The Pim-1 kinase stimulates maturation of TCRß-deficient T cell progenitors: implications for the mechanism of Pim-1 action
Isabelle Leduc,
Holger Karsunky1,
Noëlle Mathieu,
Thorsten Schmidt1,
Christophe Verthuy,
Pierre Ferrier and
Tarik Möröy1
Centre d'Immunologie INSERM-CNRS de Marseille-Luminy, Case 906, 13288 Marseille Cedex 9, France
1 Institut für Zellbiologie (Tumorforschung), IFZ, Universitätsklinikum Essen, Virchowstrasse 173, D-45122 Essen, Germany
Correspondence to:
P. Ferrier and T. Möröy
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Abstract
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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 TCRß gene enhancer-deleted mice) in which production of a functionally rearranged TCRß 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 TCRß gene recombination (i.e. allelic exclusion). While overexpression of Pim-1 positively affects cell cycle progression in selected CD4CD8 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 ß-selected pre-T cells.
Keywords: ß selection, cell proliferation, T lymphocytes
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Introduction
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The cellular and molecular events underlying early T cell development in the murine thymus have been dissected by studying subpopulations of CD4CD8 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 TCRß gene recombination and ß 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 TCRß 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 TCRß gene rearrangements. These ß-selected `L' cells (~15% of the CD25+CD44,lo DN population) represent the immediate precursors of the ultimate CD25CD44,lo DN cells which, in turn, rapidly up-regulate CD4 and CD8, and develop into the major TCR
ß+ double-positive (DP) thymocyte population.

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Fig 1. The Eµ pim-1 transgenes induces maturation of DN thymocytes lacking TCRß chains. (A) Schematic representation of thymocyte development. Surface markers that define subpopulations are indicated as well as the cell types where TCRß rearrangement or ß-selection occur. (B) Total number of cells (x106) 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, Eß/ (n = 4); 6, Eµ pim-1 Eß/ (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.
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We have previously noted a strong up-regulation in the expression of the Pim-1 cytoplasmic serine/threonine kinase in ß-selected CD25CD44,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 ß 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- (CD25CD44,lo) T cells that lack TCRß 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.
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Methods
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Mice
Transgenic Eµ pim-1 (4) and p14-TCRß (5) mice, knock-out Rag-1-deficient (Rag-1/) (6) and TCRß gene enhancer deleted Eß/ (7) mice, as well as combinatorial transgenic and knockout Eµ pim-1 Eß/ 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 TCRß gene rearrangements by LRPCR assays, using genomic DNA templates and locus-specific primers (i.e. specific for sequences within the Vß5, Vß11, Vß14 and Cß2 genes, and 5' of the Dß2 segment), was performed as previously described (7). The reverse primer (homologous to a sequence 3' of Jß2.7) used for amplification of fragments encompassing the unrearranged Dß2/Jß2 gene segments and the Dß2Jß2 or VßDJß2 rearrangements was as follows: 5'-TGAGAGCTGTCTCCTACTATCGAT-3'.
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Results
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Pim-1 overexpression rescues DP cell development in mice lacking a functionally rearranged TCRß 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 ß selection, we have introduced the Eµ pim-1 transgene into two different strains of engineered mutant mice that lack functionally rearranged TCRß 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 TCRß gene enhancer (Eß/ mice). These mice exhibit a specific inhibition of TCRß gene recombination and, consequently, express no TCRß 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 Eß/ animals, the Eµ pim-1 transgenic mice, and the combinatorial transgenic Eµ pim-1 Rag-1/ and Eµ pim-1 Eß/ 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 x 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).

