The Transcription Factor NF-ATc1 Regulates Lymphocyte Proliferation and Th2 Cytokine Production

Immunity, Vol. 8, 115–124, January, 1998, Copyright 1998 by Cell Press

The Transcription Factor NF-ATc1 Regulates Lymphocyte Proliferation and Th2 Cytokine Production Hiroki Yoshida,*† Hiroshi Nishina,*† Hiroaki Takimoto,*† Luc E. M. Marenge`re,*† Andrew C. Wakeham,*† Denis Bouchard,*† Young-Yun Kong,*† Toshiaki Ohteki,† Arda Shahinian,*† Martin Bachmann,† Pamela S. Ohashi,† Josef M. Penninger,*† Gerald R. Crabtree,‡ and Tak W. Mak*†§ * The Amgen Institute † Ontario Cancer Institute Departments of Medical Biophysics and Immunology University of Toronto Toronto, Ontario Canada M5G 2C1 ‡ Departments of Pathology and Developmental Biology Howard Hughes Medical Institute Beckman Center for Molecular and Genetic Medicine Stanford University School of Medicine Stanford, California 94305

Summary NF-ATc1 is a member of a family of genes that encodes the cytoplasmic component of the nuclear factor of activated T cells (NF-AT). In activated T cells, nuclear NF-AT binds to the promoter regions of multiple cytokine genes and induces their transcription. The role of NF-ATc1 was investigated in recombination activating gene-1 (RAG-1)–deficient blastocyst complementation assays using homozygous NF-ATc12/ 2 mutant ES cell lines. NF-ATc12/2/RAG-12/2 chimeric mice showed reduced numbers of thymocytes and impaired proliferation of peripheral lymphocytes, but normal production of IL-2. Induction in vitro of Th2 responses, as demonstrated by a decrease in IL-4 and IL-6 production, was impaired in mutant T cells. These data indicate that NF-ATc1 plays roles in the development of T lymphocytes and in the differentiation of the Th2 response.

Introduction Nuclear factor of activated T cells (NF-AT) was first described as a transcriptional regulatory complex critical for the expression of the T cell cytokine interleukin-2 (IL-2) (Shaw et al., 1988). Subsequently, NF-AT has been shown to play a role in the induction of multiple genes encoding cytokines and cell surface receptors such as CD40L and FasL in activated lymphocytes (Durand et al., 1988; Cockerill et al., 1993; Goldfeld et al., 1993; Rooney et al., 1995; Hodge et al., 1996; Tsytsykova et al., 1996). In activated lymphocytes, NF-AT translocates from the cytoplasm into the nucleus (Shibasaki et al., 1996; Timmerman et al., 1996; Liu et al., 1997) after cyclosporin-sensitive dephosphorylation by calcineurin § To whom correspondence should be addressed (e-mail: t.mak@

oci.utoronto.ca).

(Flanagan et al., 1991; McCaffrey et al., 1993a, 1993b; Ho et al., 1996; Beals et al., 1997) and, coupled with the inducible nuclear component composed of AP-1 transcription factors (Flanagan et al., 1991; Jain et al., 1992), binds to the promoter regions of multiple genes and induces their transcription. The family of cytoplasmic components of NF-AT consists of NF-ATc1 (NFATc), NF-ATc2 (NF-ATp), NF-ATc3 (NF-AT4 or NF-ATx), and NF-ATc4 (NF-AT3) (McCaffrey et al., 1993a; Northrop et al., 1994; Ho et al., 1995; Hoey et al., 1995; Masuda et al., 1995; Rao et al., 1997). Although these members share approximately 70% homology within a region distantly related to the Rel homology domain of the nuclear NF-kB transcription factors, the differential expression of NF-AT proteins in tissues and the differences in their binding preferences for recognition sites in cytokine genes suggest that each NF-AT protein controls the expression of a distinct set of genes in vivo (Rao et al., 1997). NF-ATc2, a member of the family, is expressed constitutively in resting immune cells. Disruption of the NFATc2 gene revealed its role in the regulation of IL-4 production and the establishment of T helper-2 (Th2) responses (Hodge et al., 1996; Xanthoudakis et al., 1996). NF-ATc1 is expressed in thymus and in lymphoid organs, and its transcripts are markedly increased in stimulated T cells and thymocytes (Northrop et al., 1994). NF-ATc1 has also been shown to contribute to the production of IL-2. The expression of full-length NF-ATc1 cDNA activates the IL-2 promoter in non–T lymphocytes, whereas expression of the dominant negative form of NF-ATc1 blocks the IL-2 promoter. These data have suggested a function for NF-ATc1 in the development and activation of T lymphocytes as well as in the production of IL-2 by T cells. To address whether NF-ATc1 plays specific roles in lymphocyte development and activation, we generated embryonic stem (ES) cell lines with a homozygous mutation in the NF-ATc1 gene and analyzed the development and function of lymphocytes in recombination activating gene-1 (RAG-1)–deficient blastocyst complementation assays (Chen et al., 1993, 1994a). Mutant lymphocytes exhibited decreased proliferation compared to control cells and showed impaired production of Th2-type cytokines in an in vitro differentiation assay. These results indicate that NF-ATc1 is required for the development of T lymphocytes and the differentiation of Th2 responses. Results Generation of NF-ATc1 Mutant Cell Lines and NF-ATc12/2/RAG-12/2 Chimeric Mice Mutant mouse ES cell lines in which exon 3 of the NFATc1 gene was disrupted by homologous recombination were generated (unpublished data). Homozygous NF-ATc1 2/ 2 mutant mice die before day 14.5 of embryogenesis from congestive heart failure (unpublished data), preventing the analysis of NF-ATc function in lymphocytes of these animals. To circumvent this situation,

