The JI PBL Intereron Source
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mora, A. L.
Right arrow Articles by Boothby, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mora, A. L.
Right arrow Articles by Boothby, M.
The Journal of Immunology, 2001, 167: 5628-5635.
Copyright © 2001 by The American Association of Immunologists

Inefficient ZAP-70 Phosphorylation and Decreased Thymic Selection In Vivo Result from Inhibition of NF-{kappa}B/Rel1

Ana L. Mora*, Sarah Stanley*, Wade Armistead*, Andrew C. Chan{dagger} and Mark Boothby2,*

* Department of Microbiology and Immunology, Vanderbilt University Medical School, Nashville, TN 37232; and {dagger} Washington University School of Medicine, St. Louis, MO 63110


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Signaling from the TCR regulates T lymphoid survival, deletion by apoptosis, and selective clonal expansion. One set of signaling pathways activated during thymic selection leads to degradation of a cytosolic retention protein, the inhibitor of {kappa}B (I{kappa}B){alpha}, followed by nuclear translocation of the NF-{kappa}B/Rel family of transcription factors. It has been found previously that NF-{kappa}B proteins mediate a pathway signaling the survival of mature T cells and protection of thymocytes against TNF-induced apoptosis. In contrast, we show in this study that a transgenic inhibitor of NF-{kappa}B/Rel signaling interferes with the negative selection of immature thymocytes by endogenous MHC ligands in vivo. Positive selection of the H-Y TCR also was diminished. This attenuation of thymic selection efficiency was associated with decreased ZAP-70 phosphorylation and TCR signaling of CD69 induction. These findings demonstrate that the NF-{kappa}B transcriptional pathway plays an important role in normal processes of clonal deletion and they indicate that the NF-{kappa}B/I{kappa}B axis can regulate the efficiency of TCR signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The development and maintenance of stable T cell populations are tightly regulated by thymic mechanisms (1, 2, 3, 4). During T cell development in the thymus, functionally competent cells are positively selected from immature precursors, whereas those with a high TCR affinity for self-MHC ligands are removed by negative selection (5). In the periphery, the size and turnover of mature T cell populations reflect thymic production rates (3, 4), MHC-dependent survival of resting and activated cells, and the clonal expansion of mature lymphocytes (6, 7). Thus, the role of specific signal transduction and transcriptional mechanisms in regulating the response of thymocytes to TCR engagement is a critical determinant of T cell repertoire and population sizes.

Stimulation of the TCR on thymocytes activates a signal transduction/transcription cascade culminating in the nuclear translocation of members of the NF-{kappa}B transcription factor family (8, 9). To investigate the role of NF-{kappa}B proteins during the development and survival of T lineage cells, we generated transgenic (Tg)3 mice in which these cells express the I{kappa}B{alpha}({Delta}N), a mutant form of I{kappa}B{alpha} (10). Expression of this mutant, which lacks sequences required for signal-induced degradation, inhibits the nuclear induction of c-Rel and RelA in thymocytes and T cells (10, 11). These Tg mice exhibited a decrease in the pool of TCRhigh CD8 single-positive (SP) thymocytes accompanied by a modest reduction of CD4+ cells and a dramatically decreased population of mature CD8{alpha}{beta}+ T cells in the periphery (10, 12). Other transgenic lineages subjected to Tg inhibition of NF-{kappa}B have yielded similar results (13, 14, 15, 16). Additional analyses indicated that mature T cells expressing the I{kappa}B{alpha}({Delta}N) transgene exhibit increased apoptosis and decreased proliferation after TCR cross-linking with anti-CD3 (10), consistent with evidence of increased apoptotic susceptibility of Jurkat T cells subjected to similar inhibition of NF-{kappa}B (17). Moreover, the decreased number of CD8{alpha}{beta} cells in I{kappa}B{alpha}({Delta}N) mice was ameliorated by expression of an antiapoptotic Bcl-XL transgene in T cells (12). Taken together, these observations indicated that the NF-{kappa}B/Rel pathway mediates the induction of survival signals in mature T cells. However, there is evidence that the signals transduced by a TCR stimulus in immature thymocytes may differ from those in mature T cells (18, 19, 20). Moreover, the survival of T lineage cells may also depend on the transduction and integration of signals from receptors such as CD28, CD30, Fas, and TNFR, and the roles of these NF-{kappa}B-coupled receptors during thymic selection differ from their effects on survival in the periphery (5, 21, 22, 23, 24, 25, 26, 27). Thus, we hypothesized that NF-{kappa}B/Rel transcriptional pathways may promote thymic negative selection rather than survival.

To investigate this hypothesis, we have used TCR transgenes to measure the effect of the I{kappa}B{alpha}({Delta}N) transgene on negative selection by endogenous MHC Ags in the thymus. Our data provide evidence that nuclear induction of NF-{kappa}B/Rel proteins promotes negative selection of immature thymocytes. This role in immature thymocytes, and an additional effect on positive selection, was associated with impaired TCR signaling in thymocytes, including decreases in ZAP-70 phosphorylation. Thus, although NF-{kappa}B mediates a survival signal in mature T cells, decreased activity of the NF-{kappa}B/Rel pathway leads to inhibition of thymic negative selection. Moreover, our findings indicate that the NF-{kappa}B/I{kappa}B{alpha} system regulates aspects of proximal TCR signaling, thereby suggesting the existence of a novel feedback pathway in which a transcription factor can regulate early MHC-induced signaling events.


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

I{kappa}B{alpha}({Delta}N) Tg mice in which expression of a stable inhibitor of NF-{kappa}B/Rel activation (11) is targeted to the T cell lineage using the lck promoter and CD2 locus control region have been described previously (10). Two lines of I{kappa}B{alpha}({Delta}N) Tg mice on C57BL/6 (H-2b) and BALB/c (H-2d) backgrounds (backcross > 4 in each case) were bred with DO.11.10 TCR Tg BALB/c mice (H-2d) to generate H-2bxd DO-11.10-positive pups expressing or lacking the I{kappa}B{alpha}({Delta}N) Tg molecule. These DO11 H-2bxd-positive mice were subsequently bred with BALB/c DO-11.10 (H-2dxd) mice to generate litters of H-2bxd and H-2dxd DO-11.10 mice, expressing or not the I{kappa}B{alpha}({Delta}N) transgene, that were used for analysis of positive and negative thymic selection in littermates. I{kappa}B{alpha}({Delta}N) Tg mice on the C57BL/6 background (as above) were also bred with B6 H-Y TCR Tg (28) and OT-I (29). Genotypes of the DO-11.10, HY, and OT-I TCR transgenes were performed by PCR using the following primer sets: DO-11.10-TCR, 5'-GCTGTAATCAGACTAATAACCACAACAACAT-3' and 5'-CAACTGTGAGTCTGGTTCCTTTACCAA-3'; HY-TCR, 5'-ACAAGGTGGCAGTAACAGGA3', 5'-ACAGTCAGTCTGGTTCCTGA-3'; and OT-I- TCR 5'-AAGGTGGAGAGAGACAAAGGATTC-3' and 5'-TTGAGAGCTGTCTCC-3'. MHC haplotypes were determined by PCR amplification of the I-A{alpha} gene using the primer set 5'-ACCAACGGGACGCAGCGCAT-3' and 5'-CCTCGTAGTTGTGTCTGCAC-3', followed by resolution on agarose genes and Southern blot hybridizations using probes specific for Ab or Ad, respectively: 5'-ATACGATATGTGACCAGATA-3', and 5'-ATACGGCTCGTGACCAGATA-3'.

