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• W2047709368 abstract "Article6 October 2005free access Loss of c-Cbl RING finger function results in high-intensity TCR signaling and thymic deletion Christine BF Thien Christine BF Thien School of Surgery and Pathology, University of Western Australia, Crawley, Australia Search for more papers by this author Frøydis D Blystad Frøydis D Blystad School of Surgery and Pathology, University of Western Australia, Crawley, AustraliaPresent address: Institute of Pathology, University of Oslo, Rikshospitalet, Norway Search for more papers by this author Yifan Zhan Yifan Zhan The Walter and Eliza Hall Institute of Medical Research, Royal Parade, Melbourne, Australia Search for more papers by this author Andrew M Lew Andrew M Lew The Walter and Eliza Hall Institute of Medical Research, Royal Parade, Melbourne, Australia Search for more papers by this author Valentina Voigt Valentina Voigt Centre for Experimental Immunology, The Lions Eye Institute, Nedlands, Australia Search for more papers by this author Christopher E Andoniou Christopher E Andoniou Centre for Experimental Immunology, The Lions Eye Institute, Nedlands, Australia Search for more papers by this author Wallace Y Langdon Corresponding Author Wallace Y Langdon School of Surgery and Pathology, University of Western Australia, Crawley, Australia Search for more papers by this author Christine BF Thien Christine BF Thien School of Surgery and Pathology, University of Western Australia, Crawley, Australia Search for more papers by this author Frøydis D Blystad Frøydis D Blystad School of Surgery and Pathology, University of Western Australia, Crawley, AustraliaPresent address: Institute of Pathology, University of Oslo, Rikshospitalet, Norway Search for more papers by this author Yifan Zhan Yifan Zhan The Walter and Eliza Hall Institute of Medical Research, Royal Parade, Melbourne, Australia Search for more papers by this author Andrew M Lew Andrew M Lew The Walter and Eliza Hall Institute of Medical Research, Royal Parade, Melbourne, Australia Search for more papers by this author Valentina Voigt Valentina Voigt Centre for Experimental Immunology, The Lions Eye Institute, Nedlands, Australia Search for more papers by this author Christopher E Andoniou Christopher E Andoniou Centre for Experimental Immunology, The Lions Eye Institute, Nedlands, Australia Search for more papers by this author Wallace Y Langdon Corresponding Author Wallace Y Langdon School of Surgery and Pathology, University of Western Australia, Crawley, Australia Search for more papers by this author Author Information Christine BF Thien1, Frøydis D Blystad1, Yifan Zhan2, Andrew M Lew2, Valentina Voigt3, Christopher E Andoniou3 and Wallace Y Langdon 1 1School of Surgery and Pathology, University of Western Australia, Crawley, Australia 2The Walter and Eliza Hall Institute of Medical Research, Royal Parade, Melbourne, Australia 3Centre for Experimental Immunology, The Lions Eye Institute, Nedlands, Australia *Corresponding author. School of Surgery and Pathology, University of Western Australia, Crawley, WA 6009, Australia. Tel.: +61 8 9346 2939; Fax: +61 8 9346 2891; E-mail: [email protected] The EMBO Journal (2005)24:3807-3819https://doi.org/10.1038/sj.emboj.7600841 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Signaling from the T-cell receptor (TCR) in thymocytes is negatively regulated by the RING finger-type ubiquitin ligase c-Cbl. To further investigate this regulation, we generated mice with a loss-of-function mutation in the c-Cbl RING finger domain. These mice exhibit complete thymic deletion by young adulthood, which is not caused by a developmental block, lack of progenitors or peripheral T-cell activation. Rather, this phenotype correlates with greatly increased expression of the CD5 and CD69 activation markers and increased sensitivity to anti-CD3-induced cell death. Thymic loss contrasts the normal fate of the c-Cbl–/– thymus, even though thymocytes from both mutant mice show equivalent enhancement in proximal TCR signaling, Erk activation and calcium mobilization. Remarkably, only the RING finger mutant thymocytes show prominent TCR-directed activation of Akt. We show that the mutant c-Cbl protein itself is essential for