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The Journal of Immunology, 2007, 179, 103-110
Copyright © 2007 by The American Association of Immunologists, Inc.
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Selective Role of NFATc3 in Positive Selection of Thymocytes1

Kirsten Canté-Barrett*, Monte M. Winslow2,{dagger} and Gerald R. Crabtree3,*,{dagger}

* Departments of Developmental Biology and Pathology, Howard Hughes Medical Institute and {dagger} Program in Immunology, Stanford University, Stanford, CA 94305


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The four Ca2+-dependent NFATc proteins are both signal transducers and transcription factors that reside in the cytoplasm until dephosphorylation by calcineurin. Dephosphorylation exposes nuclear import sequences and sends NFATc proteins into the nucleus where they assemble with nuclear partners into NFAT transcription complexes. Recent genetic studies have indicated that calcineurin-NFAT signaling is a major determinant of vertebrate morphogenesis and development. Mice lacking calcineurin activity show a complete block in positive selection of CD4 and CD8 double-positive thymocytes, yet the role of the NFATc proteins in T cell development has been controversial. In this study, we address the requirement for NFATc3 in T cell development by generating NFATc3 conditional knockout mice. We show that specific deletion of NFATc3 in thymocytes causes a partial block at the double-negative stage 3 and also a partial block in positive selection. Furthermore, the defect does not become more pronounced when NFATc2 is also absent, consistent with the fact that NFATc2-null mice do not have a T cell developmental defect. Expression of a nuclear (and constitutively active) NFATc1 even at subphysiological levels can rescue the transition of double-negative to double-positive thymocytes in RAG-null mice, but is unable to rescue development of CD4 and CD8 single-positive cells. In addition to NFATc3, this suggests a role for NFATc1 in T cell development. Our studies indicate that the signals that direct positive selection likely use both NFATc1 and NFATc3 downstream of calcineurin.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Nuclear factor of activated T cells transcriptional complexes play crucial roles in many developmental processes specific to vertebrates. The Ca2+-dependent phosphatase calcineurin dephosphorylates cytoplasmic NFATc family members, which then translocate into the nucleus and regulate transcription in a complex with nuclear partners (1). Calcineurin-NFAT signaling was initially discovered in T cells (2, 3, 4) but recent genetic studies have demonstrated its crucial role in the development of the immune (5, 6), musculoskeletal (7, 8), cardiovascular (9, 10), neural (11), pulmonary (12), cutaneous (13), and endocrine (14) systems. During development, calcineurin-NFAT signaling often controls the expression of extracellular ligands (cytokines, chemokines, growth factors, wnts, and neurotrophins) or cell-cell interaction proteins (CD40L, FasL, IL-2R, Frizzled, and others). Thus, this pathway in general is thought to choreograph the social lives of cells allowing them to function together during vertebrate organogenesis and morphogenesis.

In the immune system, T cell development begins when a common lymphoid precursor enters the thymus. There it proceeds through at least two discernable CD4 and CD8 double-negative (DN)4 stages before the rag genes are activated and TCR recombination begins, providing commitment to the T cell lineage. After rearrangement and surface expression of TCRbeta in the DN3 stage, thymocytes express the mature {alpha}beta-TCR and become CD4 and CD8 double positive (DP). At the DP stage, positive or negative selection occurs upon TCR triggering, distinguished based on TCR-MHC affinity/avidity and subsequent signal "strength" (15). Negative selection deletes self-reactive thymocytes while positive selection allows progression from the DP to either the CD4 or CD8 single-positive (SP) stage. Ultimately, these SP thymocytes populate peripheral lymphoid organs as mature T cells. The role of calcineurin-NFAT signaling in T cell development, as well as in peripheral T cell activation, has been intensely studied. However, because neither NFATc homologs nor an adaptive immune system exists in invertebrates, it was not until the generation of knockout mice that specific NFATc functions could be dissected. Despite the creation of several NFATc knockout mice, only a minor defect in T cell development has been revealed in the absence of NFATc1 (16, 17). NFATc2 knockout mice (18, 19) have not had detectable defects in T cell development and the reduction of CD4 and CD8 SP thymocytes in the absence of NFATc3 was attributed to increased apoptosis (20). NFATc4 appears to be largely restricted to the CNS and NFATc4-null mice have not shown immune defects. In contrast, specific deletion of the calcineurin regulatory B1 subunit from developing thymocytes results in a complete block in positive selection (5). The apparent discrepancy is likely caused by redundancy among NFATc family members or by some other calcineurin substrate. Therefore, the contribution of NFATc1, NFATc2, and NFATc3 to thymocyte development is still unclear.