Abs, fluorochrome-conjugated reagents, and flow cytometric analyses

Biotinylated and fluorochrome-conjugated Abs against CD8 (biotin, FITC, or R-PE), CD4 (biotin, FITC, or R-PE), CD3{epsilon} (FITC), TCR{alpha}{beta} (Cy-Chr), and TCR V{beta}8 (R-PE) were obtained from BD PharMingen (San Diego, CA). Anti-clonotypic Abs against the DO-11.10 and H-Y TCRs were purified from the appropriate hybridomas (KJ1.26 and T3.70, respectively) and were biotinylated (30). Streptavidin-PerCP was obtained from BD Biosciences (Mountain View, CA). 7-Amino actinomycin D for detection of death cells was obtained from Molecular Probes (Eugene, OR). Flow cytometry was performed as described previously.

Cell preparation and in vitro negative selection assays

Single-cell suspensions were prepared from thymus, spleen, or lymph nodes as described previously (10, 12), and splenocytes were plated in RP-10 (2.5 x 106 cells/ml). For in vitro negative selection, thymocytes from H-Y female Tg mice were resuspended in RPMI 1640 medium containing 5% FBS and 10-5 M 2-ME (2 x 106 cells/ml). EL4 cells (H-2b/b; 5 x 104/ml in 24-well plates with 1 ml/well) pulsed for 2 h with H-Y peptide (KCSRNRQYL) at concentrations from 10-11–10-5 M before the addition of thymocytes were used as APCs. Thymocytes (2 x 106/well) were added to the APCs, cultured for 24 h, and stained with anti-CD4, anti-CD8, and 7-actinomycin. The percentage of survival of CD8+CD4+ thymocytes was calculated using the formula 100 x [1 - (% CD4+CD8+ with HY peptide)/(% CD4+CD8+ without HY peptide)] (31). AKR-DP-603, a CD3+CD4+CD8+ thymocyte lymphoma of unknown TCR specificity, was obtained from Dr. E. Richie (Research Division, M. D. Anderson Cancer Center, Smithville, TX) and was described previously as a negative control in Ref. 32 . For detection of phosphorylated and total ZAP-70 and TCR-{zeta}, thymocytes from wild-type and I{kappa}B{alpha}({Delta}N) Tg or AKR603 cells were resuspended at 30 x 106 cells/ml in RPMI 1640 medium supplemented with 0.2% BSA. Cells were incubated in medium for 5–30 min, alone or after addition of 5 µg/ml of anti-CD3{epsilon} Ab (2C11). Alternatively, thymocytes obtained from TCR Tg mice, expressing or not the I{kappa}B{alpha}({Delta}N) transgene, were MHC-peptide stimulated. Syngeneic wild-type APCs (H-2b/b) were prepared by complement-mediated lysis of Thy1+ splenocytes and were pulsed for 2 h with OT-I peptide (SIINFEKL) at a concentration of 10-5 M. Thymocytes (1 x 107/sample) were then added to the APC (2 x 107/sample) and were incubated for 5 min at 37°C.

Immunoprecipitation and Western blot analyses

For measurements of I{kappa}B{alpha}, thymocytes were used to prepare whole cell extracts and probe immunoblots as described previously (10, 12). Thymocytes (resting or stimulated) were washed once with cold PBS and placed in lysis buffer containing 1% Triton X-100, 50 mM HEPES (pH 7.5), 5% glycerol, 100 mM NaCl, 1 mM Na3VO4, and complete protease inhibitor mixture (Boehringer Mannheim, Mannheim, Germany) for 25 min on ice. Following removal of nuclear debris, the resultant supernatants were analyzed by SDS-PAGE before or after immunoprecipitation using polyclonal antisera against ZAP-70 or anti-TCR-{zeta} mAbs (clone 6B10; Zymed Laboratories, San Francisco, CA). After incubation of cell lysates and Ab for 3 h, immune complexes were collected with protein A-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA), washed in lysis buffer, eluted in SDS-PAGE sample buffer, and analyzed by SDS-PAGE and immunoblotting. Immunoblots were developed using the RC-20-HRP mAb to phosphotyrosine (BD Transduction Laboratories, San Diego, CA) and ECL (Amersham, Arlington Heights, IL). Membranes were then stripped and reprobed with an anti-ZAP-70 mAb (BD Transduction Laboratories) or anti-TCR-{zeta} (Zymed Laboratories). Cell extracts were also subjected to Western blot analysis with Abs against extracellular signal-regulated kinase (ERK), p-ERK, c-Jun N-terminal kinase (JNK), p-JNK, p38, p-p38 (New England Biolabs, Beverly, MA and Santa Cruz Biotechnology).

Gel mobility shift analyses

Parental and I{kappa}B{alpha}({Delta}N)-transduced AKR-DP-603 cells were resuspended at 5 x 106 cells/ml in RPMI 1640 medium supplemented with 10% FBS. Cells were incubated for 30 min alone or after the addition of PMA plus ionomycin. Whole cell extracts were prepared using high-salt extraction in the presence of protease inhibitors as previously described (10, 11). These extracts were then used in gel mobility shift assays of NF-{kappa}B/Rel proteins as described previously (10, 12).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Impairment of thymic negative selection in TCR Tg with defective NF-{kappa}B/Rel signaling

Mature T cells and their immature progenitors in the thymus differ in the ability of a given level of Ag receptor engagement to promote an apoptotic response (18, 33, 34). Moreover, there may be developmentally regulated differences between double-positive (DP) thymocytes and mature T cells in the sensitivity of their TCRs to ligands (19). However, relatively little is known about the role of transcription factors in normal thymocyte deletion by endogenous MHC ligands, or about differences in TCR signaling that reflect changes in the activity of other transduction pathways. Therefore, we explored the role of NF-{kappa}B/Rel signaling in thymocyte fates after Ag receptor signaling in vivo. To do so, the transdominant inhibitor I{kappa}B{alpha}({Delta}N) was expressed using T lineage-specific control elements. Biochemical analysis of thymocytes from Tg detected expression of the mutant protein at a level 2- to 3-fold higher than that of endogenous I{kappa}B{alpha} in wild-type cells (Fig. 1Go). This increased expression was sufficient to suppress expression of the endogenous I{kappa}B{alpha} gene, leading to a far higher ratio of I{kappa}B{alpha}({Delta}N) vs wild-type protein and few NF-{kappa}B dimers associated with endogenous I{kappa}B{alpha} (Fig. 1Go). To determine the impact of NF-{kappa}B on thymic negative selection by endogenous MHC ligands, I{kappa}B{alpha}({Delta}N) Tg were crossed with mice bearing well-characterized class I (H-Y) and class II (DO-11.10) MHC-restricted TCR transgenes (28, 35). Negative selection was quantified by measuring thymocyte populations on a negatively selecting background as compared with one promoting positive selection. Because negative selection of each of the TCR transgenes leads to the emergence of a population of double-negative (DN; CD4-CD8-), clonotype-positive T cells in the periphery, we also measured these cells.



