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The Pim-1 kinase stimulates maturation of TCRβ-deficient T cell progenitors: implications for the mechanism of Pim-1 action

The Pim-1 kinase stimulates maturation of TCRβ-deficient T cell progenitors: implications for the... Abstract We demonstrate that overexpression of Pim-1, a cytoplasmic serine/threonine kinase of poorly defined function, results in the development of substantial numbers of CD4+CD8+ double-positive thymocytes in two independent knock-out mouse models (i.e. the RAG-1-deficient and TCRβ gene enhancer-deleted mice) in which production of a functionally rearranged TCRβ gene (hence the pre-TCR) is impaired. This activity of Pim-1, however, does not affect signaling through the Ras/Raf/MAP kinase cascade nor signaling which mediates suppression of TCRβ gene recombination (i.e. allelic exclusion). While overexpression of Pim-1 positively affects cell cycle progression in selected CD4–CD8– double-negative precursors, it did not affect expression of components of the cell cycle machinery, with the exception of the G1-specific phosphatase Cdc25A upon antigen receptor stimulation. We propose that Pim-1 acts downstream, or in parallel, to pre-TCR-mediated selection as one factor involved in the proliferative expansion of β-selected pre-T cells. β selection, cell proliferation, T lymphocytes Con A concanavalin A, DN double negative, DP double positive, Eβ TCRβ gene enhancer, LR-PCR long-range PCR, PE phycoerythrin, SP single positive Introduction The cellular and molecular events underlying early T cell development in the murine thymus have been dissected by studying subpopulations of CD4–CD8– double-negative (DN) precursors delineated according to the expression of various cell surface markers, including the CD44 and CD25 molecules (1) (Fig. 1A). Of note, completion of TCRβ gene recombination and β selection take place within the CD25+CD44–,lo DN compartment. As first shown by Hayday and colleagues (2), this subpopulation can be further divided into two distinct subsets of unequal size (schematized in Fig. 1A). One subset is comprised of unselected, small resting cells carrying mostly random TCRβ gene rearrangements. These are called `E' cells and represent ~85% of the CD25+CD44–,lo DN compartment. The second subset contains larger, actively dividing `blastoid like' cells carrying a high proportion of in-frame TCRβ gene rearrangements. These β-selected `L' cells (~15% of the CD25+CD44–,lo DN population) represent the immediate precursors of the ultimate CD25–CD44–,lo DN cells which, in turn, rapidly up-regulate CD4 and CD8, and develop into the major TCRαβ+ double-positive (DP) thymocyte population. We have previously noted a strong up-regulation in the expression of the Pim-1 cytoplasmic serine/threonine kinase in β-selected CD25–CD44–,lo thymocytes (3). In addition, we found that mice carrying a pim-1 transgene under the control of the Ig heavy chain enhancer [Eμ pim-1 mice (4)] contained increased numbers of cycling `L cells' in their thymus. Altogether, these data suggest a possible role for Pim-1 in β selection events. In support of this hypothesis, we now report that overexpression of Pim-1 indeed stimulates maturation of DN pro- (CD25+CD44–,lo) and pre- (CD25–CD44–,lo) T cells that lack TCRβ chain expression. This activity appears to be distinct from those of p56lck and does not affect members of the Ras/Raf/MAP kinase pathway. These results, together with the herein documented effect on the Cdc25A phosphatase, suggest a possible mechanism of action for Pim-1 in regulating cell cycle progression at this critical early T cell developmental checkpoint. Methods Mice Transgenic Eμ pim-1 (4) and p14-TCRβ (5) mice, knock-out Rag-1-deficient (Rag-1–/–) (6) and TCRβ gene enhancer deleted Eβ–/– (7) mice, as well as combinatorial transgenic and knockout Eμ pim-1 Eβ–/– and Eμ pim-1 Rag-1–/– mice, were maintained on a C57BL/6J genetic background and sacrificed for analysis between 6 and 8 weeks of age. Antibodies FITC- and phycoerythrin (PE)-conjugated mAb against CD8 (53-6.7), CD4 (H129.19), CD44 (Pgp-1) and CD25 (7D4), were purchased from PharMingen (San Diego, CA). Antibodies against phospho Erk1/2 were from New England Biolabs (Schwalbach/Taunus, Germany); anti-p27KIP1 (C-19) and Cdc25A (144) were from Santa Cruz Biotechnology (Santa Cruz, CA). Flow cytometry analysis and cell sorting Lymphocyte preparation, cell staining with saturating levels of mAb and purification by cell sorting were carried out according to published protocols (e.g. 3,7). Stimulation and immunoblots Thymocytes from transgenic and control mice were stimulated for 15 min by incubating the cells on 10 μg/ml anti-CD3 (2C11; PharMingen) mAb-coated plates or by treating the cells with concanavalin A (Con A) (2 μg/ml) or the phorbol ester phorbol myristate acetate (1 ng/ml) both purchased from Sigma (Deisenhofen, Germany). Immunoblot analyses were performed as previously described (3). Long range (LR)-PCR assays Analysis of TCRβ gene rearrangements by LR–PCR assays, using genomic DNA templates and locus-specific primers (i.e. specific for sequences within the Vβ5, Vβ11, Vβ14 and Cβ2 genes, and 5′ of the Dβ2 segment), was performed as previously described (7). The reverse primer (homologous to a sequence 3′ of Jβ2.7) used for amplification of fragments encompassing the unrearranged Dβ2/Jβ2 gene segments and the Dβ2–Jβ2 or Vβ–DJβ2 rearrangements was as follows: 5′-TGAGAGCTGTCTCCTACTATCGAT-3′. Results Pim-1 overexpression rescues DP cell development in mice lacking a functionally rearranged TCRβ gene Previous studies have demonstrated that the Eμ pim-1 transgene mediates significant Pim-1 overexpression in lymphoid cells, including thymocytes, resulting presumably in increased Pim-1 kinase activity (4). To investigate for a possible role for Pim-1 in β selection, we have introduced the Eμ pim-1 transgene into two different strains of engineered mutant mice that lack functionally rearranged TCRβ gene expression, due to a V(D)J recombination defect. In addition to Rag-1-deficient (Rag-1–/–) animals (6), we used mice carrying an homozygous deletion of the TCRβ gene enhancer (Eβ–/– mice). These mice exhibit a specific inhibition of TCRβ gene recombination and, consequently, express no TCRβ chains (7) (I. L. and P. F., unpublished results). Figure 1(B and C) shows a comparison of thymic cellularity and thymic cell development (as defined by CD4/CD8 cytofluorometric analysis) between wild-type and single knock-out Rag-1–/– and Eβ–/– animals, the Eμ pim-1 transgenic mice, and the combinatorial transgenic Eμ pim-1 Rag-1–/– and Eμ pim-1 Eβ–/– mice. Compared to the wild-type controls, the Eμ pim-1 mice have a ~2-fold increase in total thymocyte number and a slightly higher percentage of DP cells (Fig. 1B, lanes 1, 2; C, leftmost panels). As expected, the Rag-1–/– mice exhibit a very low number (<2 × 106) of essentially all DN thymocytes which do not develop beyond the CD25+CD44–,lo stage (Fig. 1B, lane 3; C, top and middle panel; see also Fig. 2). In contrast, an approximately up to 10-fold increase in total thymocytes was observed in Eμ pim-1 Rag-1–/– mice, together with the production of DP cells up to a percentage which is similar to that found in wild-type thymuses (Fig. 1B, lane 4; C, bottom and middle panel), demonstrating that expression of the Eμ pim-1 transgene can bypass the Rag developmental block. Intriguingly, this rescue of DP cell development varied among the Eμ pim-1 Rag-1–/– animals (e.g. percentages of DP thymocytes varied from <5 to >85% depending on the individual; data not shown). Significantly, however, Western blot analysis of thymic cell extracts demonstrated a direct correlation between the level of Pim-1 overexpression and percentage of DP cells found in the individual mice (Fig. 1 D). A similar rescue of DP cell development was also observed upon analysis of the Eβ-deleted animals. Although the Eβ–/– mice exhibit reduced thymic cellularity, they still develop a small number of DP cells representing, on average ~50% of total thymic cells (Fig. 1B, lane 5; C, upper right panel). Independent studies have demonstrated that the vast majority Eβ–/– DP thymocytes develop due to early expression of TCRδ chains and yet do not express any TCR on their surface (I.L. and P.F., unpublished data). In the Eμ pim-1 Eβ–/– mice, however, thymocyte numbers were also increased with a concurrent increase in the relative proportion of DP cells (Fig. 1B, lane 6; C, lower right panel; note that, contrary to the Eμ pim-1 Rag-1–/– situation, every single Eμ pim-1 Eβ–/– mouse was found to harbor >85% DP thymocytes). Although we have not verified whether these DP cells, similar to those in the Eβ–/– mice, have been selected based on in-frame TCRδ gene rearrangement (e.g. by PCR–RFLP analyses), our results demonstrate that Pim-1 exerts an effect on the rescue of developmental progression to the DP cell stage in two pre-TCR-deficient mouse models of different origin. Remarkably, neither Eμ pim-1 Rag-1–/– nor Eμ pim-1 Eβ–/– animals showed develoment of CD4+ or CD8+ single-positive (SP) cells, indicating that Pim-1 does not operate at the DP–SP cell stage transition. Of note, while the rescue of DP cell development by Pim-1 is significant, its efficiency in increasing cell number is lower than that of the constitutively active mutant p56lckF505 or the active forms of Ras or Raf (8–10). Pim-1 overexpression promotes `E'–`L' DN cell transition To obtain a more precise picture of the effect of the Eμ pim-1 transgene on early thymocyte development, we analyzed the CD44 versus CD25 profiles of DN thymocytes from the different mouse strains used in this study. In addition, by gating on CD25+CD44–,lo DN cells (gate `R1') and measuring cell counts against forward scatter, we investigated the relative proportion of `L' cells within this compartment. In agreement with our previous