To better understand the role of NFATc proteins in thymocyte development, we created an NFATc3 conditional knockout mouse. Our studies suggest that NFATc1 and NFATc3 have redundant roles during pre-TCR-induced differentiation and during positive selection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Generation of NFATc3 conditional knockout mice

A clone from a 129/SvEv embryonic stem (ES) cell genomic library was used to generate the NFATc3 targeting vector. Three loxP sites flank exon 3 of NFATc3, which includes the core residues of the DNA-binding domain. After transfection, ES cells positive for homologous recombination of the targeting vector were selected for injection into C57BL/6 blastocysts or for a secondary transfection with a plasmid expressing Cre recombinase (pMC-CreN; a gift from Dr. F. Alt, Harvard Medical School, Boston, MA) to remove the neomycin-resistance cassette. Chimeric mice were outcrossed and backcrossed to MeuCre40 mice (21), which express Cre recombinase in a mosaic, early embryonic, and ubiquitous manner. This created an allele lacking the neomycin cassette (NFATc3f) and one with complete deletion of exon 3 (NFATc3{Delta}). The null allele creates a frame shift in the transcript resulting in an early stop codon in exon 4. ES cell recombination and germline transmission were analyzed by PCR using the following oligos (arrows in Fig. 1A): 5'-CTGGTGATGGTAGTGTAC-3', 5'-GCAAGAACAGCAAGTGTAC-3', and 5'-TTGACCTCAACATTCTGGAG-3'.


Figure 1
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  		FIGURE 1. Creation of NFATc3 conditional knockout mice. A, Schematic representation of the wild-type NFATc3 allele (NFATc3+), targeting vector, targeted "tri-lox" allele (NFATc3tri-lox) and alleles after Cre-mediated deletion of the neomycin cassette (NFATc3f) and complete deletion of exon 3 (NFATc3{Delta}). Arrows indicate location of primers used for genotyping by PCR. B, Southern blots with the external, 5' probe (left) and internal, 3' probe (right) showing correctly targeted allele. C, PCR analysis on tail DNA from six mice with different genotypes. D, Western blot analysis of NFATc3 protein levels in thymocyte lysates from NFATc3 wild-type, heterozygote, and null mice, as well as from NFATc3f/f mice expressing the lck-Cre transgene. Actin was detected to show equal loading (D and E). E, Western blot analysis of NFATc1, NFATc2, and NFATc3 protein levels in thymocyte lysates from NFATc3f/f mice with and without the lck-Cre transgene. F, RT-PCR analysis using total RNA from mouse thymus and lungs (control) to determine the presence of NFATc1, NFATc2, NFATc3, and NFATc4 mRNA.

 
Mice

MeuCre40 mice (21) were a gift from Dr. M. Holzenberger (Hôpital Saint-Antoine, Paris, France). Lck-Cre mice (22) were a gift from Dr. C. Wilson (University of Washington, Seattle, WA) and Lck-Bcl-xL mice (23) were a gift from Dr. S. Korsmeyer (Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA). RAG2-null mice (Taconic Farms), NFATc2-null mice (19), Bim-null mice (Ref. 24 ; The Jackson Laboratory), and NFATc1nuc mice (8) have been described. All mice were maintained in the animal facility of Stanford University in accordance with federal and institutional guidelines. In all experiments, sets of littermates were used between 4 and 12 wk of age.