activating this pathway by recruiting the p85 regulatory subunit of PI 3-kinase. This study provides a unique model for analyzing high-intensity TCR signals that cause thymocyte deletion and highlights multiple roles of c-Cbl in regulating this process. Introduction The generation of T cells that respond to foreign antigens, but not self-antigens, is carried out in the thymus by a process initiated by T-cell receptor (TCR) engagement and the activation of intracellular signaling cascades. The amplitude and duration of these signaling responses are initially determined by the affinity of the TCR for antigen/MHC complexes and the total number of receptor interactions. After antigen engagement, signaling pathways are controlled by an array of intracellular enzymes, regulatory proteins, adaptors and transcription factors. The strength and kinetics of these signaling responses are key factors determining whether thymocytes survive or are actively deleted, by positive and negative selection respectively (Ohashi, 2003; Palmer, 2003; Starr et al, 2003). Perturbations to signaling molecules functionally involved in these outcomes can alter the fate of thymocytes and result in the development of anergy or autoimmunity. Thus, the ability to generate a functional T-cell repertoire is of great importance and requires precise regulation of ligand engagement and signal transduction. A key regulator of TCR and CD3 levels, and the activity of signaling proteins downstream, is c-Cbl (Thien and Langdon, 2001; Liu and Gu, 2002; Dikic et al, 2003; Liu, 2004). c-Cbl, and its close homologue Cbl-b, principally functions as an E3 ubiquitin ligase by virtue of a RING finger domain, which recruits ubiquitin conjugating enzymes (E2s), and a tyrosine kinase binding (TKB) domain involved in substrate targeting. The best-characterized substrates for c-Cbl- and Cbl-b-directed ubiquitylation are receptor tyrosine kinases; however, other classes of receptors, cytoplasmic protein tyrosine kinases (PTKs), adaptor proteins and regulatory proteins have also been identified as targets. Studies of c-Cbl knockout (KO) mice found elevated TCR and CD3 levels on the surface of CD4+CD8+ double positive (DP) thymocytes, increased levels of the Src family kinases Lck and Fyn and increased activity of the ZAP-70 tyrosine kinase (Murphy et al, 1998; Naramura et al, 1998; Thien et al, 1999). Indeed, CD3 signaling in c-Cbl KO thymocytes is enhanced to an extent where ZAP-70 activation is uncoupled from a requirement for CD4 coreceptor ligation, although this effect is independent of TKB domain function (Thien et al, 1999, 2003). Despite these perturbations that increase the intensity and duration of TCR signals, c-Cbl KO thymi develop with apparent normality. However, the absence of c-Cbl enhanced positive selection of CD4+ thymocytes in MHC class II-restricted TCR transgenic mice (Naramura et al, 1998). These findings are consistent with roles for c-Cbl in negatively regulating TCR signals involved in determining the fate of thymocytes. To better understand the mechanisms involved in this regulation, we generated mice with a loss-of-function mutation in the c-Cbl RING finger domain. This substitution of an alanine for the amino-terminal cysteine in the C3HC4 RING domain at position 379 (C381 in human c-Cbl) has been well characterized and abolishes c-Cbl's interaction with E2s and its function as an E3 ubiquitin ligase (Joazeiro et al, 1999; Levkowitz et al, 1999; Ota et al, 2000; Thien et al, 2001). Unlike the mouse with a loss-of-function mutation in the c-Cbl TKB domain (Thien et al, 2003), we find that the RING finger mutant mouse resembles the c-Cbl KO in many respects, such as equivalently enhanced levels of CD3, TCR and Lck in DP thymocytes. However, the RING finger mutation induces additional phenotypic changes that are more severe than those observed in c-Cbl KO mice, notable among these being a progressive loss of the thymus. Results Generation of mice with a loss-of-function mutation in the c-Cbl RING finger Mice with a Cys to Ala substitution at position 379 (C379A) were generated and genotyped as outlined in Figure 1A and B. Matings of heterozygous C379A c-Cbl mice (termed +/A) produced significantly less than expected homozygous mutant (A/A) offspring (5% compared to expected 25%; Figure 1C) with the majority of these dying in utero after E14 (22% A/A detected at E14, 7% at E16 and 4% at E19). In addition, ∼25% of A/A mice born did not survive the first 24 h (Figure 1C). Interestingly, this severe developmental effect does not occur in either the c-Cbl or Cbl-b KO mice. To overcome the scarcity of A/A mice, we generated c-Cbl A/− mice. These had improved, although still reduced, viability (Figure 1C), indicating that a single copy of the mutated allele has a less severe effect on survival. Importantly, c-Cbl A/− mice appear to be indistinguishable from homozygous c-Cbl A/A mice for all other phenotypic perturbations identified to date. Figure 1.Generation and identification of c-Cbl(C379A) mice. (A) Genomic organization of the mouse c-Cbl gene showing the region targeted for homologous recombination to introduce the C379A mutation (indicated by a large asterisk). Targeted ES cell clones were identified by Southern blotting using 5′ and 3′ probes as indicated. B, BamHI; H, HindIII; X, XbaI; X*, XbaI sites present in the 129Sv/J but not C57BL/6 strain. The loxP-flanked pGKNeo cassette was removed by Cre-mediated excision in vivo, leaving a single loxP site (solid triangle). (B) PCR genotyping of c-Cbl(C379A) mice prior to Cre-mediated deletion using primers p1 and p2 to detect the wt allele (∼600 bp product), and p1 and p3 to detect the C379A targeted allele (∼450 bp product). (C) Genotype frequencies and survival statistics. The percentage of pups of each genotype that die within the first 24 h of birth is also shown. (D) Expression of the C379A allele does not alter c-Cbl protein levels. Thymocyte lysates from c-Cbl +/A, A/A, A/− and +/− mice were immunoblotted with antibodies to c-Cbl, ZAP-70 or Erk1/2. (E) Expression of the C379A allele enhances coat color in black and agouti mice. Comparisons of a black c-Cbl(C379A) homozygous mutant mouse (A/A) with a wt C57BL/6 mouse (left panel), c-Cbl +/+ and A/A agouti littermates (middle), and agouti c-Cbl–/–, A/A and A/− mice (right) show that c-Cbl(C379A) knockin mice have darker coats, paws, ears and tails than wt or c-Cbl KO mice. Download figure Download PowerPoint Western blotting showed that c-Cbl protein levels in thymocytes are not affected by the C379A mutation (Figure 1D). Interestingly, A/A and A/− mice had darker coats, feet and tails than wild-type (wt) or KO mice (Figure 1E). This phenotype occurred in both black and agouti mice and is not evident in other Cbl mutant mice generated to date. The mechanism for the dark coloration was not examined but may be linked to enhanced activity of c-Kit, which is negatively regulated by Cbl proteins (Zeng et al, 2005) and is required for melanocyte development. Progressive thymic loss in the c-Cbl RING finger knockin mouse Examination of organs revealed a striking phenotype of c-Cbl C379A mice, namely the progressive loss of thymi as they approach adulthood. In the few A/A mice analyzed, a decrease in thymus size was evident by 2 weeks while a 40-day-old A/A mouse contained fewer than 1% of total thymocytes compared to its +/A littermate (Figure 2A). Comparison of over 100 A/− and +/− littermates shows that this progressive loss of the thymus was similarly observed in A/− mice (Figure 2B and C). This phenotype was unexpected since c-Cbl KO mice do not show this thymic loss and indeed exhibit a slight increase in thymic cellularity as young adults (Murphy et al, 1998). Figure 2.Thymocyte loss in the c-Cbl(C379A) knockin mouse is not caused by a developmental block. (A) Total thymocyte numbers from five pairs of c-Cbl A/A and +/A littermates at various ages. The difference is tabulated as the percentage decrease in thymocyte numbers from the c-Cbl A/A mouse compared to its +/A littermate. (B) Photograph and weights of spleens and thymi showing the greatly reduced thymi but enlarged spleens in c-Cbl A/− mice compared to their +/− littermates. (C) Total thymocyte numbers and spleen weights of c-Cbl A/− (red circles) and +/− littermates (black triangles) killed between 1 and 10 weeks of age. (D) Flow cytometric analysis of CD4 and CD8 on thymocytes from 19- and 36-day-old c-Cbl+/−, A/− or –/– mice. Percentages of DN, DP, and CD4 or CD8 SP populations are indicated