results (3), the proportion of CD25+CD44–,lo DN cells was reduced, whereas that of `L' cells was increased, in the Eμ pim-1 thymus compared to the wild-type control (Fig. 2A and B). Strikingly, this effect was amplified in the Rag-1–/– background where the presence of the Eμ pim-1 transgene resulted in dramatically higher percentages of both the CD25–CD44–,lo DN cells and the `L' subset (respectively from 0.2 to >50% and from <4 to >30%, Fig. 2C and D). Finally, the percentage of the `L' subset was also found to be greater for the Eμ pim-1 Eβ–/– thymocytes as compared to their non-transgenic homologues (from <10 to >20%), although a parallel increase for the CD25–CD44–,lo DN cells was not observed (Fig. 2E and F). This finding can be explained assuming that, in the Eβ–/– and Eμ pim-1 Eβ–/– mice, most CD25+CD44–,lo cells directly differentiate to the DP stage, as described in several mouse mutants where thymic cell development is relatively inefficient (11,12). The above results are compatible with the hypothesis that the Eμ pim-1 transgene affects early T cell development by impacting upon β selection-associated processes. While this report was in preparation, Jacobs et al. reported concurrent work also demonstrating that this transgene enables Rag-2-deficient pro-T cells to bypass the pre-TCR controlled checkpoint, yielding from 1–2 to 100–200 × 106 DP thymocytes (13). The variations in the rescued DP cell numbers observed in the latter and this study may be related to differences in the mouse strains and/or age at which animals have been analyzed. As opposed to Jacobs et al. (13), however, we did not observe a strong age dependency in the Pim-1-induced DP cell expansion (data not shown). Pim-1 overexpression does not affect the Ras/Raf/Erk1,2 pathway Similar to these observations with Pim-1, expression of activated p21Ras, active Raf or MAP kinase has been shown to control DN–DP thymocyte differentiation (8–10,14; reviewed in 15). Thus, we wanted to test whether the effect of Pim-1 can be linked to the signaling pathway initiated by Ras. To this end, thymocytes from the Eμ pim-1 transgenics and wild-type controls were stimulated with anti-CD3 mAb, phorbol ester or Con A, as it is known that these stimuli also activate the Ras/Raf signaling cascade and, subsequently, the downstream MAP kinase effectors such as, for example, Erk1/2—hence mimicking pre-TCR signals (16). Extracts from the stimulated thymocytes were then compared for expression of Erk1/2 and its phosphorylated (and thus activated) form by Western blotting. Purified (i.e. sorted) DN cells were also analyzed. These assays, however, did not demonstrate an alteration in Erk1/2 expression or activation by the expression of the pim-1 transgene (Fig. 3A and data not shown). Although it might be argued that activation of Erk by phorbol ester is not absolutely dependent on Ras activation and could be mediated by alternative pathways (e.g. via protein kinase C), the fact that no change in Erk1/2 expression was similarly observed when using ConA or anti-CD3 stimuli is best explained assuming that Pim-1 is unlikely to affect the activity of members of the Ras/Raf/Mek/Erk1,2 signal transduction pathway. Along the same lines, transient co-transfection assays of T (EL-4) and fibroblast (NIH-3T3) cells using a Pim-1-expressing construct and a reporter gene driven by the basal TK promoter and the SRE element which allows transcriptional transactivation upon stimulation of the Ras/Raf/Mek/Erk signaling cascade (e.g. 17) also gave negative results (data not shown) which suggests that the Ras-dependent cascade is not dependent on Pim-1. Taking into account all our data from experiments with three different stimuli and from the findings with the SRE-driven reporter gene construct, we feel confident to conclude that Pim-1 is unlikely to affect the activity of members of the Ras/Raf/Mek/Erk1,2 signal transduction pathway. Pim-1 overexpression does not affect allelic exclusion All of the major events associated with β selection, including developmental progression, proliferation and the suppression of TCRβ rearrangement [e.g. to mediate allelic exclusion, see (1)], can be induced by the introduction of a TCRβ transgene or an allele expressing lckF505 (12). Recent evidence, however, suggests that p56lck promotes thymocyte differentiation and expansion via the Ras/Raf/Map kinase pathway but that TCRβ allelic exclusion is mediated by effector pathways downstream of p56lck that may be independent of this cascade (9,10). To examine whether Pim-1 might be involved with this particular pathway, we tested the effect of Pim-1 on TCRβ rearrangement. We used LR–PCR to assess the levels of vβ–DJβ and Dβ–Jβ rearrangements (e.g. 7) in Eμ pim-1 transgenic mice as opposed to those in control wild type or TCRβ transgenic mice. As expected, Dβ2–Jβ2 rearrangements were readily detected at similar frequencies in wild-type, p14–TCRβ and Eμ pim-1 thymocytes (Fig. 4, top and left panel). In contrast, Vβ5–, Vβ11– and Vβ14–Jβ2 rearrangements were all severely reduced in the p14-TCRβ transgenic T cells (Fig. 4, right panel). Strikingly, such a diminution in Vβ–DJβ rearrangement was not observed using DNA from the Eμ pim-1 thymocytes (Fig. 4, cf. lanes 6 and 7). We conclude that overexpression of the Pim-1 kinase does not extinguish TCRβ gene rearrangement and, therefore, that Pim-1 generated signals do not mediate allelic exclusion at the TCRβ locus. High levels of Pim-1 in DN cells promote cell cycle progression The mechanisms by which Pim-1 acts in early T cells are not known. Several experimental systems demonstrate a protective effect of Pim-1 overexpression on cell death as well as a role in growth factor independence (some involving signaling pathways downstream of cytokine receptors) in different cell types (13,18–21). Pim-1 could also exert a positive effect on cell cycle progression. Indeed, we found that Eμ pim-1 transgenic mice reproducibly contain more cycling CD25+CD44–,lo DN cells than wild type controls (a total of seven transgenics have been tested, Fig. 3B) (also see 3). To gain further insight into the mechanisms underlying enhanced cell cycle progression in Eμ pim-1 DN thymocytes, we have analyzed the expression levels of the cell cycle inhibitor p27KIP as well as several other factors known to interfere with cell cycle control (the G1 cyclins D3, A and E as well as CDK2, CDK6, pRb and p130 have been tested) (for a review, see 22) but did not find any alterations, with the exception of the G1-specific phosphatase Cdc25A (Fig. 3C and data not shown). Interestingly, the expression level of Cdc25A rose dramatically only in Eμ pim-1 thymocytes, but not in those from wild-type controls, when either were stimulated with anti-CD3. This up-regulation of Cdc25A is likely due to stabilization of the protein, rather than transcriptional activation of the cdc25a gene, as the anti-CD3 treatment lasted only 15 min. Therefore, the high level of Pim-1 correlates with increased expression of Cdc25A upon CD3-mediated signaling which may, in turn, be responsible for the enhanced cell cycle progression observed in the CD25+CD44–,lo DN population. Discussion Here, we have shown that the serine threonine kinase Pim-1, whose functional role in cellular processes has so far not been exactly delineated, is able to rescue thymocyte development in two separate models of mutant mice that lack TCRβ chain expression. In our experiments, we have used combinatorial mutant mice that express a pim-1 transgene at high levels in T cells [Eμ pim-1 (4)] and either lack V(D)J recombination activity (Rag-1–/–) or the TCRβ enhancer element (Eβ–/–). In addition, using the Eμ pim-1 transgenics, we have tested the cell cycle distribution of DN, `L'-cell-containing, CD25+CD44–,lo thymocytes and demonstrate higher number/percentage of cells in the S/G2/M phases, strongly suggesting that Pim-1 accelerates passage through the cell cycle. Despite the similar effect of the Eμ pim-1 transgene on Rag-1–/– and Eβ–/– thymocyte development (e.g. Fig. 1B and C), we noted an interesting difference. While the Eμ pim-1 Eβ–/– animals consistently exhibited a high percentage of DP cells (85–90%), values in the Eμ pim-1 Rag-1–/– mice varied noticeably amongst individual animals (from <5 to ~85%). Moreover, in the Eμ pim-1 Rag-1–/– mice, the variations in the efficiency of DP cell rescue were found to correlate with variations in the level of Pim-1 thymic expression as mice which exhibited the highest thymic cellularity and highest percentage of DP cells also had the highest level of thymic Pim-1, whereas Pim-1 was hardly detected using thymocytes from Eμ pim-1 Rag-1–/– animals in which DP cells barely developed (e.g. Fig. 1D). As discussed further below, these differential effects may be related to the different number of `L' cells that incidentally develop in the thymus of the individual mice and have implications for the mechanism by which Pim-1 may function during early T cell differentiation. Recent experiments demonstrate a physical association between Pim-1 and Cdc25A and the activation of Cdc25A through phosphorylation by Pim-1 (23). It is known that Cdc25 phosphatases have important functions in the regulation of cell cycle progression (24,25). These proteins remove the phosphate groups from cyclin-dependent kinases and thereby ensure their activation. A recent study on Cdc25A points out that the activation of Cdc25A occurs in the late G1 phase at about the same time when cyclin E/CDK2 and cyclin A/CDK2 are active, suggesting that Cdc25A is directly responsible for the activation of both kinase complexes (25). Moreover, Cdc25A overexpression was shown to induce CDK2 dephosphorylation and to accelerate G1/S progression. These findings, together with our current data showing Cdc25A overexpression in stimulated pre-T cells in the presence of increased Pim-1 expression, offer a first explanation for the observed