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ES cell clone were injected into RAG-12/2 blastocysts. Because the E14 ES cells used for gene targeting are derived from a strain 129 mouse background, agematched 129/J mice were used as wild-type controls. Both heterozygous and homozygous ES cell clones were able to reconstitute the T and B cell compartments of RAG-1 2/ 2 mice. The lymph nodes and spleens of NFATc12/2 chimeras had normal cell numbers (Table 1). To confirm that mature lymphocytes in the chimeras were derived from the injected ES cells, CD41 thymocytes were cell-sorted and genotyped by the polymerase chain reaction (PCR) technique (Figure 1B). The absence of NF-ATc1 mRNA in activated T cells from NFATc12/2/RAG-12/2 chimeras was confirmed by Northern blot analysis (Figure 1C). Genotyping of the organs of the chimeras showed that the injected ES cell clones were able to differentiate into multiple organs (Figure 1D). All chimeric mice appeared to be healthy and showed no anomalies in gross anatomical examinations.

Figure 1. Generation of ES Cell Lines Homozygous for the NF-ATc1 Mutation and Generation of NF-ATc12/2/RAG-12/2 Chimeric Mice (A) Genomic analysis of embryonic stem cells. Genomic DNA was isolated from NF-ATc11/1 (1/1), NF-ATc11/2 (1/2) and NF-ATc12/2 (2/2) ES cells; digested with EcoRI; and analyzed by Southern blotting using a 59 flanking probe described elsewhere (J. L. de la Pompa et al., submitted). Relative molecular mass markers and wildtype (WT) and mutant (MT) bands are indicated. (B) Homozygous mutation of NF-ATc1 in T cells. CD41 thymocytes from 129/J mice or NF-ATc12/2 chimeric mice were cell-sorted and analyzed for wild-type (WT) or mutant (MT) alleles using PCR. (C) Absence of NF-ATc1 transcripts in T cells of chimeric mice. Total RNA was extracted from ConA-activated peripheral T cells. Northern blots were hybridized to NF-ATc1 cDNA or b-actin cDNA probes. (D) Differentiation of mutant ES cells into multiple organs. DNA was extracted from thymus, spleen, heart muscle, lung, liver, kidney, intestine, skeletal muscle, and skin of the chimeric (Ch) mice. DNA was also extracted from wild-type (WT) spleen and thymus. Presence of the mutant allele was analyzed using PCR.

we generated NF-ATc12/2 ES cell lines from NF-ATc1 1/ 2 ES cells by selection at an increased concentration of G418. Homozygous mutation of the NF-ATc1 gene was confirmed by Southern blot analysis of ES cell clone genomic DNA digested with EcoRI, as shown for one ES line in Figure 1A. NF-ATc12/2 somatic chimeras were generated by injecting NF-ATc12/ 2 mutant ES cells into blastocysts from RAG-1-deficient (RAG-12/2) mice, constituting a RAG-1–deficient blastocyst complementation assay. Since RAG-1–deficient (or RAG-2–deficient) mice do not produce any mature lymphocytes because of a block in the initiation of V(D)J recombination, mature lymphocytes in the somatic chimeras must be derived from the injected NF-ATc12/2 ES cells (Mombaerts et al., 1992; Shinkai et al., 1992; Chen et al., 1994b). NF-ATc12/2 ES cell clones and a parental NF-ATc1 1/2