in the respective quadrants. (E) Bar graph representing the mean percentages (±SEM) of CD4 and CD8 DN, DP and CD4 or CD8 SP thymocytes from 23 c-Cbl+/− and 28 A/− littermates. Statistically significant differences between A/− and +/− DN, CD4 and CD8 SP populations were detected using unpaired t-test (**P<0.05; ***P<0.001). (F) Percentages of DN and DP thymocytes from +/− and A/− littermates of varying thymus size. The data reveal that the smallest A/− thymi show reduced proportions of DP thymocytes and a corresponding increase in the DN population. (G) Flow cytometric analysis of DN subpopulations. Thymocytes from 19-, 28- and 36-day-old c-Cbl A/− and +/− littermates were stained with FITC-conjugated antibodies to CD4, CD8, CD3, TER119, B220 and Gr1, PE-conjugated anti-CD25 and APC-conjugated anti-CD44. Analysis of CD44 and CD25 expression was performed on gated FITC-negative cells. The percentage of cells found in each quadrant is indicated. Download figure Download PowerPoint A consequence of thymocyte loss in RING finger mutant mice was that lymph nodes contained 50% fewer CD4+ and CD8+ T cells and a higher proportion of B cells compared to normal littermates and c-Cbl–/– mice (Supplementary Figure 1A and B). A greater proportion of CD4+ T cells expressing higher levels of the activation markers CD44 and CD25 was evident in A/− mice; however, the absolute number of cells with this phenotype was decreased and CD62L levels, a marker for memory T-cell populations, were equivalent between CD4+ T cells from c-Cbl–/– and A/− mice (Supplementary Figure 1C). Spleens from A/A and A/− mice were increased 2.5- to 3.5-fold in size compared to a 1.2- to 2-fold increase in c-Cbl–/– mice and is caused by a greater expansion of the red pulp with increased numbers of red blood cells, megakaryocytes, megakaryoblasts and myelocytes (Figure 2B and C and data not shown). However, there is no evidence of lymphocyte hypertrophy in A/− spleens and indeed the proportion of splenic T cells is generally reduced by 80% compared to normal littermates analyzed between 4 and 7 weeks of age. Thymic loss is not due to a developmental block The effect of the C379A mutation in inducing thymic loss prompted us to investigate whether this was due to a block in thymocyte development. However, representative analyses of CD4 and CD8 expression on C379A knockin thymocytes detected double negative (DN), DP and single positive (SP) populations in near-normal proportions (Figure 2D). Thus, there is no major block in the DN to DP transition, or in the selection of DP to SP thymocytes. We observed slight increases in the proportion of DN thymocytes from RING finger mutant mice (Figure 2E), which became pronounced when thymic loss was greatest and was accompanied by a corresponding decrease in the proportion of DP thymocytes (Figure 2F). However, analysis of A/− thymi using CD44 and CD25 antibodies revealed no marked effects on the four major developmental stages of the DN population, aside from a tendency toward an increased proportion of DN2/3 cells in older mice (Figure 2G). The proportion of mature CD4 and CD8 SP thymocytes was also reduced, by approximately 40% (Figure 2D and E). This suggests that the C379A mutation also perturbs signaling events that determine SP selection, presumably because of changes to TCR and coreceptor signal strength. Thymic loss is not due to limiting numbers of progenitors We also investigated whether thymic loss was due to limiting numbers of progenitors reseeding the thymus. This was tested by repopulating lethally irradiated B6 CD45.1 congenic mice with bone marrow from wt (CD45.1) and c-Cbl+/−, A/− or –/– (CD45.2) donors mixed in 1:1 or 4:1 ratios. Mice reconstituted with marrow from single donors confirmed that >98% repopulation of the thymus with donor progenitors occurs by 3 weeks post-transfer. At this time, thymi of all reconstituted mice were of a similar size (Figure 3A); however, by 4 weeks, the mouse receiving A/− marrow alone had 59 and 80% fewer thymocytes than recipients of wt or c-Cbl–/– marrow, respectively (data not shown). By 5 weeks, thymic deletion was nearly complete, with the A/− recipient having only 4 × 106 thymocytes in contrast to 268 × 106 and 334 × 106 thymocytes in recipients of wt and