positive effect of Pim-1 on cell cycle progression in CD25+CD44–,lo DN pre-T cells. Furthermore, these data support a model in which Pim-1 activity is linked to cell cycle regulatory pathways at the G1/S border in pre-TCR-mediated selection processes. The exact mechanism(s) by which the level of Cdc25A increases in pre-T cells upon anti-CD3 stimulation and Pim-1 overexpression remains unresolved. This may involve, for example, a regulatory effect on the transcriptional rate and/or, as suggested by the previously documented binding of Pim-1 to Cdc25A (23), on protein stabilization. Studies are underway to further clarify the latter issues. A model in which Pim-1 may require pre-TCR signaling (as provided here by anti-CD3 treatment) to exert its positive effect on cell cycle progression is consistent with the finding that low thymic cellularity in CD3 γ knock-out mice cannot be rescued by co-expression of a pim-1 transgene (13). Given our results that Pim-1 is unlikely to affect the activity of members of the Ras/Raf/Map kinase signal transduction pathway (Fig. 3), which are known to control the expansion of β-selected DN thymocytes downstream of the pre-TCR complex (8–10,14; reviewed in 15), the alternative possibility that Pim-1 acts positively on cell cycle progression, during the DN–DP cell transition, by effecting efficient mediation of cytokine signaling has to be considered. Other studies have emphasized the strong potential of Pim-1 in compensating defects in IL-7/IL-3 signaling (13,21). Of note, such a mode of action of Pim-1 in the crosstalk between cytokine and (pre-) TCR signaling does not preclude a role for Pim-1 at an earlier stage of T cell development (e.g. the pro-T cell stage) where the proliferation stimulating/anti apoptosis effect(s) of Pim-1 may also be required (13). Assuming that the lack of TCRβ chain expression does not totally prohibit passage through the β selection checkpoint, possibly due to the stochastic activation of CD3 or downstream signaling molecules, this model of Pim-1-dependent stimulation of cell cycle progression would also offer an attractive explanation for the findings reported here. Indeed, a positive role of Pim-1 in cell cycle progression may explain the variations in DP cell numbers and levels of Pim-1 thymic expression amongst Eμ pim-1 Rag-1–/– individuals (see above). RAG-deficient mice have no DP cells and almost no CD25+CD44–,lo cells that develop beyond the `E' stage. However, Pim-1 overexpression could be responsible for the proliferative expansion and DP maturation of the few (presumably variable) `L' and CD25–CD44–,lo cells that still appear in the Rag–/– background (2). Furthermore, this scenario would explain the difference that we observed between the Eμ pim-1 Rag-1–/– and Eμ pim-1 Eβ–/– mice, with respect to the relative percentages of rescued DP cells. It is clear (e.g. Fig. 2) that substantially more `L' and CD25–CD44–,lo cells are present in Eβ–/– mice which can be targeted and expanded by Pim-1, whereas these populations are barely represented in Rag-1–/– mice which would leave less chance for the effect of Pim-1 to fully unfold. In summary, we provide evidence that expression of Pim-1 in thymocytes lacking a pre-TCR can rescue the developmental block normally associated with pre-TCR loss. This effect is most likely unrelated to the weak oncogenic activity of Pim-1 overexpression, as a low (~6%) percentage of Eμ pim-1 animals develop lymphomas and only after a long (>6 months) latency period (4) (we have analyzed 6- to 8-week-old mice). In addition, we detected no expansion of oligo/monoclonal T cells in these animals (e.g. Fig. 4). Rather, our complementary analyses which support a role for Pim-1 notably in T cell proliferation, together with our previous finding of a steep up-regulation of the endogenous protein correlating with the `E'–`L' cell transition (3), provide a strong argument that Pim-1 is an integral molecule for β selection-associated events in early developing T cells. Note added in proof Data referred to as “(I. L. and P. F., unpublished results)” have now been published: Leduc, I., Hempel, W. M., Mathieu, N., Verthuy, C., Bouvier, G., Watrin, F. and Ferrier, P. 2000. T cell development in TCRβ enhancer-deleted mice: Implications for αβT cell lineage commitment and differentiation. J. Immunol. 165:1364. Fig 1. View largeDownload slide The Eμ pim-1 transgenes induces maturation of DN thymocytes lacking TCRβ chains. (A) Schematic representation of thymocyte development. Surface markers that define subpopulations are indicated as well as the cell types where TCRβ rearrangement or β-selection occur. (B) Total number of cells (×106) per thymus for wild-type C57Bl/6J animals [1, number of analyzed mice (n) = 4], and for several single and combinatorial transgenic and knock-out mice: 2, Eμ pim-1 (n = 3); 3, Rag-1–/– (n = 4); 4, Eμ pim-1 Rag-1–/– (n = 4); 5, Eβ–/– (n = 4); 6, Eμ pim-1 Eβ–/– (n = 4). (C) Flow cytometry analysis of total thymocytes from one animal of each indicated genotype, using FITC- and PE-conjugated mAb against CD8 and CD4 respectively. Quandrant percentages are indicated in the lower right corner. The analyses shown are representative of at least three independent experiments, with the exception of the Eμ pim-1 Rag-1–/– mice which demonstrated substantial heterogeneity in response to the presence of the Eμ pim-1 transgene. (D) Western blot analysis of Pim-1 protein levels in thymocyte extracts from nine independent Eμ pim-1 Rag-1–/– combinatorial mutant mice showing variable degrees of DP thymocyte differentiation. The percentage of DP cells found in the thymus from the individual animals is indicated below the corresponding autoradiogram. Fig 1. View largeDownload slide The Eμ pim-1 transgenes induces maturation of DN thymocytes lacking TCRβ chains. (A) Schematic representation of thymocyte development. Surface markers that define subpopulations are indicated as well as the cell types where TCRβ rearrangement or β-selection occur. (B) Total number of cells (×106) per thymus for wild-type C57Bl/6J animals [1, number of analyzed mice (n) = 4], and for several single and combinatorial transgenic and knock-out mice: 2, Eμ pim-1 (n = 3); 3, Rag-1–/– (n = 4); 4, Eμ pim-1 Rag-1–/– (n = 4); 5, Eβ–/– (n = 4); 6, Eμ pim-1 Eβ–/– (n = 4). (C) Flow cytometry analysis of total thymocytes from one animal of each indicated genotype, using FITC- and PE-conjugated mAb against CD8 and CD4 respectively. Quandrant percentages are indicated in the lower right corner. The analyses shown are representative of at least three independent experiments, with the exception of the Eμ pim-1 Rag-1–/– mice which demonstrated substantial heterogeneity in response to the presence of the Eμ pim-1 transgene. (D) Western blot analysis of Pim-1 protein levels in thymocyte extracts from nine independent Eμ pim-1 Rag-1–/– combinatorial mutant mice showing variable degrees of DP thymocyte differentiation. The percentage of DP cells found in the thymus from the individual animals is indicated below the corresponding autoradiogram. Fig. 2. View largeDownload slide Pim-1 overexpression correlates with altered frequencies of DNA subpopulations and a higher number of `L' cells in the CD25+CD44–,lo DN subset. CD4–CD8– DN thymic cell populations from 6- to 8-week-old wild-type, or the indicated single and combinatorial transgenic and/or knock-out mice, were analyzed by flow cytometry for the expression of CD44 and CD25, as previously described (3). Quadrant percentages are indicated in the upper right corner. To determine the percentage of `L' cells, CD25+CD44–,lo DN thymocytes were gated (gate `R1') and analyzed for cell size by forward angle light scattering. The percentage of `L' cells within each CD25+CD44–,lo DN cell population is indicated. The boundary between `E' and `L' cells was set as described (2,3) and by comparing the CD25+CD44–,lo DN cells to the same subset from Rag-1–/– mice that have only the `E' subset. The analyses shown are representative of at least three independent experiments. Fig. 2. View largeDownload slide Pim-1 overexpression correlates with altered frequencies of DNA subpopulations and a higher number of `L' cells in the CD25+CD44–,lo DN subset. CD4–CD8– DN thymic cell populations from 6- to 8-week-old wild-type, or the indicated single and combinatorial transgenic and/or knock-out mice, were analyzed by flow cytometry for the expression of CD44 and CD25, as previously described (3). Quadrant percentages are indicated in the upper right corner. To determine the percentage of `L' cells, CD25+CD44–,lo DN thymocytes were gated (gate `R1') and analyzed for cell size by forward angle light scattering. The percentage of `L' cells within each CD25+CD44–,lo DN cell population is indicated. The boundary between `E' and `L' cells was set as described (2,3) and by comparing the CD25+CD44–,lo DN cells to the same subset from Rag-1–/– mice that have only the `E' subset. The analyses shown are representative of at least three independent experiments. Fig. 3. View largeDownload slide Pim-1 effects on cell cycle progression in developing thymocytes. (A) Immunoblotting analysis of total and phosphorylated Erk1/2 in protein extracts prepared from unstimulated thymocytes (lanes 1 and 4), thymocytes stimulated by anti-CD3 (lanes 2 and 5) or from purified DN thymocytes (lanes 3 and 6) of the indicated wild-type or Eμ pim-1 transgenic animals. Equal loading of the gels was controlled by staining the filter with Ponceau S. (B) CD25+CD44–,lo (DN/CD25+CD44–) cells were purified by cell sorting using thymocytes from wild-type transgenic Eμ pim-1 mice and were stained with propidium iodide. The relative percentages of cells in the S/G2/M phases are indicated. The histogram shown is representative of data obtained in independent cell cycle analyses of sorted cells from seven individual Eμ pim-1 animals. (C) Immunoblotting analysis of