Reduction in the Number of Thymocytes and Impaired Proliferation of the Thymocyte Precursor Population in the Mutant Chimeric Mice The thymi of NF-ATc12/2 chimeric mice were more than twice as small as thymi of age-matched 129/J mice or NF-ATc11/2 chimeras (Table 1; all mice examined were 5–12 weeks of age). The reduction in thymus size was due to a significant decrease in the population of CD41CD81 double-positive (DP) immature thymocytes and CD41 mature thymocytes (Table 1 and Figure 2A). Expression of the TcRab/CD3 complex and of thymocyte maturation markers such as CD69 and CD5 was comparable in NF-ATc12/2 chimeric and 129/J mice (data not shown). Immature CD42CD82 double-negative (DN) thymocytes can be classified into four distinct phenotypic precursor populations by the differential expression of CD25 and CD44, providing a maturational sequence of CD252CD441→ CD251CD441→ CD251CD442→ CD252 CD442. Since the CD252CD442 population is an immediate precursor to DP thymocytes (Godfrey and Zlotnik, 1993; Pe´nit et al., 1995; Zu´n˜iga-Pflu¨cker and Lenardo, 1996), the expression of CD25 and CD44 on CD42, CD82, CD32, B2202, and Mac-12 (lineage marker–negative) thymocytes was investigated. Compared with 129/J or NF-ATc11/ 2 chimeric mice, DN thymocytes of NFATc12/2 chimeric mice exhibited a lower percentage of CD251CD442 cells, concomitant with an increased frequency of CD252CD44 2 cells (Figure 2B). Moreover, the mutant chimeric mice had fewer CD25lowCD442 cells (Pe´nit et al., 1995) than wild-type or heterozygous mice (Figure 2B). Normal CD25lowCD442 cells and CD252 CD442 cells are cycling and contain a high percentage of blastoid cells (Pe´ nit et al., 1995). However, as shown in Figure 2B (bottom), the CD25 lowCD442 and CD252CD442 populations of NF-ATc12/2 chimeric mice contained relatively low frequencies of blastoid thymocytes as determined by forward light scatter profiling, indicating a loss of blastoid thymocytes in these compartments. In agreement with this finding, lineage marker–negative thymocytes of NF-ATc12/ 2 chimeric mice (Lin2 in Figure 2C) showed a relative increase of cells in the G01G1

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Table 1. Numbers of Lymphoid Cells in NF-ATc12/2 Chimeric Mice Absolute Numbers 6 SEM (3 1026 ) Organ Thymus CD42CD82 CD41CD81 CD41CD82 CD42CD81

NF-ATc11/1

NF-ATc11/2

6 6 6 6 6

6 6 6 6 6

80.5 4.0 68.1 10.7 2.0

13.2 0.9 10.0 1.6 0.7

95.0 4.8 82.2 14.8 3.7

8.0 0.2 3.1 0.6 0.1

Percentages 6 SEM NF-ATc12/2

NF-ATc11/1

36.0 6 7.4 6 19.7 6 5.0 6 1.4 6

5.6 78.7 12.4 3.5

NF-ATc11/2

NF-ATc12/2

a

9.0 1.5a 3.5 a 1.5a 0.8

Spleen

3.8 6 1.1

4.3 6 1.5

4.6 6 1.8

Lymph nodes

2.1 6 0.8

1.8 6 0.5

2.3 6 1.1

6 6 6 6

1.3 3.2 1.8 1.7

6.1 76.4 12.7 4.9

6 6 6 6

0.8 1.1 0.1 1.7

19.5 59.8 15.7 5.1

6 6 6 6

7.4 8.6 3.5 3.4

Total thymocytes, total spleen cells, and total axillar, inguinal, and mesenteric lymph node cells from NF-ATc11/1 (129/J) (n 5 8), NF-ATc11/2 chimera (n 5 6), and NF-ATc12/2 chimera (n 5 8) were isolated, counted, and analyzed for their surface marker expression. All mice analyzed were 5–12 weeks of age. a Statistically significant difference (Student’s t test, P , 0.001) between NF-ATc12/2 and NF-ATc11/1 mice.

phase of the mitotic cell cycle compared to wild type (83% vs. 67%), and a reduction of cells in the S and G21M phases (17% and 0.0% in the mutant, respectively, vs. 33% and 0.2% in the wild type). These results are consistent with a partial G1 block of DN thymocytes in NF-ATc12/ 2 chimeric mice. Lineage marker–positive

thymocytes from NF-ATc12/2 chimeric mice (Lin1 in Figure 2C) showed cell cycle profiles similar to those of thymocytes from wild-type mice. Reverse transcriptase PCR analysis of wild-type cells showed that NF-ATc1 is expressed in both CD251CD442 and CD25 2CD44 2 DN thymocytes (Figure 2D), suggesting that NF-ATc is