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 1. Suppression of endogenous I{kappa}B{alpha} expression resulting from overexpression of the signal-resistant mutant I{kappa}B{alpha}({Delta}N). A, Whole cell extracts (10 µg) of freshly isolated thymocyte suspensions from I{kappa}B{alpha}({Delta}N)-Tg and wild-type (WT) littermates were resolved by SDS-PAGE, directly or after immunoprecipitation (IP) using anti-FLAG Abs, as indicated. Resolved I{kappa}B{alpha}({Delta}N) proteins were visualized by immunoblotting with anti-I{kappa}B{alpha} antisera reactive with both mouse and human proteins as in Refs. 10 and 43 . Due to truncation of 36 aa from the human I{kappa}B{alpha} protein, the slower migration of I{kappa}B{alpha}({Delta}N) (upper arrow) is only slightly resolved from that of endogenous mouse I{kappa}B{alpha} (wild type). The different forms are better resolved in the anti-FLAG immunoprecipitate (lane 4) and in the supernatant of proteins remaining after anti-FLAG immunoprecipitation (wild type, lane 5 and Tg, lane 6). B, The level of endogenous mouse I{kappa}B{alpha} protein in wild-type samples was compared with serial dilutions of thymocyte extracts from I{kappa}B{alpha}({Delta}N) mice. Based on these dilutions, it can be estimated that I{kappa}B{alpha}({Delta}N) is present at levels ~3-fold higher than I{kappa}B{alpha} (wild type) in samples from nontransgenic and >10-fold higher than the endogenous I{kappa}B{alpha} (wild type) of Tg samples.

 
The class II-restricted DO-11.10 TCR is positively selected on I-Ad, whereas negative selection predominates in H-2bxd heterozygotes due to alloreactivity of the receptor with I-Ab (36). The expression of I{kappa}B{alpha}({Delta}N) exerted a relatively modest effect on the development of T cell populations under conditions of positive selection (Fig. 2GoA). In contrast, inhibition of NF-{kappa}B led to a significant increase in DP and CD4 SPcells in H-2bxd heterozygous pups (2.5- and 4.5-fold increased, respectively). This effect was observed for the bulk thymocyte population as well as after gating on the population of cells expressing the TCR clonotype recognized by the anti-idiotypic mAb KJ1–26. This evidence of decreased negative selection was accompanied by a striking diminution in the DN, clonotype-positive population in both the thymus and the periphery (Fig. 2GoB). Moreover, the prevalence of mature clonotype-positive, CD4+ cells was increased significantly. Thus, the inhibitory transgene I{kappa}B{alpha}({Delta}N) imposes a significant block to negative selection of a TCR by endogenous MHC class II molecules.



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 2. I{kappa}B{alpha}({Delta}N) inhibits negative selection of DO-11.10 T cells by I-Ab. Cells from thymus and lymph nodes of I{kappa}B{alpha}({Delta}N) mice or littermate controls, all expressing the DO-11.10 TCR and homozygous for I-Ad (A) or I-Abxd heterozygous (B), were counted and analyzed by FACS after staining for the indicated markers. Representative profiles from the viable cells gate (top panels), or viable KJ1–26+ cells, are shown. Numbers indicate relative percentage of positive cells within a quadrant. Cellularity of I-Adxd thymi: wild type (W.T.), 79.3 (±6.8) x 106 cells; Tg 87.5 (±39.3) x 106 cells. Mean thymocyte counts in heterozygous I-Abxd mice were 34.0 (±7.4) x 106 (n = 13, wild type vs 44.8 (±7.3) x 106 n = 11; I{kappa}B{alpha}({Delta}N)).

 
Our original analyses of I{kappa}B{alpha}({Delta}N) Tg showed that the NF-{kappa}B/Rel pathway plays distinct roles in CD4 compared with CD8{alpha}{beta} cells (10, 12). Thus, the population of peripheral CD8{alpha}{beta}+ T cells was 5- to 10-fold more severely decreased than the CD4+ subset, and in the thymus, we detected a modest decrease in TCRhigh CD8 SP cells, whereas all other populations were normal. To determine whether the decrease in negative selection of an MHC-II-restricted TCR applies to deletion by MHC class I molecules, we used the class I-restricted H-Y TCR, which undergoes vigorous negative selection of thymocytes in H-2 Db males, leaving mostly DN thymocytes and reduced thymic cellularity (28). Transgenic inhibition of NF-{kappa}B led to a substantial increase in the DP population (Fig. 3Go). Negative selection of the H-Y TCR leads to the accumulation of a DN population in the periphery together with a comparable number of CD8+ T cells (28). The negative selection-induced DN population in lymph nodes was blocked by I{kappa}B{alpha}({Delta}N) (Fig. 3GoA, histograms to right). Consistent with the previous findings of decreased TCRhigh, CD8 SP thymocytes and markedly decreased CD8+ T cells in mice lacking TCR transgenes (10, 13, 14, 15, 16), and the rescue of these populations was more modest. Taken together, these findings indicate that I{kappa}B{alpha}({Delta}N) provided protection against negative selection by class I as well as class II MHC proteins.



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 3. Decreased negative selection of the H-Y TCR. A, Thymus and lymph node from wild-type (WT) and I{kappa}B{alpha}({Delta}N) male HY-TCR+ mice were counted and analyzed by FACS after staining for the indicated markers. Representative profiles of viable cells in thymus or viable V{beta}8 TCR+ cell gates in lymph node are shown. Similar results were obtained using the T3.70 mAb to stain HY TCR-positive cells in a smaller independent set of mice. B, Total cell numbers in thymocyte subsets were determined after FACS analysis and were calculated according to the percentage of cells CD4-CD8-, CD4+CD8+, CD4+CD8-, or CD4-CD8+ for wild-type ({square}) and I{kappa}B{alpha}({Delta}N) ({blacksquare}) HY-TCR males. Results are presented as the mean number (±SEM) of thymocytes positive or negative for the indicated markers.