Flow cytometry

Thymocytes, splenocytes, and lymph node cells were obtained by disaggregating the whole organ through a 70-µm nylon cell strainer. Abs for surface markers and flow cytometry analysis were obtained from BD Biosciences and staining was performed according to manufacturer’s recommendations. For intracellular staining, cells were fixed at room temperature with 4% formaldehyde in PBS. All subsequent steps were performed on ice and in 0.3% (permeabilization step) or 0.1% saponin (wash and incubation steps), 5% FBS, and 10 mM HEPES (pH 7.4) in PBS. Cells were permeabilized for 30 min followed by an incubation of 1 h with anti-Bim (StressGen Biotechnologies), anti-Bcl-xL (BD Transduction Laboratories), anti-Mcl-1 (Chemicon International), and FITC-conjugated anti-Bcl-2 (eBioscience), respectively, and a secondary incubation (except for Bcl-2) of 30 min with PE-conjugated anti-rabbit IgG (The Jackson Laboratory).

Western blotting

Total cell lysates were prepared on ice in radioimmunoprecipitation assay buffer and loaded on 4–12% Bis-Tris NuPage gels (Invitrogen Life Technologies). The Abs used were: mouse anti-NFATc1 (7A6), rabbit anti-NFATc3 (both generated in our laboratory), mouse anti-NFATc2 (Santa Cruz Biotechnology), and rabbit anti-actin (Sigma-Aldrich). Signal was detected with ECL followed by exposure to autoradiograph film.

RT-PCR

After reverse transcription of total RNA, the following oligos were used for PCR: 5'-TGTGCAGCTACACGGTTACTTGGA-3' and 5'-AGTTATGGCCAGACAGCACCATCT-3' (NFATc1, 482-bp product); 5'-ACAACATGAGAGCCACCATCGACT-3' and 5'-CTGTAGTCTTCTCCATGAACACAACC-3' (NFATc2, 330-bp product); 5'-ACCTCATTGGGAGGCTGAAGGAAA-3' and 5'-TATGCTGGCTGCACTTGACAAAGC-3' (NFATc3, 342-bp product); 5'-GAAGCTACCCTCCGGTACAGAG-3' and 5'-GCTTCATAGCTGGCTGTAGCC-3' (NFATc4, 441-bp product).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Creation of NFATc3 conditional knockout mice

To selectively delete NFATc3 in developing thymocytes, we generated conditional knockout mice in which exon 3 is flanked by loxP sites (Fig. 1A). Exon 3 contains the core DNA-binding domain and was deleted in the germline by Meu-Cre40 and in developing thymocytes by Lck-Cre. Wild-type (NFATc3+), "tri-lox" (NFATc3tri-lox), floxed (NFATc3f), and null (NFATc3{Delta}) alleles are indicated. Correct recombination and genotypes were verified by Southern blot and PCR (Fig. 1, B and C). Total thymocytes from NFATc3+/+, NFATc3{Delta}/+, NFATc3{Delta}/{Delta}, NFATc3f/f, and NFATc3f/f;Lck-Cre+ mice were analyzed for NFATc3 protein expression. First, the genomic deletion in NFATc3{Delta}/{Delta} mice results in complete absence of NFATc3 protein from the thymus and all other organs (Fig. 1D and data not shown). Notably, heterozygous mice have less NFATc3 protein than wild-type mice, suggesting haploid insufficiency. Second, the conditional allele is functional because NFATc3 protein is specifically absent in thymocytes from NFATc3f/f;Lck-Cre+ mice (Fig. 1, D and E). We next analyzed the expression of other NFATc proteins and found that NFATc1 and NFATc2 protein levels are comparable between NFATc3f/f and NFATc3f/f;Lck-Cre+ thymocytes (Fig. 1E). Furthermore, NFATc1 and NFATc2 expression in thymocytes is low relative to NFATc3 expression (25). It is therefore unlikely that increased protein expression of either NFATc1 or NFATc2 compensates in the absence of NFATc3. The fourth NFATc family member, NFATc4, is not expressed in the thymus (Fig. 1F).