Figure 2. Flow Cytometric Analysis and Cell Cycle Analysis of Thymocytes (A) Thymocytes were isolated from 129/J (NF-ATc11/1), NF-ATc11/2 chimeric mice or NF-ATc12/2 chimeric mice and stained with anti-CD4, anti-CD8, and anti-Ly-9.1 antibodies. Ly-9.11 cells were analyzed for their expression of CD4 and CD8. Percentages of positive cells within a quadrant are indicated. (B) Thymocytes were stained with anti-CD4, anti-CD8, anti-TcRab, anti-B220, anti-Mac-1 (lineage markers), anti-CD25, anti-CD44, and antiLy-9.1. Ly-9.11 lineage marker–negative populations were analyzed for their expression of CD25 and CD44. Percentages of CD251CD442, CD25low CD442, and CD252CD442 cell populations are indicated. Forward light scatter profiles of CD25low/ 2CD442 cellsin Ly-9.11 lineage marker–negative populations are also shown. (C) Ly-9.11 lineage marker–negative (Lin -) thymocytes were sorted and stained with propidium iodide for cell cycle analysis. Percentages of cells in G01G1, S, and G21M phases are indicated. Ly-9.11 lineage marker–positive (Lin1) thymocytes were also analyzed as a control. (D) CD25 1CD442 (CD251) or CD252CD442 (CD25 2) thymocytes were isolated from lineage marker–negative thymocytes from 129/J mice by cell-sorting. Expression of the NF-ATc1 gene was analyzed using reverse transcriptase PCR analysis. The reaction was performed in the presence (RT1) or absence (RT2) of reverse transcriptase. Expression of the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene was analyzed as a positive control.

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Figure 4. Flow Cytometric Analysis of Lymph Node, Spleen, and Bone Marrow Cells Single-cell suspensions of lymph node cells, spleen cells, or bone marrow cells were stained with anti-CD4 versus anti-CD8; anti-B220 (B cell marker) versus anti-Thy1.2 (T cell marker); or anti-B220 versus anti-CD43, respectively, and analyzed for their surface expression by flow cytometry. Percentages of positive cells within a quadrant are indicated. Experiments were repeated three times with similar results.

(A) Single-cell suspensions of intraepithelial cells were prepared as described in Experimental Procedures. Cells were stained with FITC-conjugated anti-TcRb and PE-conjugated anti-TcRd, or FITCconjugated anti-CD8a and PE-conjugated anti-CD8b. (B) Liver MNC were prepared as described in Experimental Procedures. Cells were stained with FITC-conjugated anti-TcRb and PEconjugated anti-IL-2Rb. The numbers correspond to the proportion of the total number cells within quadrants. Experiments were repeated three times with similar results.

T cells or NK11 T cells (IL-2Rb1 TcRabint cells) in the thymus were observed between mutant and wild-type or heterozygous chimeric mice (data not shown). There also were no significant differences in the numbers of intestinal intraepithelial TcRgd T and TcRab T cells (Figure 3A). The mutant chimeric mice also had comparable numbers of NK11 T cells in the liver (Figure 3B). Thus, the absence of NF-ATc1 did not significantly affect the development of T lineage or related cells other than that of TCRab thymocytes.

involved in the expansion of CD25low/2CD442 DN thymocytes. Analysis of the apoptosis of ex vivo mutant thymocytes stimulated with anti-CD95 (Fas) antibodies, antiCD3 6 anti-CD28 antibodies, dexamethazone, or heat shock (418C, 30 min) did not reveal any significant differences from the wild type (data not shown), excluding the possibility that the reduced thymocyte number was due to stress-induced cell death. We examined whether the development of unconventional T cell subsets was affected by the absence of NFATc1. No significant differences in the numbers of TcRgd

Impaired Proliferation of Peripheral Lymphocytes No differences in the numbers of lymph node cells or spleen cells (Table 1) or in the ratio of CD41 to CD81 cells in the lymph nodes or spleen (Figure 4 and data not shown) were observed between NF-ATc12/2 chimeric mice and control mice. The expression of IL-2Ra (CD25), CD69, CTLA-4, CD28, FasL, and CD40L as analyzed by flow cytometric analysis was normal following mutant T cell activation (data not shown). However, the proliferation of peripheral T cells from the chimeric mice in response to phorbol myristate acetate (PMA) plus calcium ionophore, concanavalin A (ConA), the soluble

