Total T3.70+     5.1 ± 0.7     4.9 ± 2.4          6.6 ± 4.3 14.3 ± 9.3
T3.70+ DN*     3.9 ± 0.4     2.6 ± 1.8     0.9 ± 0.6     0.8 ± 0.9
T3.70+ DP*     0.5 ± 0.4     1.0 ± 0.7     4.0 ± 2.5 11.0 ± 7.6

Data are given in 106 cells as means ± SEM rounded to the nearest whole number. T3.70+ thymocyte subpopulations were calculated by multiplying the total T3.70+ thymocyte number by the percent of cells in a given quadrant as depicted in Fig. 2.
*  Double negative = CD4-CD8-; DP = CD4+CD8+; CD8 SP = CD4-CD8+.


Fig. 4. Analysis of LN T cells from H-Y+/zeta Tg mice. Immunofluorescence and multicolor FCM analysis of LN cells from adult female and male H-Y+/ zeta -3 ITAM Tg or H-Y+/zeta -0 ITAM Tg mice. Also shown are LN cells from a nontransgenic C57BL/6 (H-Y-/zeta +/+) mouse. Threecolor FCM was performed on total LN cells after staining with FITC- anti-H-Y clonotypic antibody (T3.70), anti-CD8-PE and anti-CD4- biotin, followed by streptavidin-red 670. CD4 versus CD8 two-color profiles are displayed on total (left) or gated T3.70+ (right) cells. Numbers in the regions reflect the percentage of total cells found in that region.
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Fig. 5. Positive selection in MHC class II-restricted alpha /beta TCR transgenic mice. AND+/zeta -/- mice were generated by mating and then reconstituted with transgenes encoding either a full-length zeta  chain (zeta -3 ITAM Tg), a zeta  chain containing a single (third) ITAM (zeta -1 ITAM Tg), or a zeta  chain that lacks ITAMs (zeta - 0 ITAM Tg). Data show immunofluorescence and multicolor FCM analysis of thymocytes from adult H-2Db mice. FCM was performed on cells stained with FITC-conjugated antibody specific for the transgenic Valpha chain (Valpha 11), anti-CD8-PE and antiCD4-biotin, followed by streptavidin-red 670. Two-color plots show software-gated Valpha 11+ thymocytes. Numbers in the quadrants reflect the percentage of total thymocytes in that quadrant. Single-color profiles (solid lines) depict Valpha 11 staining on total thymocytes. Dotted lines reflect staining with negative control antibody.
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Despite the near absence of T3.70+, CD8+ LN T cells, large numbers of T3.70-, CD8 SP thymocytes and T cells were detected in H-Y+/zeta -0 ITAM Tg females (Figs. 3 A and 4). Since these T3.70- CD8 SP cells expressed alpha /beta -TCRs composed of transgenic TCR-beta chains paired with non- transgenic, endogenously derived TCR-alpha chains, their TCR specificity was not restricted to the H-Y antigen. Therefore, the reduction in T3.70+, CD8 SP thymocytes and T cells in H-Y+/zeta -0 ITAM Tg mice specifically reflected the failure of the H-Y TCR to promote positive selection of T3.70+ DP thymocytes.

To assess the role of the zeta  ITAMs in negative selection, we next examined H-Y+/zeta Tg male mice. Both H-Y+/zeta +/+ males and H-Y+/zeta -3 ITAM males had small thymi with few (<107) thymocytes which were predominantly CD4- CD8- (Fig. 3 B, Table 1 B). The marked reduction in DP thymocytes in these mice is consistent with previous data indicating that negative selection occurs before the DP stage in H-Y Tg males (25). In contrast to mice expressing full-length zeta  chain, H-Y+/zeta -0 ITAM Tg males exhibited a very different phenotype. Thymi from these mice were three- to sixfold larger than H-Y+/zeta -3 ITAM Tg males (Table 1 B) and contained predominantly T3.70+, DP thymocytes (Fig. 3 B). Again, the phenotype exhibited by both lines of H-Y+/zeta -1 ITAM Tg mice was intermediate between H-Y+/zeta -3 ITAM Tg males and H-Y+/zeta -0 ITAM Tg males. For example, thymi from H-Y+/zeta -1 ITAM Tg males contained lower percentages of DP thymocytes than did H-Y+/zeta -0 ITAM Tg males, but contained higher percentages of CD4-CD8low cells which are thought to be the immediate precursors of DP thymocytes (26-28; Fig. 3 B, Table 1 B). Thus, the early negative selection of H-Y clonotypic (T3.70+) thymocytes was attenuated in H-Y+/zeta -0 ITAM Tg and H-Y+/zeta -1 ITAM Tg males.