 
To measure negative selection of DP cells bearing the H-Y TCR across a range of presumed densities of MHC-peptide complexes, we quantified the loss of cells in vitro in peptide dose-response curves using cells from female mice (Fig. 4Go). The results confirmed that inhibition of NF-{kappa}B was associated with weaker negative selection, and indicated that the dose-response curve for TCR-mediated deletion was shifted to the right. Thus, deletion of DP thymocytes from I{kappa}B{alpha}({Delta}N) mice required higher concentrations of a peptide recognized by the H-Y TCR as compared with wild-type thymocytes (37). This finding suggests that a role of NF-{kappa}B in mediating thymic selection is in determining the efficiency with which the TCR transduces signals in response to MHC-peptide complexes. If this possibility were true, then positive selection might also be diminished in I{kappa}B{alpha}({Delta}N) Tg thymi. Indeed, inhibition of NF-{kappa}B decreased the efficiency of positive selection when a thymic lymphoma cell line was stimulated with APCs and peptide in vitro (38). To test whether NF-{kappa}B/Rel signaling also contributes to positive selection in vivo, we measured the number of CD8 SP cells in female H-2b mice. Consistent with a contribution of NF-{kappa}B to positive as well as negative selection, the I{kappa}B{alpha}({Delta}N) transgene led to a decrease in positively selected cells in the thymus (Fig. 5Go). We conclude that impairment of NF-{kappa}B/Rel signaling by this trans-dominant inhibitor protects DP thymocytes against negative selection, but also can diminish positive selection, thereby accounting for the normal cellularity of TCRhigh thymocytes in Tg-expressing mutant I{kappa}Bs (10, 14, 15).



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 4. Reduced-efficiency deletion of the H-Y TCR in vitro. Data from a representative experiment in which thymocytes from female H-Y TCR-Tg bearing or lacking the I{kappa}B{alpha}({Delta}N) transgene were cultured in the presence of the H-2b cell line EL-4 and either no peptide (100% survival) or the indicated concentrations of H-Y-specific peptide. After overnight culture, DP thymocyte survival was quantified as described.

 


View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 5. Decreased positive selection of the H-Y TCR. Thymi from female H-Y TCR Tg bearing or lacking the I{kappa}B{alpha}({Delta}N) transgene were analyzed by FACS, and total numbers were calculated according to the percentage of cells CD4-CD8-, CD4+CD8+, CD4+CD8-, or CD4-CD8+ for wild-type (WT, {square}) and I{kappa}B{alpha}({Delta}N) ({blacksquare}) HY females. Results are presented as the mean number (±SEM) of thymocytes positive or negative for the indicated markers and the data presented, as in Fig. 3Go.

 
Inhibition of NF-{kappa}B leads to diminished ZAP-70 phosphorylation in thymocytes

Because changes in the expression of relevant T cell and APC molecules in thymic populations might contribute to the impairment of negative and positive selection observed in I{kappa}B{alpha}({Delta}N) mice, we measured the expression of TCR{alpha}{beta}, CD3{epsilon}, and MHC molecules in control and Tg mice. Flow cytometry detected comparable level expression of DO-11.10 and H-Y TCRs on CD4-CD8-, CD4+CD8+, CD4+CD8-, and CD4-CD+ thymocytes from I{kappa}B{alpha}({Delta}N) Tg as compared with wild-type controls (Fig. 6Go). A diminution in both positive and negative selection might result from blocking NF-{kappa}B signaling if this primary defect led to a decrease in the efficiency of some aspect of TCR signaling. To explore this possibility, we measured CD69 induction on thymocytes and T cells because this early response gene is induced by TCR engagement. Thymocytes and mature T lymphocytes from wild-type and I{kappa}B{alpha}({Delta}N) Tg expressing the DO.11.10 TCR transgene were stimulated with increasing concentrations of OVA peptide, and CD69 expression levels on CD4+CD8+ thymocytes and CD4+ KJ1–26+ T cells were monitored. Inhibition of NF-{kappa}B in thymocytes was associated with decreased expression of this inducible gene (Fig. 7GoA). To determine whether proximal or distal TCR signaling events were responsible for the alteration in CD69 expression, this parameter was also quantified after stimulation with PMA plus ionomycin. These pharmacologic agents can bypass early signaling events, but NF-{kappa}B induction in thymocytes is potently blocked under these conditions (39). This treatment led to normal induction of CD69 on CD4+CD8+ thymocytes from I{kappa}B{alpha}({Delta}N) Tg (Fig. 7GoA), indicating that decreased CD69 induction is not a direct consequence of the defective NF-{kappa}B/Rel pathway. Instead, the finding suggests that inhibition of the NF-{kappa}B/Rel pathway in thymocytes leads to a proximal defect in a TCR signaling pathway.



View larger version (48K):
[in this window]
[in a new window]
  		FIGURE 6. Normal expression of Tg TCRs in I{kappa}B{alpha}({Delta}N) Tg mice. Thymocytes from I{kappa}B{alpha}({Delta}N) mice or littermate controls, all expressing the DO-11.10 TCR (I-Ad) (A and B), or females expressing the HY TCR (C), were analyzed by FACS after staining for the indicated markers. Representative profiles gated on CD4-CD8-, CD4+CD8+, CD4+CD8-, or CD4-CD8+ thymocyte populations are shown for the anti-clonotypic mAb KJ1–26 (DO-11.10 TCR) (A), the invariant TCR component CD3{epsilon} (B), and the anti-clonotypic mAb T3.70 (HY-TCR; C). y axis scales differ among the single-parameter histograms to adjust for differences in the numbers of cells in various thymic subsets. WT, wild type.

 


View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 7. Dependence of TCR signaling on the status of NF-{kappa}B/Rel inducibility. A, DO-11.10 TCR (I-Ad) cells from thymi of I{kappa}B{alpha}({Delta}N) mice or littermate controls were cultured for 16 h with increasing concentrations of OVA323–339 peptide or PMA (50 ng/ml) plus ionomycin (1 µg/ml). The percentage of CD69-positive cells was determined by flow cytometry after gating on CD4+CD8+ thymocytes. Data from a representative experiment are presented. B, Cells from thymi of wild-type (WT) and I{kappa}B{alpha}({Delta}N) Tg were cultured in medium alone or with anti-CD3 (5 µg/ml) added to cell suspensions at t = 0 (leading to an appearance of slower kinetics of ZAP-70 phosphorylation. Similar results were obtained with more rapid kinetics after binding anti-CD3 to cells for 30 min at 4°C before warming to 37°C at t = 0). Tyrosine phosphorylation of ZAP-70 was visualized by antiphosphotyrosine immunoblotting (WB) of whole cell extracts subjected to immunoprecipitation (IP) using an antiserum against ZAP-70. To determine levels of ZAP-70 in the samples, membranes were stripped and reprobed using an anti-ZAP-70 mAb.