Because NFATc3-null mice have been previously generated and analyzed (20), we focused on characterizing NFATc3f/f mice in the presence or absence of the Lck-Cre transgene. It is noteworthy, though, that consistent with the previously reported germline deletion of NFATc3 (20), NFATc3{Delta}/{Delta} mice are born at less than the expected Mendelian ratio of 25% from heterozygous parents. From 15 litters with 78 pups, 12 were NFATc3{Delta}/{Delta} (15%), 43 NFATc3{Delta}/+ (55%), and 23 NFATc3+/+ (30%). Knockout animals are viable, fertile, and have no obvious abnormalities, although their litter size is generally smaller with an average of five pups per litter. In contrast, NFATc3f/f animals are indistinguishable from wild-type and breed well with normal litter sizes.

A partial block in positive selection of NFATc3f/f;Lck-Cre+thymocytes

Initial characterization of the lymphoid organs in NFATc3f/f;Lck-Cre+ mice showed reduced thymus and lymph node cellularity when compared with NFATc3f/f littermates (Fig. 2A). Thymocytes from sets of littermates (NFATc3f/f;Lck-Cre+ and NFATc3f/f control) between 4 and 12 wk of age were counted (n = 21). NFATc3-deficient thymi have approximately half the number of total thymocytes of littermate controls. Similar results were obtained for total cell numbers of both inguinal lymph nodes (n = 11). Several NFATc3{Delta}/{Delta} mice were analyzed and although lymph node numbers were lower, thymocyte numbers were not different when compared with control littermates (Fig. 2A), indicating that acute deletion of NFATc3 from thymocytes has a more severe effect on total thymocyte numbers.


Figure 2
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  		FIGURE 2. A partial block at stage DN3 and in positive selection in NFATc3f/f;Lck-Cre+ mice results in decreased numbers of peripheral T cells. A, Ratios of total thymus and inguinal lymph nodes (LN) cell numbers expressed as an average percentage ± SEM from sets of littermates 4–12 wk of age. Ratios are: NFATc3f/f;Lck-Cre+ to NFATc3f/f thymocytes (n = 21), lymph nodes (n = 11), and NFATc3{Delta}/{Delta} to NFATc3f/f thymocytes (n = 4), lymph nodes (n = 3). B, Flow cytometry analysis of NFATc3f/f and NFATc3f/f;Lck-Cre+ thymocytes (left) and DP thymocytes (right, histograms). C, Flow cytometry analysis of NFATc3f/f and NFATc3f/f;Lck-Cre+ lineage negative (CD4–, CD8–, B220–, Ly-6G–, CD11b–, and TER119–) thymocytes. Development of DN thymocytes progresses as follows: CD44+CD25– (DN1), CD44+CD25+ (DN2), CD44–CD25+ (DN3), CD44–CD25– (DN4) (top plots) and from icTCRbeta–CD25+ to icTCRbeta+CD25+ to icTCRbeta+CD25– expression (bottom plots). D, Flow cytometry analysis of NFATc3f/f and NFATc3f/f;Lck-Cre+ lymph node cells (top) and splenocytes (bottom).

 
When we analyzed thymocyte development in NFATc3f/f;Lck-Cre+ mice, we first noticed a consistent decrease in CD4 SP (6.6 vs 10.6% in control) and CD8 SP (1.08 vs 2.06% in control) thymocyte populations, with the DP subset unchanged (Fig. 2B, left panels). In addition, NFATc3-deficient thymocytes have reduced TCRbetahigh DP cells (3.3 vs 5.1% in control) and CD69high DP cells (5.7 vs 10.9% in control, Fig. 2B, right histograms). These data indicate a T cell-intrinsic defect in positive selection.