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Fig. 2. Pim-1 overexpression correlates with altered frequencies of DNA subpopulations and a higher number of `L' cells in the CD25+CD44,lo DN subset. CD4CD8 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.
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A similar rescue of DP cell development was also observed upon analysis of the Eß-deleted animals. Although the Eß/ 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 Eß/ DP thymocytes develop due to early expression of TCR
chains and yet do not express any TCR on their surface (I.L. and P.F., unpublished data). In the Eµ pim-1 Eß/ 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 Eß/ mouse was found to harbor >85% DP thymocytes). Although we have not verified whether these DP cells, similar to those in the Eß/ mice, have been selected based on in-frame TCR
gene rearrangement (e.g. by PCRRFLP 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 Eß/ animals showed develoment of CD4+ or CD8+ single-positive (SP) cells, indicating that Pim-1 does not operate at the DPSP 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 (810).
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 CD25CD44,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 Eß/ thymocytes as compared to their non-transgenic homologues (from <10 to >20%), although a parallel increase for the CD25CD44,lo DN cells was not observed (Fig. 2E and F
). This finding can be explained assuming that, in the Eß/ and Eµ pim-1 Eß/ 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 ß 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 12 to 100200 x 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 DNDP thymocyte differentiation (810,14; 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, Erk1/2hence 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.

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Fig. 3. 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.
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Pim-1 overexpression does not affect allelic exclusion
All of the major events associated with ß selection, including developmental progression, proliferation and the suppression of TCRß rearrangement [e.g. to mediate allelic exclusion, see (1)], can be induced by the introduction of a TCRß 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 TCRß 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 TCRß rearrangement. We used LRPCR to assess the levels of vßDJß and DßJß rearrangements (e.g. 7) in Eµ pim-1 transgenic mice as opposed to those in control wild type or TCRß transgenic mice. As expected, Dß2Jß2 rearrangements were readily detected at similar frequencies in wild-type, p14TCRß and Eµ pim-1 thymocytes (Fig. 4
, top and left panel). In contrast, Vß5, Vß11 and Vß14Jß2 rearrangements were all severely reduced in the p14-TCRß transgenic T cells (Fig. 4
, right panel). Strikingly, such a diminution in VßDJß 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 TCRß gene rearrangement and, therefore, that Pim-1 generated signals do not mediate allelic exclusion at the TCRß locus.

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Fig. 4. Overexpression of the Pim-1 kinase does not suppress VßDJß rearrangements. PCR amplification autoradiograms of Dß2Jß2 rearrangements, of Vß5, Vß11 and Vß14DJß2 rearrangements, and of a DNA fragment within the Cß2 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-TCRß and Eµ pim-1 single transgenic mice. Lanes 24 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 Dß2Jß2-containing fragment in the germline configuration. The PCR products marked by arrowheads represent TCRß rearrangements. A schematic representation of the murine TCRß locus is shown above the autoradiograms, indicating the relative location of the PCR oligonucleotide primers (horizontal arrows) which have been used.
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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 (13,1821). 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.
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Discussion
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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 TCRß 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 TCRß enhancer element (Eß/). 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 Eß/ thymocyte development (e.g. Fig. 1B and C
), we noted an interesting difference. While the Eµ pim-1 Eß/ animals consistently exhibited a high percentage of DP cells (8590%), 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
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 ß-selected DN thymocytes downstream of the pre-TCR complex (810,14; reviewed in 15), the alternative possibility that Pim-1 acts positively on cell cycle progression, during the DNDP 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 TCRß chain expression does not totally prohibit passage through the ß 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 CD25CD44,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 Eß/ mice, with respect to the relative percentages of rescued DP cells. It is clear (e.g. Fig. 2
) that substantially more `L' and CD25CD44,lo cells are present in Eß/ 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 ß selection-associated events in early developing T cells.
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Note added in proof
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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 TCRß enhancer-deleted mice: Implications for
ßT cell lineage commitment and differentiation. J. Immunol. 165:1364.
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Acknowledgments
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We thank Dr William M. Hempel for comments on the manuscript and Dr H. Pircher for the gift of the p14-TCRß 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'.
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Abbreviations
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Con A concanavalin A |
DN double negative |
DP double positive |
Eß TCRß gene enhancer |
LR-PCR long-range PCR |
PE phycoerythrin |
SP single positive |
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Notes
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The first two authors contributed equally to this work
Transmitting editor: L. Du Pasquier
Received 12 April 2000,
accepted 14 June 2000.
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