Figure 3. Flow Cytometric Analysis of IEL and IL-2Rb 1 TcRab1 Cells in the Liver

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Figure 5. Proliferation and Function of Peripheral Lymphocytes (A) Proliferation of lymph node T cells. Cells were stimulated with PMA plus Ca2 1 ionophore A23617, soluble anti-CD3e, soluble antiCD28, or ConA in the presence or absence of IL-2 for 48 or 72 hr. The incorporation of [3H]thymidine during the last 8 hr of culture was measured. Experiments were performed in triplicate; shown are the mean 1 SD for each sample. Open bars, wild-type control; filled bars, NF-ATc2/2 chimera. (B) Culture supernatants of the cell cultures described in (A) were assayed in triplicate for IL-2 production by ELISA. Experiments were repeated four times with similar results. Bars as in (A). (C) Proliferation of spleen B cells. Spleen cells depleted of red blood cells were stimulated with PMA plus Ca21 ionophore A23617, antiIgM, anti-CD40, or LPS in the presence or absence of IL-4 for 48 or 72 hr. The incorporation of [3H]thymidine during the last 8 hr of culture was measured. Experiments were performed in triplicate and repeated three times. Bars as in (A). (D) Basal serum immunoglobulin concentrations. The serum concentrations of different immunoglobulin subclasses were determined by ELISA. Open squares, heterozygous chimera; closed squares, homozygous chimera. Experiments were performed in duplicate and repeated twice using two mice per group per experiment. (E) Immunoblot analysis of STAT5a, STAT5b, and IRF-4 expression. Spleen cells from two heterozygous (1/2) or two homozygous (2/2) mutant chimeras were cultured for 48 hr with anti-CD3 (0.5 mg/ml) in the absence of cytokines (shown as CD3), or in the presence of 50 U/ml IL-2 (CD3 1 IL-2) or 400 U/ml IL-4 (CD3 1 IL-4), or without treatment (None). Cell lysates were prepared as described in Experimental Procedures and immunoblot analyses were performed using specific antibodies.

form of anti-CD3 6 anti-CD28, SEB, or allogeneic stimulation reached only approximately 70% of the level of proliferation of cells from wild-type or heterozygous chimeric mice (Figure 5A and data not shown). This proliferation defect of mutant T lymphocytes was not fully rescued by the addition of exogenous IL-2 (50 U/ml), although some response was observed. Moreover, stimulated T cells from homozygous chimeric and wild-type mice showed no differences in the level of IL-2 secreted in supernatants in these primary stimulating cultures (Figure 5B). These data show both that NF-ATc1 is not essential for the production of IL-2 and that the defect

resulting in reduced proliferation of mutant T lymphocytes is intrinsic to the cells themselves and is not due to impaired IL-2 production. Development of B cells in the bone marrow and spleen of the chimeric mice appeared normal (Figure 4). No anomalies in the total numbers or percentages of B2201 B cells in the spleen or lymph nodes were observed (Figure 4 and data not shown). However, in response to PMA plus calcium ionophore, anti-immunoglobulin M (anti-IgM), or anti-CD40, B cells from NF-ATc12/ 2 chimeric mice showed only approximately 50%–60% of the proliferation of wild-type or heterozygous B cells (Figure

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5C). The impaired response was not rescued by the addition of exogenous IL-4 (400 U/ml). There was no significant difference in the proliferation of mutant B cells in response to lipopolysaccharide (LPS) in the absence or presence of IL-4. Serum concentrations of total immunoglobulin were comparable between NF-ATc1 2/ 2 and NF-ATc11/2 chimeric mice (data not shown). However, the homozygous mutant chimeric mice showed slightly elevated levels of IgM and IgG2a compared with heterozygous mutant chimeric mice, but slightly reduced levels of IgG1 (Figure 5D). The NF-ATc12/2 chimeric mice produced neutralizing antibodies (IgM and IgG) against vesicular stomatitis virus at levels similar to those in wild-type mice (data not shown). The chimeric mice also developed comparable cytotoxic T lymphocyte responses to the virus (data not shown). NF-ATc1 is thus not essential for the helper function of T cells in vivo. To investigate further the mechanism underlying the IL-2–resistant defect in the proliferation of mutant T lymphocytes, we examined the expression of the transcription factors STAT5a and STAT5b (signal transducers and activators of transcription 5a and 5b), molecules involved in the IL-2–mediated signal transduction pathway (Gilmour et al., 1995; Hou et al., 1995; Lin et al., 1996). The expression of STAT5a and STAT5b was comparable in anti-CD3–stimulated lymphocytes of heterozygous and homozygous mutant chimeric mice (Figure 5E) and was not altered in the presence of IL-2 or IL-4. We also examined the expression of the transcription factor interferon regulatory factor-4 (IRF-4), since the disruption of the IRF-4 gene resulted in a similar but more profound impairment of T and B cell proliferation that also could not be rescued by the addition of exogenous IL-2 or IL-4 (Mittrucker et al., 1997). No significant differences in the induction or expression of IRF-4 were recorded after the stimulation of mutant T cells with anti-CD3 (Figure 5E).