c-Cbl–/– marrow, respectively (Figure 3A). Figure 3.Thymocyte loss is not due to limiting numbers of progenitors. (A) Thymi from irradiated mice 3 and 5 weeks after bone marrow transfer showing thymic loss in irradiated mice receiving c-Cbl A/− bone marrow, either alone or as a mix with wt marrow. (B) Flow cytometric profiles of CD4+ CD8+ DP thymocytes from lethally irradiated mice 3, 4 and 5 weeks after mixed bone marrow reconstitution. The contribution to DP thymocytes from wt donor marrow was determined by CD45.1 staining and the contribution from c-Cbl+/−, A/− or –/– marrow by CD45.2 staining. Similar proportions of donor contribution were observed in DN and SP populations. (C) Thymi from irradiated B6.CD45.1 or GK/2.43 transgenic mice 3 and 5 weeks following bone marrow reconstitution show that equivalent thymic loss in A/− bone marrow recipients occurs in the presence (B6.CD45.1) or absence (GK/2.43) of peripheral T cells. The number of thymocytes (× 106) isolated from each thymus are shown. (D) Flow cytometric profiles of cell surface TCR on spleen and mesenteric lymph node cells showing the absence of peripheral T cells in GK/2.43 reconstituted mice 5 weeks after bone marrow transfer. Download figure Download PowerPoint In mixed bone marrow experiments, the relative contribution of each donor reconstituting the DP thymocyte population was determined by anti-CD45.1 (+/+) and anti-CD45.2 (A/− or +/−) staining. From such experiments, it was clear that A/− thymic progenitors were not limiting and indeed could repopulate the thymus with greater efficiency than either c-Cbl +/+, +/− or –/– marrow (Figure 3B and data not shown). A time-course analysis showed that at 3 weeks after transfer, equivalent contributions were evident among all groups receiving the 1:1 mixes (Figure 3B, first three panels, top row). Remarkably, by 4 weeks, 99% of thymocytes in the 1:1 mix of +/+ and A/− marrow originated from the A/− donor (Figure 3B), although the thymus had not yet diminished in size compared to the recipient of the +/+:+/− mix (188 and 194 × 106 thymocytes, respectively). Thymic depletion became apparent at 5 weeks, indicating that the A/− contribution had completely overwhelmed the +/+ contribution to the extent that the +/+ thymocytes did not have an opportunity to rescue the thymus (Figure 3A, lower middle panel). The competitive advantage of A/− marrow is shown even more clearly when the repopulating mix was biased 4–1 in favor of +/+ (CD45.1) marrow (last two columns of Figure 3B). This slowed but did not prevent the dominance of A/− thymocytes, with a 95% contribution from the c-Cbl A/− donor detected after 5 weeks (lower right panels of Figure 3B) at which time the thymus was reduced 50% in size compared to the 4:1 +/+:+/− control. Thus, even when diluted four-fold, the A/− thymocytes were able to outcompete +/+ thymocytes and prevent thymic rescue. These results demonstrate that thymic progenitors are not limiting in the c-Cbl A/− mouse and that thymic deletion is due to an inherent perturbation of c-Cbl A/− thymocytes, and not from an altered stroma. The marked repopulating bias by A/− marrow may be a property of multipotential progenitors since similar increases in A/− derived cells were seen in the B lymphoid and myeloid lineages (Supplementary Figure 2 and data not shown). The prominence of A/− thymocytes does not appear to be due to a growth advantage, as all three genotypes showed equivalent numbers of BrdU-positive thymocytes after injection with APC-labeled BrdU (data not shown). Similarly, cell cycle analysis of wt, c-Cbl A/− and –/– thymocytes revealed similar proportions in S and G2/M phases (data not shown). Thymic loss is not due to peripheral T-cell activation Peripheral T-cell activation can cause nonspecific thymocyte death by eliciting a ‘cytokine storm’ (Martin and Bevan, 1997; Brewer et al, 2002; Zhan et al, 2003). To determine if this is the cause of thymic loss in the c-Cbl A/− mouse, we transferred c-Cbl A/− bone marrow into lethally irradiated GK/2.43 mice, which lack peripheral T cells. GK/2.43 mice are doubly transgenic for anti-CD4 (GK1.5) and anti-CD8 (2.43) antibodies that deplete CD4+ and CD8+ T cells in the periphery yet do not affect thymocyte development (Zhan et al, 2000b, 2003; Y Zhan