A partial block at stage DN3 of NFATc3f/f;Lck-Cre+thymocytes

In addition to the defect in positive selection, we observed higher DN thymocyte percentages in NFATc3f/f;Lck-Cre+ mice (Fig. 2B, left panels). Therefore, we examined DN cells in more detail and found that NFATc3-deficient thymocytes have a partial, but consistent DN development defect (Fig. 2C). DN thymocytes (CD4–, CD8–, B220–, Ly-6G–, CD11b–, TER119–) were analyzed for CD44 and CD25 expression (Fig. 2C, top plots). In addition, these DN thymocytes were fixed and analyzed for intracellular (ic) TCRbeta (Fig. 2C, bottom plots). Development of DN thymocytes progresses as follows: CD44+CD25– (DN1), CD44+CD25+ (DN2), CD44–CD25+ (DN3), CD44–CD25– (DN4) (top plots) and from icTCRbeta–CD25+ to icTCRbeta+CD25+ to icTCRbeta+CD25– expression (bottom plots). CD44 and CD25 analysis reveals an increased percentage of CD44–CD25+ DN thymocytes (termed DN3, lower right quadrant) in NFATc3f/f;Lck-Cre+ mice (63.2 vs 51.8% in control). Correspondingly, the icTCRbeta– CD25+ population is increased (43.3 vs 31.7% for control) and the icTCRbeta+CD25– population decreased (34.4 vs 49.7% for control, Fig. 2C, bottom plots). Because DN thymocytes accumulate at stage DN3 where they receive pre-TCR signals, we conclude that NFATc3-deficient thymocytes do not properly develop after pre-TCR signaling, resulting in a relatively smaller DN4 population and reduced total thymic cellularity.

Consistent with the requirement for NFATc3 in proper DN development and positive selection, NFATc3f/f;Lck-Cre+ mice have lower total cell numbers in lymph nodes (Fig. 2A) and decreased percentages of CD4 and CD8 T cells in lymph nodes (Fig. 2D, top panels) as well as spleen (bottom panels) compared with control mice.

NFATc3f/f;Lck-Cre+DP thymocytes have increased cell death but normal Bim, Bcl-xL, Mcl-1, and Bcl-2 levels

In addition to the partial blocks in DN development and positive selection, reduced viability of NFATc3-deficient thymocytes could also contribute to the reduction of total thymocyte numbers. Therefore, we analyzed thymocyte viability and found that NFATc3f/f;Lck-Cre+ mice have more DP thymocytes undergoing apoptosis in vivo as detected by annexin V (Fig. 3A). Although NFATc3-deficient DP thymocytes have more cell death at time = 0, the rate of viability loss in culture over time is comparable to control cells (Fig. 3B, solid symbols). The fact that the rate of cell death in culture (in the absence of positive selection) is similar between NFATc3-deficient and control thymocytes indicates that NFATc3 plays a role specifically in the survival of thymocytes in vivo, which is not recapitulated in vitro.


Figure 3
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  		FIGURE 3. NFATc3f/f;Lck-Cre+ DP thymocytes have increased cell death but normal Bim, Bcl-xL, Mcl-1, and Bcl-2 levels. A, Annexin V flow cytometry analysis of NFATc3f/f and NFATc3f/f;Lck-Cre+ DP thymocytes. B, Time course of NFATc3f/f (circles) and NFATc3f/f;Lck-Cre+ (triangles) DP thymocyte viability, without (solid symbols) and with (open symbols) anti-CD3 plus anti-CD28 activation-induced cell death to mimic negative selection. Viability is measured by flow cytometry and expressed as the annexin V-negative percentage of DP-gated thymocytes. The experiment was done in quadruplicate and presented as mean ± SD. C, Intracellular staining of Bim, Bcl-xL, Mcl-1, and Bcl-2 and flow cytometry analysis of NFATc3f/f and NFATc3f/f;Lck-Cre+ DP thymocytes. Each plot shows a histogram in light gray as a control for staining.