Impaired Production of Th2 Cytokines by NF-ATc12/2 T Cells NF-ATc2, a member of the cytoplasmic NF-AT family, appears to be involved in Th2 development, since disruption of the NF-ATc2 gene results in augmented production of IL-4 by T cells that had differentiated into Th2 cells (Hodge et al., 1996), or the accumulation of eosinophils accompanied by an elevated level of IgE (Xanthoudakis et al., 1996). To investigate the role of NF-ATc1 in the development of Th cell responses, we examined the response of NF-ATc12/ 2 T cells to two different stimuli known to induce the differentiation of T cells into either the Th1 or Th2 type. NF-ATc12/ 2 spleen T cells stimulated with anti-CD3 in the presence of IL-4 (which normally induces Th2 differentiation) showed decreased IL-4 production compared to wild-type T cells (Figure 6). Mutant spleen cells also produced less IL-6 than wild-type cells in the same experiments. In contrast, when mutant T cells were stimulated with antiCD3 in the presence of IL-12 (which normally induces Th1 responses), there were no differences between homozygous and heterozygous chimeric cells in the production of the Th1-type cytokines interferon-g (IFNg)

and tumor necrosis factor-a (TNFa) (Figure 6). These data demonstrate that the absence of NF-ATc1 during an ongoing immune response impairs the establishment of the Th2 response but does not affect the establishment of the Th1 response. Discussion The development and responses of the immune system are coordinated by an interacting network of transcription factors involved in the expression of effector proteins such as cytokines (Crabtree, 1989; Paul and Seder, 1994; Zlotnik and Moore, 1995; Clevers and Grosschedl, 1996). The NF-AT family of transcription factors has been reported to play a critical role in this process. Our analyses of NF-ATc1 2/ 2/RAG-12/2 somatic chimeras indicate that NF-ATc1 is required for the development of T lymphocytes, the proliferation of lymphocytes, and the production of Th2 cytokines. The development of thymocytes from precursors to mature T cells is tightly regulated by the expression and cooperation of multiple molecules, the identity of which depends on a given developmental stage (Godfrey and Zlotnik, 1993; Zlotnik and Moore, 1995; Zu´n˜iga-Pflu¨cker and Lenardo, 1996). NF-ATc1 has a specific function in the proliferative expansion of CD25low/ 2CD44 2CD42 CD82CD32 triple-negative (TN) thymocytes, an immediate precursor population for DP thymocytes. This cell population is unable to produce IL-4 or IL-2 (Godfrey and Zlotnik, 1993; Zlotnik and Moore, 1995), indicating a requirement for the involvement of other molecule(s) whose transcription is controlled by NF-ATc1. Disruption of the transcription factor T cell factor-1 gene (Tcf-1) (Verbeek et al., 1995) or functional inactivation of Rho GTPase (Galandrini et al., 1997; Henning et al., 1997) in the thymus resulted in similar but more drastic impairments of the proliferative expansion of immature thymocytes. In addition, disruption of the transcription factor SOX-4 gene produced cardiac malformations similar to those observed in NF-ATc12/2 mutant embryos (unpublished data), as well as a profound defect in B cell development (Schilham et al., 1996) and compromised expansion of thymocytes in fetal thymic organ cultures (Schilham et al., 1997). Together, these data suggest that a common signaling pathway(s) that is critical for the proliferative expansion of thymocytes may exist downstream of these transcription factors. Despite the impaired proliferation of thymocyte progenitors, the development of TcRgd T cells in the thymus and the intestine, and the development of NK11 T cells in the thymus and the liver of NF-ATc12/2 chimeras appeared normal (Figures 3A and 3B and data not shown). Since CD251CD442 TN thymocytes are known to be the last precursor population that gives rise to TcRgd T cells and NK11 T cells (Godfrey and Zlotnik, 1993; Zu´ n˜igaPflu¨cker and Lenardo, 1996), the normal development of TcRgd T cells and NK11 T cells in the thymus, small intestine, and liver of homozygous chimeric mice indicates that the proliferative expansion of the CD251 CD442 TN thymocyte population is intact in NF-ATc12/2 chimeric mice. NF-ATc1 is also involved in the proliferation of peripheral T lymphocytes and B lymphocytes (Figures 5A and