p27KIP and Cdc25A, as outlined in the legend of part (A). Asterisks indicate non-specific signals that appear when using the anti-Cdc25A antibody. The Western blot analyses were repeated twice, each time using extracts from different individuals, with consistent results. Fig. 3. View largeDownload slide Pim-1 effects on cell cycle progression in developing thymocytes. (A) Immunoblotting analysis of total and phosphorylated Erk1/2 in protein extracts prepared from unstimulated thymocytes (lanes 1 and 4), thymocytes stimulated by anti-CD3 (lanes 2 and 5) or from purified DN thymocytes (lanes 3 and 6) of the indicated wild-type or Eμ pim-1 transgenic animals. Equal loading of the gels was controlled by staining the filter with Ponceau S. (B) CD25+CD44–,lo (DN/CD25+CD44–) cells were purified by cell sorting using thymocytes from wild-type transgenic Eμ pim-1 mice and were stained with propidium iodide. The relative percentages of cells in the S/G2/M phases are indicated. The histogram shown is representative of data obtained in independent cell cycle analyses of sorted cells from seven individual Eμ pim-1 animals. (C) Immunoblotting analysis of p27KIP and Cdc25A, as outlined in the legend of part (A). Asterisks indicate non-specific signals that appear when using the anti-Cdc25A antibody. The Western blot analyses were repeated twice, each time using extracts from different individuals, with consistent results. Fig. 4. View largeDownload slide Overexpression of the Pim-1 kinase does not suppress Vβ–DJβ rearrangements. PCR amplification autoradiograms of Dβ2–Jβ2 rearrangements, of Vβ5–, Vβ11– and Vβ14–DJβ2 rearrangements, and of a DNA fragment within the Cβ2 constant region gene (shown here to control for DNA loading) from total thymocytes (Th) and kidney (Kd) of a wild-type mouse and from total thymocytes of the p14-TCRβ and Eμ pim-1 single transgenic mice. Lanes 2–4 in each panel contain thymus DNA which has been diluted with kidney DNA, as indicated. Product specificity was confirmed by hybridization with an oligonucleotide probe internal to the amplified fragment. The arrow to the upper left panel indicates Dβ2–Jβ2-containing fragment in the germline configuration. The PCR products marked by arrowheads represent TCRβ rearrangements. A schematic representation of the murine TCRβ locus is shown above the autoradiograms, indicating the relative location of the PCR oligonucleotide primers (horizontal arrows) which have been used. Fig. 4. View largeDownload slide Overexpression of the Pim-1 kinase does not suppress Vβ–DJβ rearrangements. PCR amplification autoradiograms of Dβ2–Jβ2 rearrangements, of Vβ5–, Vβ11– and Vβ14–DJβ2 rearrangements, and of a DNA fragment within the Cβ2 constant region gene (shown here to control for DNA loading) from total thymocytes (Th) and kidney (Kd) of a wild-type mouse and from total thymocytes of the p14-TCRβ and Eμ pim-1 single transgenic mice. Lanes 2–4 in each panel contain thymus DNA which has been diluted with kidney DNA, as indicated. Product specificity was confirmed by hybridization with an oligonucleotide probe internal to the amplified fragment. The arrow to the upper left panel indicates Dβ2–Jβ2-containing fragment in the germline configuration. The PCR products marked by arrowheads represent TCRβ rearrangements. A schematic representation of the murine TCRβ locus is shown above the autoradiograms, indicating the relative location of the PCR oligonucleotide primers (horizontal arrows) which have been used. The first two authors contributed equally to this work Transmitting editor: L. Du Pasquier We thank Dr William M. Hempel for comments on the manuscript and Dr H. Pircher for the gift of the p14-TCRβ transgenic mice. This work was supported by institutional grants from INSERM and the CNRS, and by specific grants from the `Association pour la Recherche sur le Cancer', the Commission of the European Communities and the `Fondation Princesse Grace de Monaco' (to P. F.), as well as by the Deutsche Forschungsgemeinschaft (grant Mo 435/9-1 and 9-3) and the `Fond der chemischen Industrie' (to T. M.). I. L. was a fellow of the `Ligue Nationale Contre le Cancer'. References 1 Kisielow, P. and von Boehmer, H. 1995. Development and seletion of T cells: facts and puzzles. Adv. Immunol.  58: 87 Google Scholar 2 Hoffman, E. S., Passoni, L., Crompton, T., Leu, T. M. J., Schatz, D. G., Koff, A., Owen, M. J. and Hayday, A. C. 1996. Productive T-cell receptor β-chain gene rearrangement: coincident regulation of cell cycle and clonality during development in vivo. Genes Dev.  10: 948 Google Scholar 3 Schmidt, T., Karsunky, H., Rödel, B., Zevnik, B., Elsässer, P. and Möröy, T. 1998. Evidence implicating gfi-1 and pim-1 in pre-T-cell differentiation steps associated with beta-selection. EMBO J.  17: 5349 Google Scholar 4 van Lohuizen, M., Verbeek, S., Krimpenfort, P., Domen, J., Saris, C., Radaszkiewicz, T. and Berns, A. 1989. Predisposition to lymphomagenesis in pim-1 transgenic mice: cooperation with c-myc and N-myc in murine leukemia virus-induced tumors. Cell  56: 673 Google Scholar 5 Pircher, H., Bürki, K., Lang, R., Hengartner, H. and Zinkernagel, R. M. 1989. Tolerance induction in double specific T-cell receptor transgenic mice varies with antigen. Nature  342: 559 Google Scholar 6 Spanopoulou, E., Roman, C. A., Corcoran, L. M., Schlissel, M. S., Silver, D. P., Nemazee, D., Nussenweig, M. C., Shinton, S. A., Hardy, R. R. and Baltimore, D. 1994. Functional immunoglobulin transgenes guide ordered B-cell differentiation in RAG-1-deficient mice. Genes Dev.  8: 1030 Google Scholar 7 Bouvier, G., Watrin, F., Naspetti, M., Verthuy, C., Naquet, P. and Ferrier, P. 1996. Deletion of the mouse T-cell receptor β gene enhancer blocks αβ T-cell development. Proc. Natl Acad. Sci. USA  93: 7877 Google Scholar 8 Swat, W., Shinkai, Y., Cheng, H. L., Davidson, L. and Alt, F. W. 1996. Activated ras signals differentiation and expansion of CD4+8+ thymocytes. Proc. Natl Acad. Sci. USA  93: 4683 Google Scholar 9 Gärtner, F., Alt, F. W., Munroe, R., Chu, M., Sleckman, B. P., Davidson, L. and Swat, W. 1999. Immature thymocytes employ distinct signaling pathways for allelic exclusion versus differentiation and expansion. Immunity  10: 537 Google Scholar 10 Iritani, B. M., Alberola-Ila, J., Forbush, K. A. and Perlmutter, R. M. 1999. Distinct signals mediate maturation and allelic exclusion in lymphocyte progenitors. Immunity  10: 713 Google Scholar 11 Crompton, T., Moore, M., MacDonald, H. R. and Malissen, B. 1994. Double-negative thymocyte subsets in CD3ς chain-deficient mice: absence of HSA+CD44–CD25– cells. Eur. J. Immunol.  24: 1903 Google Scholar 12 Fehling, H. J. and von Boehmer, H. 1997. Early αβ T cell development in the thymus of normal and genetically altered mice. Curr. Opin. Immunol.  9: 263 Google Scholar 13 Jacobs, H., Krimpenfort, P., Haks, M., Allen, J., Blom, B., Demolliere, C., Kruisbeek, A., Spits, H. and Berns, A. 1999. PIM1 reconstitutes thymus cellularity in interleukin 7- and common γ chain-mutant mice and permits thymocyte maturation in rag- but not CD3γ-deficient mice. J. Exp. Med.  190: 1059 Google Scholar 14 Crompton, T., Gilmore, K. C. and Owen, M. J. 1996. The MAP kinase pathway controls differentiation from double-negative to double-positive thymocyte. Cell  86: 243 Google Scholar 15 Farrar, M. A., Doerfler, P. and Sauer, K. 1998. Signal transduction pathways regulating the development of αβ T cells. Biochim. Biophys. Acta  1377: F35 Google Scholar 16 Michie, A. M. S., Trop, S., Wiest, D. L. and Zuniga-Pflucker, J. C. 1999. Extracellular signal-regulated kinase (ERK) activation by the pre-T cell receptor in developing thymocytes in vivo. J. Exp. Med.  190: 1647 Google Scholar 17 Janknecht, R. 1996. Analysis of the ERK-stimulated ETS transcription factor ER81. Mol. Cell. Biol.  16: 1550 Google Scholar 18 Möröy, T., Grzeschiczek, A., Petzold, S. and Hartmann, K. U. 1993. Expression of a Pim-1 transgene accelerates lymphoproliferation and inhibits apoptosis in lpr/lpr mice. Proc. Natl Acad. Sci. USA  90: 10734 Google Scholar 19 Krumenacker, J. S., Buckley, D. J., Leff, M. A., McCormick, J. T., de Jong, G., Gout, P. W., Reed, P. W., Miyashita, T., Magnuson, N. S. and Buckley, A. R. 1998. Prolactin-regulated apoptosis of Nb2 lymphoma cells: pim-1, bcl-2, and bax expression. Endocrine  9: 163 Google Scholar 20 Lilly, M., Sandholm, J., Cooper, J. J., Koskinen, P. J. and Kraft, A. 1999. The PIM-1 serine kinase prolongs survival and inhibits apoptosis-related mitochondrial dysfunction in part through a bcl-2-dependent pathway. Oncogene  18: 4022 Google Scholar 21 Shirogane, T., Fukada, T., Muller, J. M. M., Shima, D. T., Hibi, M. and Hirano, T. 1999. Synergistic roles for Pim-1 and c-Myc in STAT3-mediated cell cycle progression and antiapoptosis. Immunity  11: 709 Google Scholar 22 Zhang, P. 1999. The cell cycle and development: redundant roles of cell cycle regulators. Curr. Opin. Cell Biol.  11: 655 Google Scholar 23 Mochizuki, T., Kitanaka, C., Noguchi, K., Muramatsu, T., Asai, A. and Kuchino, Y. 1999. Physical and functional interactions between Pim-1 kinase and Cdc25A phosphatase. J. Biol. Chem.  274: 18659 Google Scholar 24 Hoffman, I., Draetta, G. and Karsenti, E. 1994. Activation of the phosphatase activity of human cdc25A by a cdk2-cyclin E dependent phosphorylation at the G1/S transition. EMBO J.  13: 4302 Google Scholar 25 Blomberg, I. and Hoffman, I. 1999. Ectopic expression of Cdc25A accelerates the G1/S transition and leads to premature activation of cyclin E- and cyclin A-dependent kinases. Mol. Cel. Biol.  19: 6183 Google Scholar © 2000 Japanese Society for Immunology http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png International Immunology Oxford University Press

The Pim-1 kinase stimulates maturation of TCRβ-deficient T cell progenitors: implications for the mechanism of Pim-1 action

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Oxford University Press