 
Next, we stimulated thymocytes with plate-bound anti-CD3 and anti-CD28 to mimic negative selection. Both NFATc3-deficient and control DP thymocytes die under negative selection conditions in vitro, as shown by an increase in cell death during the first 4 h in culture (Fig. 3B, open symbols). Furthermore, the level of the proapoptotic protein Bim, which is necessary for negative selection (26), is comparable in NFATc3f/f;Lck-Cre+ and NFATc3f/f control thymocytes (Fig. 3C, top left) and increased but comparable between NFATc3-deficient and control thymocytes under anti-CD3/anti-CD28-stimulated conditions (data not shown). Therefore, we conclude that negative selection is unaffected in the absence of NFATc3. In contrast to what has been reported for mice with germline deletion of NFATc3 (20), we found no change in Bcl-2 levels between NFATc3-deficient and control DP or SP thymocytes (Fig. 3C, bottom right and data not shown). Prosurvival Bcl-2 family members Bcl-xL and Mcl-1 are also unaffected by NFATc3 deficiency (Fig. 3C, top right and bottom left histograms). Thus, we conclude that the increased apoptosis observed in NFATc3-deficient thymocytes is at least in part due to increased "death by neglect" as a result of the defect in positive selection and defect in TCR signaling and that the expression of Bcl-2 family members does not contribute to this cell death phenotype. Together, these data are consistent with the fact that the calcineurin-NFAT-signaling pathway is necessary for positive selection, but is not involved in negative selection (5, 27).

NFATc2 deficiency does not enhance the block in positive selection or the block at DN3

Because the defect in positive selection is complete in the absence of calcineurin B1 (5), but incomplete in the absence of NFATc3 (Fig. 2B), other NFAT family members must also play a role unless calcineurin has other, unknown targets. Two lines of NFATc2-null mice have been generated and it has been shown that NFATc2 is dispensable for thymocyte development (18, 19). However, it is possible that NFATc2 contributes to the signaling required for pre-TCR-induced DN developmental progression and positive selection. We therefore bred NFATc2–/– mice (19) with our NFATc3f/f mice, again in the presence or absence of the Lck-Cre transgene. We did not observe any defect in thymocyte development in NFATc2–/– mice (Fig. 4, A and B, third plot). When both NFATc2 and NFATc3 are absent from thymocytes, the defect in positive selection is not more pronounced than in NFATc3-deficient thymocytes as shown by CD4 and CD8 SP percentages (Fig. 4A, top row, second and fourth plots), TCRbetahigh and CD69high DP percentages (data not shown) and annexin V+ DP percentages (Fig. 4A, bottom row). Although NFATc2 knockout thymocytes do not exhibit initial decreased viability at t = 0, the rate of viability loss in culture of NFATc2-deficient and NFATc2/c3 double-deficient thymocytes is slightly increased compared with NFATc3-deficient or control thymocytes (Fig. 4A, bottom row and data not shown). This indicates that in contrast to NFATc3, NFATc2 contributes to thymocyte survival in culture.


Figure 4
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  		FIGURE 4. NFATc2 deficiency does not enhance the block in positive selection or the block at DN3. A, Flow cytometry analysis of NFATc3f/f and NFATc3f/f;Lck-Cre+ thymocytes in the presence or absence of NFATc2 protein (top row). Bottom row, Annexin V analysis of DP thymocytes from mice with genotypes as in the top row. B, Flow cytometry analysis as in A of lineage-negative (CD4–, CD8–, B220–, Ly-6G–, CD11b–, and TER119–) thymocytes. Development of DN thymocytes progresses as follows: CD44+CD25– (DN1), CD44+CD25+ (DN2), CD44–CD25+ (DN3), CD44–CD25– (DN4).