 
The recruitment and phosphorylation-dependent activation of ZAP-70 protein tyrosine kinase are essential for positive and negative selection of thymocytes (40, 41, 42, 43). Because our data indicated that both selection processes are attenuated in Tg-expressing I{kappa}B{alpha}({Delta}N), we determined the levels of ZAP-70 tyrosine phosphorylation after polyclonal TCR stimulation. ZAP-70 phosphorylation was decreased in thymocytes expressing I{kappa}B{alpha}({Delta}N) (Fig. 7GoB). Inasmuch as >80% of CD3+ thymocytes are CD4+CD8+, and levels of ZAP-70 phosphorylation are quantitatively comparable in DP and SP cells (19), the observed decrease in phospho-ZAP-70 reflects impairment of this signaling process within DP thymocytes. To investigate these biochemical observations further while using a homogeneous DP cell population, we used a DP thymocyte lymphoma line, AKR-DP-603. Signaling in parental cells after TCR stimulation was compared with a population expressing an I{kappa}B{alpha}({Delta}N) cDNA at levels that inhibit NF-{kappa}B activation (Fig. 8GoA). These experiments confirmed that inhibition of NF-{kappa}B by I{kappa}B{alpha}({Delta}N) was able to decrease ZAP-70 phosphorylation in response to the TCR (Fig. 8GoB). Of note, the level of each of these proteins I{kappa}B{alpha}({Delta}N) thymocytes appeared normal (Figs. 7GoB and 8B), and ZAP-70 phosphorylation in response to the phosphatase inhibitor pervanadate was normal as well (Fig. 8GoC). Consistent with the data obtained in primary thymocytes, TCR{alpha}{beta} and CD3{epsilon} expression were comparable in parental and I{kappa}B{alpha}({Delta}N)-transfected cells (Fig. 8GoD). It was of interest to explore a TCR signaling event proximal to ZAP-70 phosphorylation. TCR-{zeta} chain phosphorylation influences the association of ZAP-70 with the TCR complex and subsequent ZAP-70 activation (44, 45). Comparisons of primary thymocytes with normal or inhibited NF-{kappa}B signaling demonstrated comparable levels of p21-form phospho-TCR-{zeta} and of induced p23 TCR-{zeta} phosphorylation, using MHC-peptide or anti-CD3 as stimuli (Fig. 9Go, A and B). Total levels of TCR-{zeta} expression were also similar in wild-type and I{kappa}B{alpha}({Delta}N) thymocytes (Fig. 9GoC and data not shown). Taken together, these data indicate that a normal I{kappa}B{alpha}/NF-{kappa}B signaling axis is necessary for normal TCR signaling in immature T lymphoid cells in the thymus, regulating the induced phosphorylation of ZAP-70. In light of our finding that selection of such DP thymocytes by endogenous MHC ligands is attenuated, we propose that this alteration of TCR signaling is a mechanism by which the NF-{kappa}B/Rel transcription factors regulate thymic selection.



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 8. Decreased ZAP-70 phosphorylation in DP thymocytes subjected to inhibition of NF-{kappa}B. A, Parental (wild-type, WT) AKR-DP-603 thymocytes were retrovirally transduced with an I{kappa}B{alpha}({Delta}N) cDNA, selected with L-histidinol, and used for mobility shift assays of nuclear extracts from unstimulated and activated (PMA + ionomycin) cells as indicated. B, Parental (wild-type) AKR-DP-603 thymocytes and I{kappa}B{alpha}({Delta}N)-transduced cells were activated by cross-linking Abs against CD3{epsilon} as indicated, and lysates of these cells were subjected to immunoprecipitation (IP) using Abs against mouse ZAP-70 followed by immunoblotting (WB) using the indicated Abs. C, Independent experiments performed as in A, but using pervanadate as the stimulus. D, Parental and I{kappa}B{alpha}({Delta}N)-transduced AKR-DP-603 thymocytes cells were analyzed by FACS after staining for the indicated markers. Representative profiles for CD4 and CD8 staining and histograms gated on CD4+CD8+ cells for TCR{alpha}{beta} and CD3{epsilon} are shown.

 


View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 9. Equivalent TCR-{zeta} chain phosphorylation in wild-type (WT) and I{kappa}B{alpha}({Delta}N) thymocytes. Antiphosphotyrosine immunoblot results using extracts from activated or resting thymocytes, as indicated. A, Thymocytes from OT-1 TCR Tg (wild type and I{kappa}B{alpha}({Delta}N), as indicated), were lysed before and after stimulation with peptide-pulsed syngeneic APCs. Unfractionated lysates were then resolved and probed with the RC-20 mAb against phosphotyrosine. Similar results were obtained in three independent experiments. B, Thymocytes from wild-type and I{kappa}B{alpha}({Delta}N) Tg were activated by cross-linking Abs against CD3{epsilon} as indicated. Lysates of these cells were subjected to immunoprecipitation (IP) using Abs against mouse TCR-{zeta}, and precipitated proteins were analyzed as in A. The result shown is representative of four independent experiments. In some other experiments, results matching those of Ref. 45 were obtained for both wild-type and Tg I{kappa}B{alpha}({Delta}N) thymocytes. C, Anti-TCR-{zeta} immunoprecipitates were reprobed with anti-TCR-{zeta} Ab after stripping the antiphosphotyrosine immunoblot.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A central problem in understanding thymic selection mechanisms is to determine how specific signal transduction and transcriptional pathways influence the deletion of immature thymocytes by endogenous ligands in vivo. NF-{kappa}B/Rel complexes are present in the nuclei of resting thymocytes, albeit at low levels, and this pathway is further activated by the TCR. Thus, increased nuclear levels of members of the Rel protein family have been documented in DP thymocytes under conditions suggesting that these cells are responding to engagement of Ag receptors (8, 46). Of note, additional cell surface molecules implicated in thymic negative selection in vivo (CD28 and CD30) also trigger the NF-{kappa}B/Rel pathway (21, 22, 23). Using Tg systems to investigate the role of this transcriptional pathway in thymic selection in vivo revealed that inhibition of NF-{kappa}B led to decreased negative selection by autoreactive MHC-peptide complexes expressed as endogenous ligands. Consistent with these findings, we also found a decrease in the efficiency of peptide-induced death among DP cells expressing the transgenic inhibitor as compared with controls during in vitro assays. These data led to the conclusion that, in contrast to its role in mature T cells, the NF-{kappa}B/Rel pathway contributes to death of thymocytes by negative selection. Moreover, our data were consistent with a prior demonstration that NF-{kappa}B/Rel signaling promotes positive selection of the DPK cell line by Ag in vitro (38).

Because the data on peripheral, mature CD4+ T cells can more readily be interpreted in mice expressing NF-{kappa}B inhibitors, partial protection against negative selection is most clearly observed using an MHC class II-restricted TCR (10, 13, 14, 15, 16). There is a striking disparity between the effects of I{kappa}B{alpha}({Delta}N) on the populations of mature CD8 and CD4 SP cells, such that thymus-derived peripheral CD8{alpha}{beta} cells are dramatically underrepresented (0.05–0.1 times normal), whereas CD4 cells are decreased less drastically (10, 12, 13, 14, 15, 16). Thus, although protection against negative selection and the emergence of clonotype-positive CD4+ T cells can readily be detected for an MHC class II-restricted TCR transgene in I{kappa}B{alpha}({Delta}N) mice (Fig. 2Go), an increase in CD8 SP cells due to decreased negative selection may be counterbalanced by the mechanisms that account for a preferential effect of NF-{kappa}B inhibitors on the peripheral CD8 {alpha}{beta} population. It might also be expected that a decrease in thymic output would be counterbalanced by expansion in the periphery, but the presence of I{kappa}B{alpha}({Delta}N) strongly inhibits homeostatic and Ag-driven expansion in vivo (Ref. 12 and A. L. Mora, S. Stanley, W. Armistead, A. C. Chan, and M. Boothby, unpublished observations). In summary, our data indicate that the efficacy of both positive and negative selection depends on NF-{kappa}B, but the ability of I{kappa}B{alpha}({Delta}N) to inhibit negative selection is not absolute, and is most readily observed for an MHC class II-restricted TCR, but also influences class I-restricted thymocytes.