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© 2000 Japanese Society for Immunology
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0953-8178
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1460-2377
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10.1093/intimm/12.10.1389
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Abstract

Abstract We demonstrate that overexpression of Pim-1, a cytoplasmic serine/threonine kinase of poorly defined function, results in the development of substantial numbers of CD4+CD8+ double-positive thymocytes in two independent knock-out mouse models (i.e. the RAG-1-deficient and TCRβ gene enhancer-deleted mice) in which production of a functionally rearranged TCRβ gene (hence the pre-TCR) is impaired. This activity of Pim-1, however, does not affect signaling through the Ras/Raf/MAP kinase cascade nor signaling which mediates suppression of TCRβ gene recombination (i.e. allelic exclusion). While overexpression of Pim-1 positively affects cell cycle progression in selected CD4–CD8– double-negative precursors, it did not affect expression of components of the cell cycle machinery, with the exception of the G1-specific phosphatase Cdc25A upon antigen receptor stimulation. We propose that Pim-1 acts downstream, or in parallel, to pre-TCR-mediated selection as one factor involved in the proliferative expansion of β-selected pre-T cells. β selection, cell proliferation, T lymphocytes Con A concanavalin A, DN double negative, DP double positive, Eβ TCRβ gene enhancer, LR-PCR long-range PCR, PE phycoerythrin, SP single positive Introduction The cellular and molecular events underlying early T cell development in the murine thymus have been dissected by studying subpopulations of CD4–CD8– double-negative (DN) precursors delineated according to the expression of various cell surface markers, including the CD44 and CD25 molecules (1) (Fig. 1A). Of note, completion of TCRβ gene recombination and β selection take place within the CD25+CD44–,lo DN compartment. As first shown by Hayday and colleagues (2), this subpopulation can be further divided into two distinct subsets of unequal size (schematized in Fig. 1A). One subset is comprised of unselected, small resting cells carrying mostly random TCRβ gene rearrangements. These are called `E' cells and represent ~85% of the CD25+CD44–,lo DN compartment. The second subset contains larger, actively dividing `blastoid like' cells carrying a high proportion of in-frame TCRβ gene rearrangements. These β-selected `L' cells (~15% of the CD25+CD44–,lo DN population) represent the immediate precursors of the ultimate CD25–CD44–,lo DN cells which, in turn, rapidly up-regulate CD4 and CD8, and develop into the major TCRαβ+ double-positive (DP) thymocyte population. We have previously noted a strong up-regulation in the expression of the Pim-1 cytoplasmic serine/threonine kinase in β-selected CD25–CD44–,lo thymocytes (3). In addition, we found that mice carrying a pim-1 transgene under the control of the Ig heavy chain enhancer [Eμ pim-1 mice (4)] contained increased numbers of cycling `L cells' in their thymus. Altogether, these data suggest a possible role for Pim-1 in β selection events. In support of this hypothesis, we now report that overexpression of Pim-1 indeed stimulates maturation of DN pro- (CD25+CD44–,lo) and pre- (CD25–CD44–,lo) T cells that lack TCRβ chain expression. This activity appears to be distinct from those of p56lck and does not affect members of the Ras/Raf/MAP kinase pathway. These results, together with the herein documented effect on the Cdc25A phosphatase, suggest a possible mechanism of action for Pim-1 in regulating cell cycle progression at this critical early T cell developmental checkpoint. Methods Mice Transgenic Eμ pim-1 (4) and p14-TCRβ (5) mice, knock-out Rag-1-deficient (Rag-1–/–) (6) and TCRβ gene enhancer deleted Eβ–/– (7) mice, as well as combinatorial transgenic and knockout Eμ pim-1 Eβ–/– and Eμ pim-1 Rag-1–/– mice, were maintained on a C57BL/6J genetic background and sacrificed for analysis between 6 and 8 weeks of age. Antibodies FITC- and phycoerythrin (PE)-conjugated mAb against CD8 (53-6.7), CD4 (H129.19), CD44 (Pgp-1) and CD25 (7D4), were purchased from PharMingen (San Diego, CA). Antibodies against phospho Erk1/2 were from New England Biolabs (Schwalbach/Taunus, Germany); anti-p27KIP1 (C-19) and Cdc25A (144) were from Santa Cruz Biotechnology (Santa Cruz, CA). Flow cytometry analysis and cell sorting Lymphocyte preparation, cell staining with saturating levels of mAb and purification by cell sorting were carried out according to published protocols (e.g. 3,7). Stimulation and immunoblots Thymocytes from transgenic and control mice were stimulated for 15 min by incubating the cells on 10 μg/ml anti-CD3 (2C11; PharMingen) mAb-coated plates or by treating the cells with concanavalin A (Con A) (2 μg/ml) or the phorbol ester phorbol myristate acetate (1 ng/ml) both purchased from Sigma (Deisenhofen, Germany). Immunoblot analyses were performed as previously described (3). Long range (LR)-PCR assays Analysis of TCRβ gene rearrangements by LR–PCR assays, using genomic DNA templates and locus-specific primers (i.e. specific for sequences within the Vβ5, Vβ11, Vβ14 and Cβ2 genes, and 5′ of the Dβ2 segment), was performed as previously described (7). The reverse primer (homologous to a sequence 3′ of Jβ2.7) used for amplification of fragments encompassing the unrearranged Dβ2/Jβ2 gene segments and the Dβ2–Jβ2 or Vβ–DJβ2 rearrangements was as follows: 5′-TGAGAGCTGTCTCCTACTATCGAT-3′. Results Pim-1 overexpression rescues DP cell development in mice lacking a functionally rearranged TCRβ gene Previous studies have demonstrated that the Eμ pim-1 transgene mediates significant Pim-1 overexpression in lymphoid cells, including thymocytes, resulting presumably in increased Pim-1 kinase activity (4). To investigate for a possible role for Pim-1 in β selection, we have introduced the Eμ pim-1 transgene into two different strains of engineered mutant mice that lack functionally rearranged TCRβ gene expression, due to a V(D)J recombination defect. In addition to Rag-1-deficient (Rag-1–/–) animals (6), we used mice carrying an homozygous deletion of the TCRβ gene enhancer (Eβ–/– mice). These mice exhibit a specific inhibition of TCRβ gene recombination and, consequently, express no TCRβ chains (7) (I. L. and P. F., unpublished results). Figure 1(B and C) shows a comparison of thymic cellularity and thymic cell development (as defined by CD4/CD8 cytofluorometric analysis) between wild-type and single knock-out Rag-1–/– and Eβ–/– animals, the Eμ pim-1 transgenic mice, and the combinatorial transgenic Eμ pim-1 Rag-1–/– and Eμ pim-1 Eβ–/– mice. Compared to the wild-type controls, the Eμ pim-1 mice have a ~2-fold increase in total thymocyte number and a slightly higher percentage of DP cells (Fig. 1B, lanes 1, 2; C, leftmost panels). As expected, the Rag-1–/– mice exhibit a very low number (<2 × 106) of essentially all DN thymocytes which do not develop beyond the CD25+CD44–,lo stage (Fig. 1B, lane 3; C, top and middle panel; see also Fig. 2). In contrast, an approximately up to 10-fold increase in total thymocytes was observed in Eμ pim-1 Rag-1–/– mice, together with the production of DP cells up to a percentage which is similar to that found in wild-type thymuses (Fig. 1B, lane 4; C, bottom and middle panel), demonstrating that expression of the Eμ pim-1 transgene can bypass the Rag developmental block. Intriguingly, this rescue of DP cell development varied among the Eμ pim-1 Rag-1–/– animals (e.g. percentages of DP thymocytes varied from <5 to >85% depending on the individual; data not shown). Significantly, however, Western blot analysis of thymic cell extracts demonstrated a direct correlation between the level of Pim-1 overexpression and percentage of DP cells found in the individual mice (Fig. 1 D). A similar rescue of DP cell development was also observed upon analysis of the Eβ-deleted animals. Although the Eβ–/– mice exhibit reduced thymic cellularity, they still develop a small number of DP cells representing, on average ~50% of total thymic cells (Fig. 1B, lane 5; C, upper right panel). Independent studies have demonstrated that the vast majority Eβ–/– DP thymocytes develop due to early expression of TCRδ chains and yet do not express any TCR on their surface (I.L. and P.F., unpublished data). In the Eμ pim-1 Eβ–/– mice, however, thymocyte numbers were also increased with a concurrent increase in the relative proportion of DP cells (Fig. 1B, lane 6; C, lower right panel; note that, contrary to the Eμ pim-1 Rag-1–/– situation, every single Eμ pim-1 Eβ–/– mouse was found to harbor >85% DP thymocytes). Although we have not verified whether these DP cells, similar to those in the Eβ–/– mice, have been selected based on in-frame TCRδ gene rearrangement (e.g. by PCR–RFLP analyses), our results demonstrate that Pim-1 exerts an effect on the rescue of developmental progression to the DP cell stage in two pre-TCR-deficient mouse models of different origin. Remarkably, neither Eμ pim-1 Rag-1–/– nor Eμ pim-1 Eβ–/– animals showed develoment of CD4+ or CD8+ single-positive (SP) cells, indicating that Pim-1 does not operate at the DP–SP cell stage transition. Of note, while the rescue of DP cell development by Pim-1 is significant, its efficiency in increasing cell number is lower than that of the constitutively active mutant p56lckF505 or the active forms of Ras or Raf (8–10). Pim-1 overexpression promotes `E'–`L' DN cell transition To obtain a more precise picture of the effect of the Eμ pim-1 transgene on early thymocyte development, we analyzed the CD44 versus CD25 profiles of DN thymocytes from the different mouse strains used in this study. In addition, by gating on CD25+CD44–,lo DN cells (gate `R1') and measuring cell counts against forward scatter, we