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Figure 6. Differentiation In Vitro of T Cells into Th1 or Th2 Spleen cells (1 3 10 6/ml) were stimulated with 10 mg/ml plate-bound anti-CD3e in the presence of 1 ng/ml IL-12 for Th1 differentiation or 1000 U/ml IL-4 for Th2 differentiation. Cultured cells were fed with fresh medium after 4 days, and an equal number of viable cells (0.5 3 106/ml/sample) after 7 days were restimulated with plate-bound anti-CD3 in the absence of any additional cytokines. Supernatants were collected 24 hr later and IL-4 and IL-6 or IFNg and TNFa in the supernatants of Th2- or Th1-differentiated cells, respectively, were measured by ELISA. Shown are representative results of four replications of these experiments.

5C). The molecular basis of the role of NF-ATc1 in cell proliferation is currently unknown but cannot be totally dependent on cytokine production, since exogenous cytokines (IL-2 for T cells and IL-4 for B cells) did not rescue the proliferation defect in NF-ATc12/2 lymphocytes (Figures 5A and 5C). The expression of the transcription factors STAT5a, STAT5b, and IRF-4, all of which are involved in lymphocyte proliferation, was not altered in the mutant lymphocytes (Figure 5E). Thus, the role(s) of NF-ATc1 in the proliferation of cells may reside in a pathway distinct from those involving STAT5 or IRF-4. This study has also demonstrated that NF-ATc1 is not essential for the production of IL-2 (Figure 5B), but our results do not exclude the possibility that NFATc1 is involved in some way. Since the four members of the NF-AT family have overlapping DNA-binding and transcriptional specificities (McCaffrey et al., 1993a, 1993b; Northrop et al., 1994; Ho et al., 1995; Hoey et al., 1995; Masuda et al., 1995; Rao et al., 1997), a functional redundancy of NF-AT activities could explain the production of IL-2, at least in primary cultures, in NFATc12/2 T cells. The establishment of cytotoxic T lymphocyte responses and the T-dependent production of neutralizing antibodies against vesicular stomatitis virus infection were also intact in chimeric mice. CD41 T cells potentiate the inflammatory or humoral immune response to bacterial, parasitic, and viral infections through the Th1 or Th2 responses, respectively (Mosmann et al., 1986; Mosmann and Coffman, 1989; Scott and Kaufmann, 1991; Mosmann and Sad, 1996). The differentiation of naive CD41 T cells into Th1 or Th2 cells appears to be a multistep process controlled by cytokines produced during the immune response. In particular, IL-12 induces differentiation into Th1 cells, whereas IL-4 induces differentiation into Th2 cells (Seder et al., 1992, 1993; Paul and Seder, 1994; Paul, 1997). The molecular mechanisms by which the activation of T cells drives Th1 and Th2 effector cell differentiation are poorly understood, although the contributions of several different transcription factors have been reported (Kaplan et al., 1996; Takeda et al., 1996; Lohoff et al., 1997; Taki et al., 1997; Zheng and Flavell, 1997). In this study, the development of Th2 responses in vitro was compromised in the absence of NF-ATc1, whereas Th1 cytokine production was not altered in the mutant cells (Figure 6), providing evidence that NF-ATc1 may

be involved in the mechanism(s) that determines the Th2 response. The transcription factor GATA-3 has been shown to be selectively expressed in differentiating and effector Th2 cells and required for the transcription of all Th2 cytokine genes (Zheng and Flavell, 1997). The expression of GATA-3 in activated homozygous mutant spleen cells, however, was unaffected (data not shown). We also examined the expression of the transcription factor IRF-1, since the disruption of the IRF-1 gene resulted in compromised establishment of Th1 responses concomitant with augmented production of IL-4 by mutant cells (Lohoff et al., 1997; Taki et al., 1997), but the expression of IRF-1 in activated mutant spleen cells was also unaffected (data not shown). These data indicate that a distinct mechanism(s) of Th cell differentiation may be exploited by NF-AT members. In contrast to the impaired establishment of the Th2 response in NF-ATc12/ 2 T cells, targeted disruption of NF-ATc2 resulted in the enhancement of Th2 responses (Hodge et al., 1996; Xanthoudakis et al., 1996). Thus, the balance between NF-AT family members with distinct, nonoverlapping functions may be critical in the control of gene expression and the development of T cell function during ongoing immune responses (Rooney et al., 1994; Rincon and Flavell, 1997a, 1997b). Expression of NFATc1 as well as NF-ATc2 has been reported in NK11 T cells (Hodge et al., 1996), a population responsible for IL-4 production in response to challenge in vivo with anti-CD3 (Carding et al., 1991; Sen et al., 1994; Yoshimoto and Paul, 1994; Yoshimoto et al., 1995). These data imply that both NF-ATc1 and NF-ATc2 may be involved in the production of IL-4 and the resulting bias toward Th2 differentiation (Hodge et al., 1996). NF-ATc1 mutant chimeric mice had comparable numbers of NK11 T cells in the liver and the thymus (Figure 3B and data not shown), but the absence of NF-ATc1 may have adversely affected the ability of this population to produce IL-4. Our analyses of NF-ATc12/2 chimeric mice indicate that NF-ATc1 has critical functions in the development of T cells, the proliferation of lymphocytes, and the establishment of the Th2 response. The specific roles of other members of the NF-AT family at distinct stages of lymphocyte development and in lymphocyte activation will no doubt be clarified by the generation of mice deficient for these molecules.