Several studies concurrent with the present work pertain to the involvement of NF-{kappa}B activation in thymic selection. Inactivation of the I{kappa}B kinase complex led to a profound lack of thymocytes, which could mostly be reversed by the formation of mixed bone marrow chimeras combined with eliminating TNF-{alpha} signaling through TNFR1 (47). Although thymic selection was not specifically investigated in this system, the establishment of normal proportions of SP thymocytes is similar to observations in the various systems where NF-{kappa}B has been blocked in T lineage cells using mutant I{kappa}B{alpha} transgenes (15, 16). Another recent study of the role of NF-{kappa}B in selecting MHC class I-restricted TCRs used a Tg approach analogous to I{kappa}B{alpha}({Delta}N) (16, 48). The data from these latter studies have been interpreted as documenting a role of NF-{kappa}B in positive selection (48) and in anti-CD3-induced thymocyte apoptosis in vivo but not negative selection (16, 48). Two lines of evidence suggest that differences between the findings presented in this study and those of Hettman and Leiden (48) are related to a higher level of expression of the I{kappa}B{alpha}({Delta}N) transgene as compared with the I{kappa}B{alpha}(A32/36) transgene. Biochemical analyses indicated that I{kappa}B{alpha}(A32/36) was expressed in thymocytes at a 1:1 ratio relative to endogenous I{kappa}B{alpha}, and further showed that the level of inhibition was insufficient to suppress expression of the endogenous I{kappa}B{alpha} (47), an NF-{kappa}B-dependent gene (49). In contrast, the severalfold higher level of I{kappa}B{alpha}({Delta}N) expression was associated with suppression of endogenous I{kappa}B{alpha} (Fig. 1Go). Of note, the inhibition of NF-{kappa}B/Rel signaling is not absolute at either level of expression, as is characteristic of trans-dominant inhibitors and the biochemistry of NF-{kappa}B activation (in that some Rel dimers bind to the wild-type I{kappa}B{alpha} in Tg thymocytes and T cells). Consistent with this higher ratio of mutant to endogenous I{kappa}B{alpha}, T cells in the periphery of I{kappa}B{alpha}({Delta}N) mice are decreased substantially more than in I{kappa}B{alpha}(A32/36) mice (16). Taken together, the aggregate data are most consistent with the view that NF-{kappa}B plays a role in promoting negative selection dependent on signal intensity and on the degree to which the NF-{kappa}B/Rel pathway is inhibited. This model is consistent with the results of peptide dose-response assays of negative selection in vitro in that high doses of negatively selecting peptide ultimately can overcome the inhibition of negative selection by I{kappa}B{alpha}({Delta}N) (Fig. 4Go), and protection against negative selection is in any event not absolute. However, an alternative view is that the pathway is relatively more important for negative selection of MHC class II-restricted TCRs.

In considering the mechanism by which inhibition of NF-{kappa}B/Rel proteins leads to a decrease in negative selection, our data provide evidence of a novel role for transcription factors in the regulation of TCR signaling in thymocytes. Thymic negative selection has been proposed to process as a multistep process (50, 51, 52). In this model, the first step (dulling the expression of both the CD4 and CD8 coreceptors) can be mimicked by pharmacologic doses of PKC activators; it is followed by a deletional step requiring APCs plus Ag and new protein synthesis (50, 51). This in vitro model raises the intriguing possibility that an intact I{kappa}B{alpha}/NF-{kappa}B signaling system is needed for execution of the second step in this biphasic model of negative selection. It is not clear what transcription factors may mediate this deletional step, but the present data are consistent with the possibility that NF-{kappa}B activates part of the genetic program implied by this dependence on new protein synthesis. What might represent target genes subject to transcriptional regulation? In contrast to our findings in mature T cells (A. L. Mora, S. G. Goenka, M. Aronica, S. Stanley, B. Enerson, D. W. Ballard, and M. Boothby, manuscript in preparation), we found no differences in levels of anti-apoptotic proteins Bcl-XL and Bcl-2 when comparing wild-type and Tg thymocytes (data not shown). Thus, the critical target genes leading to a requirement for new protein synthesis to execute negative selection are not clear.

However, the data also support a novel role for NF-{kappa}B in which this transcription factor influences TCR signaling complexes by a mechanism independent of changes in Ag receptor expression and phosphorylation of TCR-{zeta}. Thus, thymocytes from I{kappa}B{alpha}({Delta}N) mice exhibited decreased ZAP-70 phosphorylation in response to TCR cross-linking, a finding replicated in a DP thymic lymphoma cell line expressing inhibitory levels of I{kappa}B{alpha}({Delta}N). It is intriguing that this effect on ZAP-70 phosphorylation applies only to thymocytes but not mature T cells (A. L. Mora, unpublished observations). In terms of the thymocyte defect, our preliminary studies suggest that the alteration of ZAP-70 phosphorylation has a functional impact in that phosphorylation of the ZAP-70 target SLP-76 also is diminished (A. L. Mora, unpublished observations). Three classes of mitogen-activated protein kinases, the ERKs, p38, and JNKs all lie downstream from the activation of ZAP-70 (53). Although a variety of data pertain to this issue, recent findings suggest several points in this regard. First, the ERK pathway may preferentially contribute to thymic positive selection, whereas p38 and JNK are more closely coupled to negative selection (reviewed in 54). Second, certain perturbations of signaling may selectively alter coupling of p38/JNK activation to the TCR, thereby leading to a defect of negative but not positive selection (55) or vice versa (56). In this regard, our finding that both positive and negative selection are diminished is consistent with preliminary findings that I{kappa}B{alpha}({Delta}N) exerts a similar effect on each of these classes of mitogen-activated protein kinase. Thus, it appears unlikely that there is any specificity to the inhibitory effects downstream from ZAP-70. There is little precedent for such feedback links between transcription factor activity and regulation of ZAP-70, but there are a few examples of regulated changes in TCR sensitivity. In one such case, the tyrosine phosphatase SHP1 seems to interfere with the activation of ZAP-70 and/or Lck (57). A serine/threonine kinase activated by Ras and considered a downstream effector of TCR signaling, ERK-1, may provide positive feedback regulation by interfering with SHP-1 recruitment to the TCR (unpublished data summarized in 58). Although the mechanism of NF-{kappa}B feedback regulation of TCR sensitivity in thymocytes remains to be established, I{kappa}B{alpha} degradation may be downstream from Ras and mitogen-activated protein kinase kinase kinase (59) and could thereby participate in positive feedback regulation of TCR signaling. Alternatively, it is conceivable that NF-{kappa}B activity regulates the level of a protein involved in the efficient formation of a signaling complex (60, 61, 62).