investigated the relative proportion of `L' cells within this compartment. In agreement with our previous results (3), the proportion of CD25+CD44–,lo DN cells was reduced, whereas that of `L' cells was increased, in the Eμ pim-1 thymus compared to the wild-type control (Fig. 2A and B). Strikingly, this effect was amplified in the Rag-1–/– background where the presence of the Eμ pim-1 transgene resulted in dramatically higher percentages of both the CD25–CD44–,lo DN cells and the `L' subset (respectively from 0.2 to >50% and from <4 to >30%, Fig. 2C and D). Finally, the percentage of the `L' subset was also found to be greater for the Eμ pim-1 Eβ–/– thymocytes as compared to their non-transgenic homologues (from <10 to >20%), although a parallel increase for the CD25–CD44–,lo DN cells was not observed (Fig. 2E and F). This finding can be explained assuming that, in the Eβ–/– and Eμ pim-1 Eβ–/– mice, most CD25+CD44–,lo cells directly differentiate to the DP stage, as described in several mouse mutants where thymic cell development is relatively inefficient (11,12). The above results are compatible with the hypothesis that the Eμ pim-1 transgene affects early T cell development by impacting upon β selection-associated processes. While this report was in preparation, Jacobs et al. reported concurrent work also demonstrating that this transgene enables Rag-2-deficient pro-T cells to bypass the pre-TCR controlled checkpoint, yielding from 1–2 to 100–200 × 106 DP thymocytes (13). The variations in the rescued DP cell numbers observed in the latter and this study may be related to differences in the mouse strains and/or age at which animals have been analyzed. As opposed to Jacobs et al. (13), however, we did not observe a strong age dependency in the Pim-1-induced DP cell expansion (data not shown). Pim-1 overexpression does not affect the Ras/Raf/Erk1,2 pathway Similar to these observations with Pim-1, expression of activated p21Ras, active Raf or MAP kinase has been shown to control DN–DP thymocyte differentiation (8–10,14; reviewed in 15). Thus, we wanted to test whether the effect of Pim-1 can be linked to the signaling pathway initiated by Ras. To this end, thymocytes from the Eμ pim-1 transgenics and wild-type controls were stimulated with anti-CD3 mAb, phorbol ester or Con A, as it is known that these stimuli also activate the Ras/Raf signaling cascade and, subsequently, the downstream MAP kinase effectors such as, for example, Erk1/2—hence mimicking pre-TCR signals (16). Extracts from the stimulated thymocytes were then compared for expression of Erk1/2 and its phosphorylated (and thus activated) form by Western blotting. Purified (i.e. sorted) DN cells were also analyzed. These assays, however, did not demonstrate an alteration in Erk1/2 expression or activation by the expression of the pim-1 transgene (Fig. 3A and data not shown). Although it might be argued that activation of Erk by phorbol ester is not absolutely dependent on Ras activation and could be mediated by alternative pathways (e.g. via protein kinase C), the fact that no change in Erk1/2 expression was similarly observed when using ConA or anti-CD3 stimuli is best explained assuming that Pim-1 is unlikely to affect the activity of members of the Ras/Raf/Mek/Erk1,2 signal transduction pathway. Along the same lines, transient co-transfection assays of T (EL-4) and fibroblast (NIH-3T3) cells using a Pim-1-expressing construct and a reporter gene driven by the basal TK promoter and the SRE element which allows transcriptional transactivation upon stimulation of the Ras/Raf/Mek/Erk signaling cascade (e.g. 17) also gave negative results (data not shown) which suggests that the Ras-dependent cascade is not dependent on Pim-1. Taking into account all our data from experiments with three different stimuli and from the findings with the SRE-driven reporter gene construct, we feel confident to conclude that Pim-1 is unlikely to affect the activity of members of the Ras/Raf/Mek/Erk1,2 signal transduction pathway. Pim-1 overexpression does not affect allelic exclusion All of the major events associated with β selection, including developmental progression, proliferation and the suppression of TCRβ rearrangement [e.g. to mediate allelic exclusion, see (1)], can be induced by the introduction of a TCRβ transgene or an allele expressing lckF505 (12). Recent evidence, however, suggests that p56lck promotes thymocyte differentiation and expansion via the Ras/Raf/Map kinase pathway but that TCRβ allelic exclusion is mediated by effector pathways downstream of p56lck that may be independent of this cascade (9,10). To examine whether Pim-1 might be involved with this particular pathway, we tested the effect of Pim-1 on TCRβ rearrangement. We used LR–PCR to assess the levels of vβ–DJβ and Dβ–Jβ rearrangements (e.g. 7) in Eμ pim-1 transgenic mice as opposed to those in control wild type or TCRβ transgenic mice. As expected, Dβ2–Jβ2 rearrangements were readily detected at similar frequencies in wild-type, p14–TCRβ and Eμ pim-1 thymocytes (Fig. 4, top and left panel). In contrast, Vβ5–, Vβ11– and Vβ14–Jβ2 rearrangements were all severely reduced in the p14-TCRβ transgenic T cells (Fig. 4, right panel). Strikingly, such a diminution in Vβ–DJβ rearrangement was not observed using DNA from the Eμ pim-1 thymocytes (Fig. 4, cf. lanes 6 and 7). We conclude that overexpression of the Pim-1 kinase does not extinguish TCRβ gene rearrangement and, therefore, that Pim-1 generated signals do not mediate allelic exclusion at the TCRβ locus. High levels of Pim-1 in DN cells promote cell cycle progression The mechanisms by which Pim-1 acts in early T cells are not known. Several experimental systems demonstrate a protective effect of Pim-1 overexpression on cell death as well as a role in growth factor independence (some involving signaling pathways downstream of cytokine receptors) in different cell types (13,18–21). Pim-1 could also exert a positive effect on cell cycle progression. Indeed, we found that Eμ pim-1 transgenic mice reproducibly contain more cycling CD25+CD44–,lo DN cells than wild type controls (a total of seven transgenics have been tested, Fig. 3B) (also see 3). To gain further insight into the mechanisms underlying enhanced cell cycle progression in Eμ pim-1 DN thymocytes, we have analyzed the expression levels of the cell cycle inhibitor p27KIP as well as several other factors known to interfere with cell cycle control (the G1 cyclins D3, A and E as well as CDK2, CDK6, pRb and p130 have been tested) (for a review, see 22) but did not find any alterations, with the exception of the G1-specific phosphatase Cdc25A (Fig. 3C and data not shown). Interestingly, the expression level of Cdc25A rose dramatically only in Eμ pim-1 thymocytes, but not in those from wild-type controls, when either were stimulated with anti-CD3. This up-regulation of Cdc25A is likely due to stabilization of the protein, rather than transcriptional activation of the cdc25a gene, as the anti-CD3 treatment lasted only 15 min. Therefore, the high level of Pim-1 correlates with increased expression of Cdc25A upon CD3-mediated signaling which may, in turn, be responsible for the enhanced cell cycle progression observed in the CD25+CD44–,lo DN population. Discussion Here, we have shown that the serine threonine kinase Pim-1, whose functional role in cellular processes has so far not been exactly delineated, is able to rescue thymocyte development in two separate models of mutant mice that lack TCRβ chain expression. In our experiments, we have used combinatorial mutant mice that express a pim-1 transgene at high levels in T cells [Eμ pim-1 (4)] and either lack V(D)J recombination activity (Rag-1–/–) or the TCRβ enhancer element (Eβ–/–). In addition, using the Eμ pim-1 transgenics, we have tested the cell cycle distribution of DN, `L'-cell-containing, CD25+CD44–,lo thymocytes and demonstrate higher number/percentage of cells in the S/G2/M phases, strongly suggesting that Pim-1 accelerates passage through the cell cycle. Despite the similar effect of the Eμ pim-1 transgene on Rag-1–/– and Eβ–/– thymocyte development (e.g. Fig. 1B and C), we noted an interesting difference. While the Eμ pim-1 Eβ–/– animals consistently exhibited a high percentage of DP cells (85–90%), values in the Eμ pim-1 Rag-1–/– mice varied noticeably amongst individual animals (from <5 to ~85%). Moreover, in the Eμ pim-1 Rag-1–/– mice, the variations in the efficiency of DP cell rescue were found to correlate with variations in the level of Pim-1 thymic expression as mice which exhibited the highest thymic cellularity and highest percentage of DP cells also had the highest level of thymic Pim-1, whereas Pim-1 was hardly detected using thymocytes from Eμ pim-1 Rag-1–/– animals in which DP cells barely developed (e.g. Fig. 1D). As discussed further below, these differential effects may be related to the different number of `L' cells that incidentally develop in the thymus of the individual mice and have implications for the mechanism by which Pim-1 may function during early T cell differentiation. Recent experiments demonstrate a physical association between Pim-1 and Cdc25A and the activation of Cdc25A through phosphorylation by Pim-1 (23). It is known that Cdc25 phosphatases have important functions in the regulation of cell cycle progression (24,25). These proteins remove the phosphate groups from cyclin-dependent kinases and thereby ensure their activation. A recent study on Cdc25A points out that the activation of Cdc25A occurs in the late G1 phase at about the same time when cyclin E/CDK2 and cyclin A/CDK2 are active, suggesting that Cdc25A is directly responsible for the activation of both kinase complexes (25). Moreover, Cdc25A overexpression was shown to induce CDK2 dephosphorylation and to accelerate G1/S progression. These findings, together with our current data showing Cdc25A overexpression in