and AM Lew, unpublished). Analysis of mice 3, 4 and 5 weeks after transfer showed that thymic deletion progressed with equivalent kinetics and severity in recipient GK/2.43 mice as that of control B6.CD45.1 mice that received A/− marrow (Figure 3C). Importantly, the GK/2.43 recipient mice lacked splenic or lymph node T cells, whereas T cells were evident in the B6.CD45.1 recipients (Figure 3D). Thymocytes from the C379A mouse are susceptible to anti-CD3-induced death Since thymic deletion cannot be explained by a developmental block, a lack of progenitors or peripheral T-cell activation, we investigated the possibility that RING finger mutant thymocytes are more susceptible to CD3-directed death signals. Induction of thymocyte apoptosis in vitro requires triggering of both CD3 and CD28 receptors, and this response is confined to the DP population and is not dependent on Fas or TNF receptor interactions (Punt et al, 1997). Consistent with this, thymocytes from all three genotypes exhibited cell death when exposed to anti-CD3+CD28 antibodies (Figure 4A). However, c-Cbl A/− thymocytes cultured with anti-CD3 also invariably showed marked induction of cell death compared to those from c-Cbl+/− littermates, which did not die in response to this level of in vitro stimulation (Figure 4A, data from five experiments). The c-Cbl KO thymocytes were also susceptible to anti-CD3-induced cell death but at approximately half the level of that observed for c-Cbl A/− thymocytes. Thus, a signaling response through CD3 alone in c-Cbl RING finger mutant thymocytes is of sufficient intensity to induce cell death. The uncoupled requirement for coreceptor signals in thymocytes and T cells is a common theme in Cbl mutant mice and these findings provide another example where a Cbl mutation promotes greater responsiveness to a suboptimal signal. Figure 4.A Bcl-2 transgene rescues the c-Cbl(C379A) thymus. (A) c-Cbl(C379A) thymocytes show enhanced susceptibility to in vitro anti-CD3-mediated cell death. Bar graphs represents the mean percentages (±SEM) from five experiments of PI-positive thymocytes above that of unstimulated controls following 24 h culture with plate-bound anti-CD3 or anti-CD3+CD28. c-Cbl+/− (white bars), A/− (black) and –/– (hatched). Statistically significant differences as measured by unpaired t-test are indicated: **P<0.001 and ***P<0.0001. The extent of cell death in unstimulated thymocyte cultures from wt, c-Cbl A/− and c-Cbl–/– mice was not significantly different. (B) The Bcl-2 transgene rescues thymic loss and anti-CD3 killing of A/− thymocytes. Four littermates of genotypes c-Cbl+/−, c-Cbl A/−, c-Cbl+/−;Vav-Bcl-2 and c-Cbl A/−;Vav Bcl-2 were examined for thymocyte numbers and in vitro killing by plate-bound anti-CD3. Dead cells were detected by flow cytometry for uptake of PI. (C) Levels of pro- and antiapoptotic proteins are not altered in c-Cbl mutant thymocytes. Thymocyte lysates from 3- and 4-week-old mice were immunoblotted with anti-Bim, Bax, Bcl-2 and Bcl-XL antibodies. The greater intensity in 4 weeks lysates is due to loading differences and not an age-related effect. Download figure Download PowerPoint Thymocyte loss in the C379A mouse is rescued by a Bcl-2 transgene We reasoned that if the thymic loss in C379A mice involved a cell death mechanism, then this phenotype would be abrogated by expression of a prosurvival molecule. Indeed, using Vav promoter-driven Bcl-2 transgenic mice (Ogilvy et al, 1999), we found that Bcl-2 expression in C379A mice can rescue in vivo thymic loss (Figure 4B, upper table) and block anti-CD3 mediated thymocyte death in vitro (Figure 4B, lower panels). In the light of these findings, we examined levels of the antiapoptotic proteins Bcl-2 and Bcl-XL, as well as two proapoptotic proteins of the Bcl-2 family, Bim and Bax (Strasser, 2005). However, analysis of these four family members in c-Cbl +/−, A/− and –/– thymocytes revealed no gross changes in their levels (Figure 4C) although a compromise in their function cannot be ruled out. Equivalent CD3 and TCR levels on c-Cbl C379A and c-Cbl KO DP thymocytes Increased levels of CD3 and TCR on DP thymocytes from c-Cbl–/– mice contribute to enhanced signal strength in these cells (Murphy et al, 1998; Naramura et al, 1998; Thien et