   Acknowledgments
 
We gratefully acknowledge expert technical assistance from Ben Enerson and Susan McCarthy. We also thank Rebecca Merica, Thore Hettman, Eugene Oltz, and Jin Chen for helpful discussions, Jin Chen, Eugene Oltz, Geraldine Miller, Derya Unutmaz, and Jacek Hawiger for critical readings of manuscript drafts, Rebecca Merica and Marc Jenkins, Hung-Shia Teh, Andrew Lichtman, and Ellen Richie for generous gifts of reagents and mouse lines, Jim Price and David McFarland for preparative and analytical flow cytometry, and the Vanderbilt Ingram Cancer Center and Diabetes Research and Training Center for tissue culture, DNA, molecular biology, and flow cytometry core functions.


   Footnotes
 
1 The Vanderbilt-Ingram Cancer and Diabetes Research and Training Centers (National Institutes of Health Grants CA68485 and P60 DK20593) provided support through core functions (FACS and oligonucleotide synthesis). M.B. was a Scholar of the Leukemia Society of America, and other funding for this work was provided by the National Institutes of Health (AI-36997, HL-61752, GM-42550, and a Vanderbilt University Discovery Grant). A.C.C. is an investigator of the Howard Hughes Medical Institute. Back

2 Address correspondence and reprint requests to Dr. Mark Boothby, Department of Microbiology and Immunology, Vanderbilt University Medical School, AA-4214 MCN, Nashville, TN 37232-2363. E-mail address: mark.boothby{at}mcmail.vanderbilt.edu Back

3 Abbreviations used in this paper: Tg, transgenic; SP, single positive; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; DP, double positive; DN, double negative. Back