stimulated pre-T cells in the presence of increased Pim-1 expression, offer a first explanation for the observed positive effect of Pim-1 on cell cycle progression in CD25+CD44–,lo DN pre-T cells. Furthermore, these data support a model in which Pim-1 activity is linked to cell cycle regulatory pathways at the G1/S border in pre-TCR-mediated selection processes. The exact mechanism(s) by which the level of Cdc25A increases in pre-T cells upon anti-CD3 stimulation and Pim-1 overexpression remains unresolved. This may involve, for example, a regulatory effect on the transcriptional rate and/or, as suggested by the previously documented binding of Pim-1 to Cdc25A (23), on protein stabilization. Studies are underway to further clarify the latter issues. A model in which Pim-1 may require pre-TCR signaling (as provided here by anti-CD3 treatment) to exert its positive effect on cell cycle progression is consistent with the finding that low thymic cellularity in CD3 γ knock-out mice cannot be rescued by co-expression of a pim-1 transgene (13). Given our results that Pim-1 is unlikely to affect the activity of members of the Ras/Raf/Map kinase signal transduction pathway (Fig. 3), which are known to control the expansion of β-selected DN thymocytes downstream of the pre-TCR complex (8–10,14; reviewed in 15), the alternative possibility that Pim-1 acts positively on cell cycle progression, during the DN–DP cell transition, by effecting efficient mediation of cytokine signaling has to be considered. Other studies have emphasized the strong potential of Pim-1 in compensating defects in IL-7/IL-3 signaling (13,21). Of note, such a mode of action of Pim-1 in the crosstalk between cytokine and (pre-) TCR signaling does not preclude a role for Pim-1 at an earlier stage of T cell development (e.g. the pro-T cell stage) where the proliferation stimulating/anti apoptosis effect(s) of Pim-1 may also be required (13). Assuming that the lack of TCRβ chain expression does not totally prohibit passage through the β selection checkpoint, possibly due to the stochastic activation of CD3 or downstream signaling molecules, this model of Pim-1-dependent stimulation of cell cycle progression would also offer an attractive explanation for the findings reported here. Indeed, a positive role of Pim-1 in cell cycle progression may explain the variations in DP cell numbers and levels of Pim-1 thymic expression amongst Eμ pim-1 Rag-1–/– individuals (see above). RAG-deficient mice have no DP cells and almost no CD25+CD44–,lo cells that develop beyond the `E' stage. However, Pim-1 overexpression could be responsible for the proliferative expansion and DP maturation of the few (presumably variable) `L' and CD25–CD44–,lo cells that still appear in the Rag–/– background (2). Furthermore, this scenario would explain the difference that we observed between the Eμ pim-1 Rag-1–/– and Eμ pim-1 Eβ–/– mice, with respect to the relative percentages of rescued DP cells. It is clear (e.g. Fig. 2) that substantially more `L' and CD25–CD44–,lo cells are present in Eβ–/– mice which can be targeted and expanded by Pim-1, whereas these populations are barely represented in Rag-1–/– mice which would leave less chance for the effect of Pim-1 to fully unfold. In summary, we provide evidence that expression of Pim-1 in thymocytes lacking a pre-TCR can rescue the developmental block normally associated with pre-TCR loss. This effect is most likely unrelated to the weak oncogenic activity of Pim-1 overexpression, as a low (~6%) percentage of Eμ pim-1 animals develop lymphomas and only after a long (>6 months) latency period (4) (we have analyzed 6- to 8-week-old mice). In addition, we detected no expansion of oligo/monoclonal T cells in these animals (e.g. Fig. 4). Rather, our complementary analyses which support a role for Pim-1 notably in T cell proliferation, together with our previous finding of a steep up-regulation of the endogenous protein correlating with the `E'–`L' cell transition (3), provide a strong argument that Pim-1 is an integral molecule for β selection-associated events in early developing T cells. Note added in proof Data referred to as “(I. L. and P. F., unpublished results)” have now been published: Leduc, I., Hempel, W. M., Mathieu, N., Verthuy, C., Bouvier, G., Watrin, F. and Ferrier, P. 2000. T cell development in TCRβ enhancer-deleted mice: Implications for αβT cell lineage commitment and differentiation. J. Immunol. 165:1364. Fig 1. View largeDownload slide The Eμ pim-1 transgenes induces maturation of DN thymocytes lacking TCRβ chains. (A) Schematic representation of thymocyte development. Surface markers that define subpopulations are indicated as well as the cell types where TCRβ rearrangement or β-selection occur. (B) Total number of cells (×106) per thymus for wild-type C57Bl/6J animals [1, number of analyzed mice (n) = 4], and for several single and combinatorial transgenic and knock-out mice: 2, Eμ pim-1 (n = 3); 3, Rag-1–/– (n = 4); 4, Eμ pim-1 Rag-1–/– (n = 4); 5, Eβ–/– (n = 4); 6, Eμ pim-1 Eβ–/– (n = 4). (C) Flow cytometry analysis of total thymocytes from one animal of each indicated genotype, using FITC- and PE-conjugated mAb against CD8 and CD4 respectively. Quandrant percentages are indicated in the lower right corner. The analyses shown are representative of at least three independent experiments, with the exception of the Eμ pim-1 Rag-1–/– mice which demonstrated substantial heterogeneity in response to the presence of the Eμ pim-1 transgene. (D) Western blot analysis of Pim-1 protein levels in thymocyte extracts from nine independent Eμ pim-1 Rag-1–/– combinatorial mutant mice showing variable degrees of DP thymocyte differentiation. The percentage of DP cells found in the thymus from the individual animals is indicated below the corresponding autoradiogram. Fig 1. View largeDownload slide The Eμ pim-1 transgenes induces maturation of DN thymocytes lacking TCRβ chains. (A) Schematic representation of thymocyte development. Surface markers that define subpopulations are indicated as well as the cell types where TCRβ rearrangement or β-selection occur. (B) Total number of cells (×106) per thymus for wild-type C57Bl/6J animals [1, number of analyzed mice (n) = 4], and for several single and combinatorial transgenic and knock-out mice: 2, Eμ pim-1 (n = 3); 3, Rag-1–/– (n = 4); 4, Eμ pim-1 Rag-1–/– (n = 4); 5, Eβ–/– (n = 4); 6, Eμ pim-1 Eβ–/– (n = 4). (C) Flow cytometry analysis of total thymocytes from one animal of each indicated genotype, using FITC- and PE-conjugated mAb against CD8 and CD4 respectively. Quandrant percentages are indicated in the lower right corner. The analyses shown are representative of at least three independent experiments, with the exception of the Eμ pim-1 Rag-1–/– mice which demonstrated substantial heterogeneity in response to the presence of the Eμ pim-1 transgene. (D) Western blot analysis of Pim-1 protein levels in thymocyte extracts from nine independent Eμ pim-1 Rag-1–/– combinatorial mutant mice showing variable degrees of DP thymocyte differentiation. The percentage of DP cells found in the thymus from the individual animals is indicated below the corresponding autoradiogram. Fig. 2. View largeDownload slide Pim-1 overexpression correlates with altered frequencies of DNA subpopulations and a higher number of `L' cells in the CD25+CD44–,lo DN subset. CD4–CD8– DN thymic cell populations from 6- to 8-week-old wild-type, or the indicated single and combinatorial transgenic and/or knock-out mice, were analyzed by flow cytometry for the expression of CD44 and CD25, as previously described (3). Quadrant percentages are indicated in the upper right corner. To determine the percentage of `L' cells, CD25+CD44–,lo DN thymocytes were gated (gate `R1') and analyzed for cell size by forward angle light scattering. The percentage of `L' cells within each CD25+CD44–,lo DN cell population is indicated. The boundary between `E' and `L' cells was set as described (2,3) and by comparing the CD25+CD44–,lo DN cells to the same subset from Rag-1–/– mice that have only the `E' subset. The analyses shown are representative of at least three independent experiments. Fig. 2. View largeDownload slide Pim-1 overexpression correlates with altered frequencies of DNA subpopulations and a higher number of `L' cells in the CD25+CD44–,lo DN subset. CD4–CD8– DN thymic cell populations from 6- to 8-week-old wild-type, or the indicated single and combinatorial transgenic and/or knock-out mice, were analyzed by flow cytometry for the expression of CD44 and CD25, as previously described (3). Quadrant percentages are indicated in the upper right corner. To determine the percentage of `L' cells, CD25+CD44–,lo DN thymocytes were gated (gate `R1') and analyzed for cell size by forward angle light scattering. The percentage of `L' cells within each CD25+CD44–,lo DN cell population is indicated. The boundary between `E' and `L' cells was set as described (2,3) and by comparing the CD25+CD44–,lo DN cells to the same subset from Rag-1–/– mice that have only the `E' subset. The analyses shown are representative of at least three independent experiments. Fig. 3. View largeDownload slide Pim-1 effects on cell cycle progression in developing thymocytes. (A) Immunoblotting analysis of total and phosphorylated Erk1/2 in protein extracts prepared from unstimulated thymocytes (lanes 1 and 4), thymocytes stimulated by anti-CD3 (lanes 2 and 5) or from purified DN thymocytes (lanes 3 and 6) of the indicated wild-type or Eμ pim-1 transgenic animals. Equal loading of the gels was controlled by staining the filter with Ponceau S. (B) CD25+CD44–,lo (DN/CD25+CD44–) cells were purified by cell sorting using thymocytes from wild-type transgenic Eμ pim-1 mice and were stained with propidium iodide. The relative percentages of cells in the S/G2/M phases are indicated. The histogram shown is representative of data obtained in independent