 
Similar to the positive selection defect, the partial block at DN3 does not become more severe in NFATc2/c3 double-deficient thymocytes (Fig. 4B). However, double-deficient thymocytes have increased DN1 and subsequently more severely decreased DN4 populations that are not observed in either NFATc2- or NFATc3-deficient thymocytes. Therefore, NFATc2 plays a redundant role in DN thymocyte development that only comes to light when both NFATc2 and NFATc3 are absent, but NFATc2 does not contribute to positive selection. This is illustrated not only by the fact that NFATc2-null mice do not have a positive selection defect, but also by the fact that NFATc2 does not compensate for the lack of NFATc3 because the observed defect in positive selection is not more severe in double-deficient thymocytes than in NFATc3-deficient thymocytes.

Active NFATc1 drives the development of DN to DP thymocytes in the absence of pre-TCR signaling

Because positive selection neither requires NFATc2 (Refs. 18 and 19 and Fig. 4) nor NFATc4 due to its absence in thymocytes (Fig. 1F), we next focused on NFATc1. Even though NFATc1–/–Rag2–/– chimeric mice (in which the lymphoid compartment completely consists of NFATc1-deficient cells) have no defect in positive selection, delayed lymphoid reconstitution and consequently lower numbers of thymocytes were observed (16, 17). Therefore, it is still unclear whether DN thymocyte development is affected in NFATc1-deficient thymocytes. In either case, NFATc1 and NFATc3 double-deficient thymocytes could very well cause the complete block in positive selection seen in calcineurin-deficient thymocytes (5). However, NFATc1-null mice are embryonic lethal due to cardiac valve defects (28, 29) and conditional alleles are not yet available. Instead, we made use of mice expressing a constitutively active, nuclear NFATc1 under the control of the tetracycline-responsive operator that were bred to Eµ-tTA mice. The tetracycline-responsive operator-driven expression of active NFATc1 can be suppressed with doxycycline. These mice have been described and are referred to as NFATc1nuc mice (8). Surprisingly, when NFATc1nuc mice were bred onto the Rag2–/– background, in which no TCR rearrangement, no TCR signaling, and consequently no thymocyte development takes place, ~4–30% DP thymocytes appeared (Fig. 5A, middle panel). These DP thymocytes express CD5 (Fig. 5B, middle panel) and CD90, but lack TCRbeta (data not shown). The development of these cells is suppressed when the mice are treated with doxycycline to turn off NFATc1nuc expression (Fig. 5, A and B, third plot). The results suggest that active NFATc1 bypasses the need for pre-TCR signaling and rescues, albeit to a limited extent, the DN to DP thymocyte transition. The fact that nuclear NFATc1 is (at least partly) sufficient for the DN-to-DP transition points to the possibility of NFATc1 responding to (pre-)TCR signaling and contributing to DN development and potentially positive selection in the absence of NFATc3, thus explaining the incomplete thymocyte development block in NFATc3f/f;Lck-Cre+ mice.


Figure 5
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  		FIGURE 5. Active NFATc1 drives the development of DN to DP thymocytes in the absence of pre-TCR signaling. A, Flow cytometry analysis of thymocytes from Rag2–/–, Rag 2–/–; NFATc1nuc, and Rag2–/–; NFATc1nuc doxycycline (DOX) suppressed mice. B, CD5 histograms of DN and DP thymocytes from mice in A.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have examined the role of the transcription factor NFATc3 in thymocyte development by specific deletion of NFATc3 from thymocytes. Given that the phosphatase calcineurin regulates NFATc nuclear translocation and is necessary for positive selection (5), it is likely that one or more NFATc transcription factors regulate thymocyte positive selection. Of the four Ca2+/calcineurin-dependent NFATc family members, three (NFATc1, NFATc2, and NFATc3) are expressed in the immune system (Ref. 30 and Fig. 1F). Because NFATc3-deficient thymocytes only exhibit a partial defect in positive selection, we hypothesized that other NFATc family members play redundant roles and may compensate for the lack of NFATc3. Genetic deletion of NFATc2 does not cause a defect in thymocyte development (18, 19) and does not enhance the defect in NFATc3-deficient thymocytes (Fig. 4). This indicates that among NFAT family members, a variety of functional differences exist, even within the lymphoid lineage. For example, while NFATc2 is not involved in thymocyte development, it controls, together with NFATc1, immune responses in peripheral T and B cells (31). In contrast, NFATc3 is expressed at its highest level in DP thymocytes (20, 25) where it is involved in positive selection (Ref. 20 and Fig. 2). Analysis of previously generated NFATc2/c3 double knockout mice revealed, among many other things, massive lymphadenopathy and splenomegaly (32), not observed in our NFATc3f/f;Lck-Cre+;NFATc2–/– mice. The difference can be explained by the fact that in our mice, NFATc3 is absent only from the T cell lineage and implicates important roles for NFATc3 in other organs as well.