Received for publication December 13, 2000. Accepted for publication September 12, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. von Boehmer, H.. 1993. The developmental biology of T lymphocytes. Annu. Rev. Immunol. 6:309.[Medline]
  2. Tanchot, C., M. Rosada, F. Agenes, A. Freitas, B. Rocha. 1997. Lymphocyte homeostasis. Semin. Immunol. 9:331.[Medline]
  3. Tanchot, C., B. Rocha. 1997. Peripheral selection of T cell repertoires: the role of continuous thymus output. J. Exp. Med. 186:1099.[Abstract/Free Full Text]
  4. Berzins, S. P., R. L. Boyd, J. F. Miller. 1998. The role of the thymus and recent thymic migrants in the maintenance of the adult peripheral lymphocyte pool. J. Exp. Med. 187:1839.[Abstract/Free Full Text]
  5. Robey, E., B. J. Fowlkes. 1994. Selective events in T cell development. Annu. Rev. Immunol. 12:675.[Medline]
  6. Rocha, B., N. Dautigny, P. Pereira. 1989. Peripheral T lymphocytes: expansion potential and homeostatic regulation of pool sizes and CD4/CD8 ratios in vivo. Eur. J. Immunol. 19:905.[Medline]
  7. Sprent, J., M. Schaefer, M. Hurd, C. D. Surh, Y. Ron. 1991. Mature murine B and T cells transferred to SCID mice can survive indefinitely and many maintain a virgin phenotype. J. Exp. Med. 174:717.[Abstract/Free Full Text]
  8. Sen, J., L. Venkataraman, Y. Shinkai, J. W. Pierce, F. W. Alt, S. J. Burakoff, R. Sen. 1995. Expression and induction of nuclear factor-{kappa}B-related proteins in thymocytes. J. Immunol. 154:3213.[Abstract]
  9. Ghosh, S., M. J. May, E. B. Kopp. 1998. NF-{kappa}B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu. Rev. Immunol. 16:225.[Medline]
  10. Boothby, M., A. L. Mora, D. Scherer, J. Brockman, D. W. Ballard. 1997. Perturbation of the T lymphocyte lineage in transgenic mice expressing a constitutive repressor of nuclear factor (NF)-{kappa}B. J. Exp. Med. 185:1897.[Abstract/Free Full Text]
  11. Brockman, J., D. Scherer, T. McKinsey, S. Hall, X. Qi, W. Lee, D. W. Ballard. 1995. Coupling of a signal response domain in I{kappa}B{alpha} to multiple pathways for NF-{kappa}B activation. Mol. Cell. Biol. 15:2809.[Abstract]
  12. Mora, A. L., D. Chen, M. Boothby, D. Rubin. 1999. Lineage-specific differences among CD8+ T cells in their dependence of NF-{kappa}B/Rel signaling. Eur. J. Immunol. 29:2968.[Medline]
  13. Esslinger, C., A. Wilson, B. Sordat, F. Beermann, C. Jongeneel. 1997. Abnormal T lymphocyte development induced by targeted overexpression of I{kappa}B{alpha}. J. Immunol. 158:5075.[Abstract]
  14. Attar, R. ., H. Macdonald-Bravo, C. Raventos-Suarez, S. Durham, R. Bravo. 1998. Expression of constitutively active I{kappa}B{beta} in T cells of transgenic mice: persistent NF-{kappa}B activity is required for T-cell immune responses. Mol. Cell. Biol. 18:477.[Abstract/Free Full Text]
  15. Ferreira, V., N. Sidenkus, N. Tarantino, P. Hubert, L. Chatenoud, F. Blasi, M. Korner. 1999. In vivo inhibition of NF-{kappa}B in T-lineage cells leads to a dramatic decrease in cell proliferation and cytokine production and to increased cell apoptosis in response to mitogenic stimuli, but not to abnormal thymopoiesis. J. Immunol. 162:6442.[Abstract/Free Full Text]
  16. Hettmann, T., J. DiDonato, M. Karin, J. Leiden. 1999. An essential role for nuclear factor {kappa}B in promoting double-positive thymocyte apoptosis. J. Exp. Med. 189:145.[Abstract/Free Full Text]
  17. Van Antwerp, D. J., S. J. Martin, T. Kafri, D. R. Green, I. M. Verma. 1996. Suppression of TNF-{alpha}-induced apoptosis by NF-{kappa}B. Science 274:787.[Abstract/Free Full Text]
  18. Pircher, H., U. H. Rohrer, D. Moskophidis, R. M. Zinkernagel, H. Hengartner. 1991. Lower receptor avidity required for thymic clonal deletion than for effector T cell function. Nature 351:482.[Medline]
  19. Lucas, B., I. Stefanova, K. Yasutomo, N. Dautigny, R. N. Germain. 1999. Divergent changes in the sensitivity of maturing T cells to structurally related ligands underlies formation of a useful T cell repertoire. Immunity 10:367.[Medline]
  20. Sun, Z., C. Arendt, W. Ellmeier, E. Schaeffer, M. J. Sunshine, L. Gandhi, J. Annes, D. Petrzilka, A. Kupfer, P. L. Schwartzberg, D. R. Littman. 2000. PKC-{theta} is required for TCR-induced NF-{kappa}B activation in mature but not immature thymocytes. Nature 404:402.[Medline]
  21. Amakawa, R., A. Hakem, T. M. Kundig, T. Matsuyama, J. Simard, E. Timms, A. Wakeham, H. Mittruecker, H. Griesser, H. Takimoto, et al 1996. Impaired negative selection of T cells in Hodgkin’s disease antigen CD30-deficient mice. Cell 84:551.[Medline]
  22. Noel, P. J., M. L. Alegre, S. Reiner, C. B. Thompson. 1998. Impaired negative selection in CD28-deficient mice. Cell. Immunol. 187:131.[Medline]
  23. Punt, J. A., W. Havran, R. Abe, A. Sarin, A. Singer. 1997. T cell receptor (TCR)-induced death of immature CD4+CD8+ thymocytes by two distinct mechanisms differing in their requirement for CD28 costimulation: implications for negative selection in the thymus. J. Exp. Med. 186:1911.[Abstract/Free Full Text]
  24. Herron, L. R., R. A. Eisenberg, E. Roper, V. Kakkanaiah, P. L. Cohen, B. L. Kotzin. 1993. Selection of the T cell receptor repertoire in Lpr mice. J. Immunol. 151:3450.[Abstract]
  25. Singer, G. G., A. K. Abbas. 1994. The fas antigen is involved in peripheral but not thymic deletion of T lymphocytes in T cell receptor transgenic mice. Immunity 1:365.[Medline]
  26. Sytwu, H. K., R. S. Liblau, H. O. McDevitt. 1996. The roles of Fas/APO-1(CD95) and TNF-{alpha} in antigen-induced programmed cell death in T cell receptor transgenic mice. Immunity 5:17.[Medline]
  27. Pfeffer, K., T. W. Mak. 1994. Lymphocyte ontogeny and activation in gene-targeted mutant mice. Annu. Rev. Immunol. 12:367.[Medline]
  28. Kisielow, P., H. Bluthmann, U. Staerz, M. Steinmetz, H. von Boehmer. 1988. Tolerance in T-cell-receptor transgenic mice involves deletion of nonmature CD4+8+ thymocytes. Nature 333:742.[Medline]
  29. Hogquist, K. A., S. C. Jameson, W. R. Heath, J. L. Howard, M. J. Bevan, F. R. Bevan. 1994. T cell receptor antagonist peptides induce positive selection. Cell 14:17.
  30. Harlow, E., D. Lane. 1988. Antibodies: A Laboratory Manual 340. Cold Spring Harbor Lab. Press, Cold Spring Harbor, NY.
  31. Kong, Y. Y., K. D. Fisher, M. F. Bachmann, S. Mariathasan, I. Kozieradzki, M. P. Nghiem, D. Bourchard, A. Bernstein, P. S. Ohashi, J. M. Penninger. 1998. Vav regulates peptide-specific apoptosis in thymocytes. J. Exp. Med. 188:2099.[Abstract/Free Full Text]
  32. Kovalik, J.-P., B. Ansari, M. Boothby. 1996. Rapid commitment to negative selection of a thymocyte line independent of CD4 co-engagement. J. Immunol. 157:5290.[Abstract]
  33. Hogquist, K. A., S. C. Jameson, M. J. Bevan. 1995. Strong agonist ligands for the T cell receptor do not mediate positive selection of functional CD8+ T cells. Immunity 3:79.[Medline]
  34. Sebzda, E., T. M. Kundig, C. T. Thomson, K. Aoki, S. Y. Mak, J. P. Mayer, T. Zamborelli, S. G. Nathenson, P. S. Ohashi. 1996. Mature T cell reactivity altered by peptide agonist that induces positive selection. J. Exp. Med. 183:1093.[Abstract/Free Full Text]
  35. Murphy, K., A. Heimberger, D. H. Loh. 1990. Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlow thymocytes in vivo. Science 250:1720.[Abstract/Free Full Text]
  36. Liu, C.-P., J. W. Kappler, P. Marrack. 1996. Thymocytes can become mature T cells without passing through the CD4+CD8+, double-positive stage. J. Exp. Med. 184:1619.[Abstract/Free Full Text]
  37. Markiewicz, M. A., C. Girao, J. T. Opferman, J. Sun, Q. Hu, A. A. Agulnik, C. E. Bishop, C. B. Thompson, P. G. Ashton-Rickart. 1998. Long-term T cell memory requires the surface expression of self-peptide/major histocompatibility complex molecules. Proc. Natl. Acad. Sci. USA 95:3065.[Abstract/Free Full Text]
  38. Ochoa-Garay, J., J. Kaye, J. E. Coligan. 1998. Nuclear factor {kappa}B is required for peptide antigen-induced differentiation of a CD4+CD8+ thymocyte line. J. Immunol. 160:3835.[Abstract/Free Full Text]
  39. Manger, B., A. Weiss, J. Imboden, T. Laing, J. D. Stobo. 1987. The role of protein kinase C in transmembrane signaling by the T cell antigen receptor complex: effects of stimulation with soluble or immobilized CD3 antibodies. J. Immunol. 139:2755.[Abstract]
  40. Arpaia, E., M. Shahar, H. Dadi, A. Cohen, C. F. Roifman. 1994. Defective T cell receptor signaling and CD8+ thymic selection in humans lacking ZAP-70 kinase. Cell 76:947.[Medline]
  41. Negishi, I., N. Motoyama, K. Nakayama, K. Nakayama, S. Senju, S. Hatakeyama, Q. Zhang, A. C. Chan, D. Y. Loh. 1995. Essential role for ZAP-70 in both positive and negative selection of thymocytes. Nature 376:435.[Medline]
  42. Wiest, D. L., J. M. Ashe, T. K. Howcroft, H. M. Lee, D. M. Kemper, I. Negishi, D. S. Dinger, A. Singer, R. Abe. 1997. A spontaneously arising mutation in the DLAARN motif of mouse ZAP-70 abrogates kinase activity and arrests thymocyte development. Immunity 6:663.[Medline]
  43. Chan, A. C., M. Dalton, R. Johnson, G. H. Kong, T. Wang, T. Thoma, T. Kurosaki. 1995. Activation of ZAP-70 kinase activity by phosphorylation of tyrosine 493 is required for lymphocyte antigen receptor function. EMBO J. 14:2499.[Medline]
  44. Van Oers, N., N. Killeen, A. Weiss. 1994. ZAP-70 is constitutively associated with tyrosine phosphorylated TCR {zeta} in murine thymocytes and lymph node T cells. Immunity 1:675.[Medline]
  45. Van Oers, N., P. E. Love, E. W. Shores, A. Weiss. 1998. Regulation of TCR signal transduction in murine thymocytes by multiple TCR {zeta}-chain signaling motifs. J. Immunol. 160:163.[Abstract/Free Full Text]
  46. Moore, N. C., J. Girdlestone, G. Anderson, J. J. Owen, E. J. Jenkinson. 1995. Stimulation