cell cycle analyses of sorted cells from seven individual Eμ pim-1 animals. (C) Immunoblotting analysis of p27KIP and Cdc25A, as outlined in the legend of part (A). Asterisks indicate non-specific signals that appear when using the anti-Cdc25A antibody. The Western blot analyses were repeated twice, each time using extracts from different individuals, with consistent results. Fig. 3. View largeDownload slide Pim-1 effects on cell cycle progression in developing thymocytes. (A) Immunoblotting analysis of total and phosphorylated Erk1/2 in protein extracts prepared from unstimulated thymocytes (lanes 1 and 4), thymocytes stimulated by anti-CD3 (lanes 2 and 5) or from purified DN thymocytes (lanes 3 and 6) of the indicated wild-type or Eμ pim-1 transgenic animals. Equal loading of the gels was controlled by staining the filter with Ponceau S. (B) CD25+CD44–,lo (DN/CD25+CD44–) cells were purified by cell sorting using thymocytes from wild-type transgenic Eμ pim-1 mice and were stained with propidium iodide. The relative percentages of cells in the S/G2/M phases are indicated. The histogram shown is representative of data obtained in independent cell cycle analyses of sorted cells from seven individual Eμ pim-1 animals. (C) Immunoblotting analysis of p27KIP and Cdc25A, as outlined in the legend of part (A). Asterisks indicate non-specific signals that appear when using the anti-Cdc25A antibody. The Western blot analyses were repeated twice, each time using extracts from different individuals, with consistent results. Fig. 4. View largeDownload slide Overexpression of the Pim-1 kinase does not suppress Vβ–DJβ rearrangements. PCR amplification autoradiograms of Dβ2–Jβ2 rearrangements, of Vβ5–, Vβ11– and Vβ14–DJβ2 rearrangements, and of a DNA fragment within the Cβ2 constant region gene (shown here to control for DNA loading) from total thymocytes (Th) and kidney (Kd) of a wild-type mouse and from total thymocytes of the p14-TCRβ and Eμ pim-1 single transgenic mice. Lanes 2–4 in each panel contain thymus DNA which has been diluted with kidney DNA, as indicated. Product specificity was confirmed by hybridization with an oligonucleotide probe internal to the amplified fragment. The arrow to the upper left panel indicates Dβ2–Jβ2-containing fragment in the germline configuration. The PCR products marked by arrowheads represent TCRβ rearrangements. A schematic representation of the murine TCRβ locus is shown above the autoradiograms, indicating the relative location of the PCR oligonucleotide primers (horizontal arrows) which have been used. Fig. 4. View largeDownload slide Overexpression of the Pim-1 kinase does not suppress Vβ–DJβ rearrangements. PCR amplification autoradiograms of Dβ2–Jβ2 rearrangements, of Vβ5–, Vβ11– and Vβ14–DJβ2 rearrangements, and of a DNA fragment within the Cβ2 constant region gene (shown here to control for DNA loading) from total thymocytes (Th) and kidney (Kd) of a wild-type mouse and from total thymocytes of the p14-TCRβ and Eμ pim-1 single transgenic mice. Lanes 2–4 in each panel contain thymus DNA which has been diluted with kidney DNA, as indicated. Product specificity was confirmed by hybridization with an oligonucleotide probe internal to the amplified fragment. The arrow to the upper left panel indicates Dβ2–Jβ2-containing fragment in the germline configuration. The PCR products marked by arrowheads represent TCRβ rearrangements. A schematic representation of the murine TCRβ locus is shown above the autoradiograms, indicating the relative location of the PCR oligonucleotide primers (horizontal arrows) which have been used. The first two authors contributed equally to this work Transmitting editor: L. Du Pasquier We thank Dr William M. Hempel for comments on the manuscript and Dr H. Pircher for the gift of the p14-TCRβ transgenic mice. This work was supported by institutional grants from INSERM and the CNRS, and by specific grants from the `Association pour la Recherche sur le Cancer', the Commission of the European Communities and the `Fondation Princesse Grace de Monaco' (to P. F.), as well as by the Deutsche Forschungsgemeinschaft (grant Mo 435/9-1 and 9-3) and the `Fond der chemischen Industrie' (to T. M.). I. L. was a fellow of the `Ligue Nationale Contre le Cancer'. References 1 Kisielow, P. and von Boehmer, H. 1995. Development and seletion of T cells: facts and puzzles. Adv. Immunol.  58: 87 Google Scholar 2 Hoffman, E. S., Passoni, L., Crompton, T., Leu, T. M. J., Schatz, D. G., Koff, A., Owen, M. J. and Hayday, A. C. 1996. Productive T-cell receptor β-chain gene rearrangement: coincident regulation of cell cycle and clonality during development in vivo. Genes Dev.  10: 948 Google Scholar 3 Schmidt, T., Karsunky, H., Rödel, B., Zevnik, B., Elsässer, P. and Möröy, T. 1998. Evidence implicating gfi-1 and pim-1 in pre-T-cell differentiation steps associated with beta-selection. EMBO J.  17: 5349 Google Scholar 4 van Lohuizen, M., Verbeek, S., Krimpenfort, P., Domen, J., Saris, C., Radaszkiewicz, T. and Berns, A. 1989. Predisposition to lymphomagenesis in pim-1 transgenic mice: cooperation with c-myc and N-myc in murine leukemia virus-induced tumors. Cell  56: 673 Google Scholar 5 Pircher, H., Bürki, K., Lang, R., Hengartner, H. and Zinkernagel, R. M. 1989. Tolerance induction in double specific T-cell receptor transgenic mice varies with antigen. Nature  342: 559 Google Scholar 6 Spanopoulou, E., Roman, C. A., Corcoran, L. M., Schlissel, M. S., Silver, D. P., Nemazee, D., Nussenweig, M. C., Shinton, S. A., Hardy, R. R. and Baltimore, D. 1994. Functional immunoglobulin transgenes guide ordered B-cell differentiation in RAG-1-deficient mice. Genes Dev.  8: 1030 Google Scholar 7 Bouvier, G., Watrin, F., Naspetti, M., Verthuy, C., Naquet, P. and Ferrier, P. 1996. Deletion of the mouse T-cell receptor β gene enhancer blocks αβ T-cell development. Proc. Natl Acad. Sci. USA  93: 7877 Google Scholar 8 Swat, W., Shinkai, Y., Cheng, H. L., Davidson, L. and Alt, F. W. 1996. Activated ras signals differentiation and expansion of CD4+8+ thymocytes. Proc. Natl Acad. Sci. USA  93: 4683 Google Scholar 9 Gärtner, F., Alt, F. W., Munroe, R., Chu, M., Sleckman, B. P., Davidson, L. and Swat, W. 1999. Immature thymocytes employ distinct signaling pathways for allelic exclusion versus differentiation and expansion. Immunity  10: 537 Google Scholar 10 Iritani, B. M., Alberola-Ila, J., Forbush, K. A. and Perlmutter, R. M. 1999. Distinct signals mediate maturation and allelic exclusion in lymphocyte progenitors. Immunity  10: 713 Google Scholar 11 Crompton, T., Moore, M., MacDonald, H. R. and Malissen, B. 1994. Double-negative thymocyte subsets in CD3ς chain-deficient mice: absence of HSA+CD44–CD25– cells. Eur. J. Immunol.  24: 1903 Google Scholar 12 Fehling, H. J. and von Boehmer, H. 1997. Early αβ T cell development in the thymus of normal and genetically altered mice. Curr. Opin. Immunol.  9: 263 Google Scholar 13 Jacobs, H., Krimpenfort, P., Haks, M., Allen, J., Blom, B., Demolliere, C., Kruisbeek, A., Spits, H. and Berns, A. 1999. PIM1 reconstitutes thymus cellularity in interleukin 7- and common γ chain-mutant mice and permits thymocyte maturation in rag- but not CD3γ-deficient mice. J. Exp. Med.  190: 1059 Google Scholar 14 Crompton, T., Gilmore, K. C. and Owen, M. J. 1996. The MAP kinase pathway controls differentiation from double-negative to double-positive thymocyte. Cell  86: 243 Google Scholar 15 Farrar, M. A., Doerfler, P. and Sauer, K. 1998. Signal transduction pathways regulating the development of αβ T cells. Biochim. Biophys. Acta  1377: F35 Google Scholar 16 Michie, A. M. S., Trop, S., Wiest, D. L. and Zuniga-Pflucker, J. C. 1999. Extracellular signal-regulated kinase (ERK) activation by the pre-T cell receptor in developing thymocytes in vivo. J. Exp. Med.  190: 1647 Google Scholar 17 Janknecht, R. 1996. Analysis of the ERK-stimulated ETS transcription factor ER81. Mol. Cell. Biol.  16: 1550 Google Scholar 18 Möröy, T., Grzeschiczek, A., Petzold, S. and Hartmann, K. U. 1993. Expression of a Pim-1 transgene accelerates lymphoproliferation and inhibits apoptosis in lpr/lpr mice. Proc. Natl Acad. Sci. USA  90: 10734 Google Scholar 19 Krumenacker, J. S., Buckley, D. J., Leff, M. A., McCormick, J. T., de Jong, G., Gout, P. W., Reed, P. W., Miyashita, T., Magnuson, N. S. and Buckley, A. R. 1998. Prolactin-regulated apoptosis of Nb2 lymphoma cells: pim-1, bcl-2, and bax expression. Endocrine  9: 163 Google Scholar 20 Lilly, M., Sandholm, J., Cooper, J. J., Koskinen, P. J. and Kraft, A. 1999. The PIM-1 serine kinase prolongs survival and inhibits apoptosis-related mitochondrial dysfunction in part through a bcl-2-dependent pathway. Oncogene  18: 4022 Google Scholar 21 Shirogane, T., Fukada, T., Muller, J. M. M., Shima, D. T., Hibi, M. and Hirano, T. 1999. Synergistic roles for Pim-1 and c-Myc in STAT3-mediated cell cycle progression and antiapoptosis. Immunity  11: 709 Google Scholar 22 Zhang, P. 1999. The cell cycle and development: redundant roles of cell cycle regulators. Curr. Opin. Cell Biol.  11: 655 Google Scholar 23 Mochizuki, T., Kitanaka, C., Noguchi, K., Muramatsu, T., Asai, A. and Kuchino, Y. 1999. Physical and functional interactions between Pim-1 kinase and Cdc25A phosphatase. J. Biol. Chem.  274: 18659 Google Scholar 24 Hoffman, I., Draetta, G. and Karsenti, E. 1994. Activation of the phosphatase activity of human cdc25A by a cdk2-cyclin E dependent phosphorylation at the G1/S transition. EMBO J.  13: 4302 Google Scholar 25 Blomberg, I. and Hoffman, I. 1999. Ectopic expression of Cdc25A accelerates the G1/S transition and leads to premature activation of cyclin E- and cyclin A-dependent kinases. Mol. Cel. Biol.  19: 6183 Google Scholar © 2000 Japanese Society for Immunology

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International Immunology – Oxford University Press

Published: Oct 1, 2000

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