NFATc1-null mice have a modest defect in the transition from DN to DP thymocytes (16, 17) and therefore NFATc1 could be involved in a redundant manner with NFATc3. Our data show that a constitutively active form of NFATc1 can drive DN thymocyte development to the DP stage in the absence of pre-TCR signaling (Fig. 5). Additionally, pre-TCR signaling has been correlated with a rise in cytosolic Ca2+ concentration and NFAT activation (33). This suggests that NFATc1, together with NFATc3, regulates thymocyte development. Thymocyte-specific deletion of both NFATc1 and NFATc3 would decisively answer the question of whether positive selection is blocked as efficiently in NFATc1/c3 double-deficient thymocytes as in calcineurin-deficient thymocytes (5).

Our work shows that freshly isolated NFATc3-deficient thymocytes initially exhibit more apoptosis (Fig. 3, A and B, at 0 h), but we find that the rate of cell death in culture is the same between NFATc3-deficient and control DP thymocytes (Fig. 3B). This discrepancy can at least partly be explained by the fact that in vivo, DP thymocytes with a TCR-signaling defect fail to undergo positive selection and die by "death by neglect." In vitro however, no positive selection takes place and all thymocytes undergo death by neglect at the same rate. If NFATc3 were a general survival factor, the NFATc3-deficient thymocytes would show an increased rate of cell death in culture. Interestingly, NFATc2-deficient thymocytes have a slightly increased rate of cell death in culture, but do not have increased cell death at t = 0 (Fig. 4A and data not shown). These observations indicate that NFATc2 plays a role in thymocyte survival in culture and are consistent with the fact that NFATc2 is not involved in positive selection.

Additionally, none of the Bcl-2 family members involved in general cell death/survival that were analyzed are differentially expressed between NFATc3-deficient and control DP thymocytes (Fig. 3C). In essence, thymocytes lacking NFATc3 are equivalent to cells without effective TCR gene recombination or effective TCR expression and hence do not receive an effective positive selection signal. We therefore conclude that decreased thymocyte viability in the absence of NFATc3 is a result of the defect in positive selection and not vice versa.


   Acknowledgments
 
We thank Lei Chen for technical assistance and Elena Gallo and Amy Radermacher for helpful discussions and for critically reviewing the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Howard Hughes Medical Institute (HHMI), National Institutes of Health Grant 1-RO1-AI-60037-01, the Boehringer Ingelheim Fonds (to K.C.-B.), and Stanford Graduate and HHMI Predoctoral Fellowships (to M.M.W.). Back

2 Current address: Massachusetts Institute of Technology Center for Cancer Research, 40 Ames Street, Cambridge, MA 02142. Back

3 Address correspondence and reprint requests to Dr. Gerald R. Crabtree, Stanford University, Beckman Center Room B211, 279 Campus Drive, Stanford, CA 94305. E-mail address: crabtree{at}stanford.edu Back

4 Abbreviations used in this paper: DN, double negative; DP, double positive; SP, single positive; ES, embryonic stem; ic, intracellular. Back

Received for publication March 13, 2007. Accepted for publication April 27, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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