We next examined LNs to determine the fate of the DP thymocytes observed in male H-Y+/zeta -0 ITAM Tg and H-Y+/zeta -1 ITAM Tg mice. Whereas LN from both H-Y+/ zeta +/+ males and H-Y+/zeta -3 ITAM Tg males contained almost no "conventional" CD4high or CD8high T cells, large numbers of both CD4high and CD8high T cells were present in H-Y+/zeta -0 ITAM Tg (Fig. 4) and H-Y+/zeta -1 ITAM Tg males (data not shown). These CD4high and CD8high T cells expressed high levels of TCR which were uniformly T3.70low/- indicating that they were composed primarily of nontransgenic TCR-alpha chains. Therefore, although negative selection of T3.70+ DP thymocytes was not abrogated in H-Y+/zeta -0 ITAM Tg and H-Y+/zeta -1 ITAM Tg males, some DP thymocytes were able to escape negative selection. Furthermore, these thymocytes were also capable of being positively selected on the basis of non-H-Y restricted TCRs generated from rearrangement and expression of endogenous TCR-alpha genes.

To study the role of zeta -mediated signals in deletion of more mature (SP) thymocytes, we examined superantigenmediated thymocyte deletion in the various transgenic lines. The superantigen SEB deletes SP thymocytes bearing TCRs that include Vbeta 8 (29). Adult mice were injected with SEB or PBS every other day for 1 wk and the percentage of TCRVbeta 8+ SP thymocytes remaining was determined by FCM analysis. At similar doses of antigen, the efficiency of SEB-induced thymocyte deletion in zeta -0 ITAM mice was significantly reduced relative to zeta -3 ITAM Tg mice (Fig. 6). Nevertheless, thymocyte deletion by SEB was detectable in zeta -0 ITAM Tg mice and increased with increasing SEB dosage (Fig. 6). The deletion of Vbeta 8+ thymocytes was specific as the percent TCRVbeta 14+ SP thymocytes (which are not deleted by SEB) was similar in zeta -0 ITAM Tg and zeta -3 ITAM Tg mice (data not shown). Therefore, in the absence of TCR-zeta signals, deletion of SP thymocytes by SEB was significantly impaired.


Fig. 6. Superantigen-mediated deletion of TCRVbeta 8+ thymocytes. Mice were injected with PBS alone, 10 µg SEB in PBS, or 20 µg SEB. Thymocytes were stained with anti-Vbeta 8-FITC or anti-Vbeta 14-FITC, followed by anti-CD3epsilon -PE. Deletion was assessed by software gating on CD3high (SP cells) and determining the percentage of cells expressing individual TCRVbeta chains. Deletion was specific for cells expressing Vbeta 8 as cells expressing Vbeta 14 were not deleted (29). Results are given as mean ± SEM and are from at least six different SEB injected mice per group compiled from two to three separate experiments.
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Discussion

In these studies, we investigated the role of the TCR-zeta chain ITAMs in thymocyte selection by fixing the specificity of the TCR and then varying the TCR signaling potential by altering the number of TCR-zeta chain ITAMs within the TCR complex. The results reveal a direct relationship between the number of zeta  chain ITAMs in the TCR complex and the efficiency of both positive and negative thymocyte selection. In addition, they suggest that the multiple zeta  chain ITAMs function primarily to amplify signals generated by the TCR during thymocyte selection. In this respect, our findings are consistent with in vitro results obtained from mature T cell clones which failed to identify a specific function for individual zeta  ITAMs, but found that triplication of a single motif resulted in a quantitative enhancement of signaling (9, 10). However, our current results do not preclude a specific role for individual TCR-zeta ITAMs in more specialized T cell functions that have yet to be examined.