al, 1999). Higher levels of these receptors in the C379A mouse could expose cells to stronger signals and possibly lead to a proapoptotic response. However, remarkably equivalent increases were seen in CD3 and TCR levels on c-Cbl–/– and A/− DP thymocytes compared to wt controls (Figure 5A). This clearly demonstrates that the increased levels of these receptors on c-Cbl–/– DP thymocytes are specifically caused by a loss of RING finger function. This finding also indicates that thymic deletion in the RING finger mutant mouse cannot be explained by antigen receptor levels on DP thymocytes exceeding those of the c-Cbl–/– mouse. Furthermore, repeated analyses showed no difference between both mutant thymocytes in the kinetics of CD3 or TCR internalization, the loss of these receptors following internalization, or TCR recycling (Supplementary Figure 3 and data not shown). Therefore, the opposing thymic outcomes in these two mutant mice cannot be attributed to differences in ligand-induced internalization or processing of the receptor that might affect the duration of signaling. Figure 5.Increased expression of surface TCR, CD3, CD5 and CD69 on DP thymocytes from c-Cbl mutant mice. DP thymocytes from age-matched sets of mice at 10 days, 3 weeks or 5 weeks were analyzed by flow cytometry for cell surface expression of TCR, CD3, CD5 and CD69. c-Cbl+/− (shaded histogram), A/− (bold) and –/– (dashed). Download figure Download PowerPoint CD5 and CD69 expression is greatly increased in RING finger mutant mice CD5 expression on DP thymocytes parallels the affinity of the positively selecting TCR–MHC–ligand interaction, suggesting that its expression fine-tunes the strength of the TCR signaling response (Tarakhovsky et al, 1995; Azzam et al, 1998). CD69 is another marker of thymocyte activation and its upregulation occurs during both positive and negative selection (Bendelac et al, 1992; Kishimoto and Sprent, 1997). Consistent with this, enhanced signaling from the TCR is accompanied by increased surface expression of CD5 and CD69 on DP thymocytes from the c-Cbl KO mouse (Naramura et al, 1998; Thien et al, 2003). Remarkably, levels of CD5 and CD69 are even more elevated in the RING finger mutant mouse (Figure 5). This increase becomes more marked with age (compare 5 weeks with 10 days), suggesting that the majority of the DP cells remaining in the diminishing thymus have encountered very strong TCR-directed signals. Indeed, the enhanced upregulation of CD5 and CD69, despite similar levels of TCR and CD3 on c-Cbl A/− and c-Cbl–/– DP thymocytes, indicates that a more potent signal is being transmitted downstream of the TCR/CD3 complex in the A/− thymus. Thymic loss is not linked to enhanced ZAP-70 or ERK activation Consequently, we sought to determine whether there is evidence of differentially enhanced signaling downstream of the antigen receptor that could account for thymic loss in the C379A knockin, but not the c-Cbl KO. Crosslinking of CD3 and CD4 rapidly induces tyrosine phosphorylation of Fyn, Lck, ZAP-70, LAT and SLP-76 and this signal is enhanced in c-Cbl–/– thymocytes compared to the wt (Murphy et al, 1998; Naramura et al, 1998; Thien et al, 1999). Consistent with the effects on receptor levels, we observed that c-Cbl A/− and –/– thymocytes showed equally enhanced protein tyrosine phosphorylation (Figure 6A) and associations between Lck and ZAP-70 (Figure 6B). Lck and Fyn levels were also elevated in thymocytes from both mutant mice (Supplementary Figure 4), providing definitive evidence that these kinases are negatively regulated by the c-Cbl RING finger. Inactivation of the RING finger did not however affect levels of ZAP-70, SLP-76, LAT, Akt, Erk, PLCγ1, p85 or Cbl-b (Figures 6 and 7 and data not shown). Figure 6.Enhanced signaling in c-Cbl mutant mice. (A) Lysates from unstimulated or anti-CD3+CD4-stimulated thymocytes from c-Cbl+/− and A/−" @default.
• W2047709368 created "2016-06-24" @default.
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• W2047709368 date "2005-10-06" @default.
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• W2047709368 title "Loss of c-Cbl RING finger function results in high-intensity TCR signaling and thymic deletion" @default.
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