Our results with two alpha /beta -TCR Tg systems (H-Y and AND) suggest that positive selection, which is thought to be mediated by low avidity TCR-ligand interactions (2, 16) is particularly dependent upon signal amplification by the TCR-zeta chain. In H-Y+/zeta -0 ITAM Tg female mice, most thymocytes that expressed only the H-Y-specific TCR failed to be positively selected, whereas a large number of thymocytes that expressed non-H-Y-specific TCRs were capable of being positively selected. We predict that the mature T cells generated in both non-alpha /beta -TCR Tg/zeta -0 ITAM Tg (15) and H-Y+/zeta -0 ITAM Tg mice are selected on the basis of TCRs with relatively high affinity for self ligands, and that these TCRs might otherwise promote negative selection in zeta +/+ or zeta -3 ITAM Tg mice where the TCR contains the full complement of ITAMs. Signal amplification by the zeta  chain ITAMs may therefore not be absolutely required for positive selection if the avidity of the positively selecting TCR-ligand interaction is sufficiently high. In these instances, signal transduction by the remaining ITAMs supplied by the CD3 chains (which are targeted to the cell surface in our model system) is apparently adequate for selection in the absence of zeta -mediated signals.

Interestingly, incremenatal reduction in the number of TCR-zeta ITAMs resulted in graded effects on positive selection. Graded or quantitative effects on positive selection have also been observed with increasing concentrations of positively selecting ligand (16). These and other (20) data suggest that a broad range of signaling responses below a certain threshold may be capable of mediating positive selection, albeit with different efficiency.

Our results demonstrate that TCR signal amplification also plays an important role in negative selection; however, in H-Y Tg mice, the impact of removing the zeta  ITAMs from the TCR complex varied depending on the developmental stage. Negative selection of early "transtitional" (CD8lowCD4-) thymocytes was abrogated in the absence of zeta -mediated signals, presumably because low CD8 surface density lowers the avidity of the ligand interaction (21, 25). These cells could give rise to T3.70+ (CD4highCD8high) DP thymocytes, but DP thymocytes were more susceptible to negative selection as demonstrated by their low numbers in H-Y+/zeta -0 ITAM Tg males. Nevertheless, negative selection was also impaired at the DP stage in H-Y+/zeta -0 ITAM Tg mice to the extent that some DP thymocytes survived, rearranged, and expressed endogenous TCR-alpha , and were positively selected on the basis of non-H-Y clonotypic TCRs. Superantigen-induced deletion of SP thymocytes was also affected in zeta -0 ITAM Tg mice, demonstrating that negative selection which occurs at later stages of thymocyte developement is also impaired in the absence of zeta -mediated signals.

That reduction in the number of TCR ITAMs quantitatively affects both positive and negative selection is consistent with the hypothesis that the outcome (positive or negative) of thymocyte selection is dictated by quantitative rather than qualitative differences in the TCR signaling response (2). Although it might have been predicted, we did not observe positive selection of T3.70+ thymocytes in H-Y+zeta 0-ITAM Tg males. However, the signaling capacity of the H-Y TCR in H-Y+zeta 0-ITAM Tg males may not have been sufficiently reduced to observe such an outcome, as negative selection, though impaired, was not abrogated in these mice. Our findings suggest that signals generated from negative selecting interactions are more difficult to attenuate than those generated during positive selection, not only because higher avidity TCR-ligand interactions are thought to promote negative selection, but also because of the effect of TCR signal amplification. Therefore, these results provide an alternative interpretation to studies suggesting that positive and negative selection are biochemically distinguishable based primarily on the finding that approaches that disrupt positive selection fail to perturb negative selection (30, 31).

Finally, it has remained unclear how relatively small differences in the affinity or avidity of TCR self-ligand interactions can differentially lead either to positive or negative selection (32). Our data suggest that the ability of the TCR to elicit such developmentally distinct cell fate decisions stems from its unique structure. Multiple ITAMs may serve to greatly magnify TCR signals and thereby translate relatively small differences in avidity into large signaling differences. Thus, the unique configuration of the TCR, with its capacity for signal amplification, plays a pivitol role in determining the ultimate fate of developing thymocytes and in shaping the mature T cell repertoire.


Footnotes

Address correspondence to Elizabeth W. Shores, Division of Hematologic Products, Center for Biologics, HFM 538, Bldg. 29A, Rm 2B23, 29 Lincoln Drive, MSC 4555, Bethesda, MD 20892-4555.

Received for publication 3 January 1997.

   1Abbreviations used in this paper: DP, double positive; FCM, flow cytometry; ITAM, immunoreceptor tyrosine-based activation motif; SEB, Staphylococcus enterotoxin B; SP, single positive.

We thank B.J Fowlkes, A.S. Rosenberg, A. Singer, and M. Vacchio for reading the manuscript and for helpful discussions.


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