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Experimental Procedures Generation of the NF-ATc12/2 ES Cell Lines The establishment of ES cell lines heterozygous for the NF-ATc1 exon 3 mutation is described elsewhere (J. L. de la Pompa et al., submitted). For the generation of NF-ATc12/2 ES cell lines, G418resistant NF-ATc11/2 ES clones were cultured at an increased concentration of G418 (2 mg/ml, Gibco–BRL, Grand Island, NY). Screening for the homozygous mutation was carried out by PCR. Primers used were 59-GAT GGT CTG CCT AGT CAT GAA A-39 and 59-CTT CCT CGT GCT TTA CGG TAT C-39, specific for the wild-type NFATc1 allele, and 59-CAC GTC GTG GAA AAA CTG TC-39, specific for the neomycin resistance gene. Genomic DNA samples were digested with EcoRI and analyzed by Southern blot. Of 48 colonies, two showed homozygous mutation of NF-ATc1 exon 3. Both homozygous mutant cell lines contributed to the reconstitution of T and B cell compartments in RAG-1–deficient blastocyst complementation assays and showed similar phenotypes. RAG-1–Deficient Blastocyst Complementation Assay For the generation of chimeric mice, two NF-ATc12/2 ES cell clones and a parental NF-ATc11/2 ES cell clone were injected into 3.5day RAG-12/2 blastocysts and were transferred to pseudopregnant foster mothers. Mice were maintained under pathogen-free conditions in accordance with the guidelines of the Canadian Medical Research Council. Peripheral blood of the chimeric mice was analyzed for the reconstitution of mature T and B lymphocytes by flow cytometric analysis. CD41 thymocytes were cell-sorted and analyzed for the presence of wild-type and mutant alleles by PCR using the primers described above. Total RNA extracted from columnpurified T cells from control or mutant chimeric mice was electrophoresed, blotted, and hybridized to an NF-ATc1 cDNA probe. Cell Preparation Mononuclear cells (MNC) from thymus, spleen, lymph node, and bone marrow were obtained by standard methods. To obtain liver MNC, the liver was pressed through stainless steel mesh and suspended in 50 ml of phosphate-buffered saline (PBS). After one wash in PBS, the cells were fractionated by discontinuous (40% and 80%) Percoll gradient centrifugation for 10 min at 900 3 g. The interface was harvested and washed with 5% fetal calf serum (FCS)–PBS prior to use in experiments. To obtain intraepithelial lymphocytes (IEL), small intestine dissected free of Peyer’s patches was incised longitudinally, washed with PBS, and cut into 5 mm pieces. These pieces were incubated in PBS with stirring for 20 min at 378C. The supernatant, containing IEL and epithelial cells, was washed with 5% FCS-PBS and fractionated as above by discontinuous Percoll gradient centrifugation for 10 min at 900 3 g. The interface was harvested and washed with 5% FCS-PBS. Flow Cytometric Analysis Single-cell suspensions of thymocytes from 129/J NF-ATc11/2 chimeric mice or NF-ATc12/2 chimeric mice were stained with phycoerythrin (PE)–conjugated anti-CD4, fluorescein isothiocyanate (FITC)– conjugated anti-CD8 and biotin-conjugated anti-Ly-9.1 antibodies (PharMingen, San Diego, CA), followed by staining with streptavidinconjugated RED670 (Gibco–BRL) and analysis of the expression of surface markers using a flow cytometer (FACScalibur, Becton Dickinson, San Jose, CA). The mutant ES cell lines used in the experiments were derived from strain 129 mice that are positive for the Ly-9.1 marker. The host RAG-12/2 cells are negative for Ly-9.1, so Ly-9.11 cells from the chimeric mice were analyzed. For the analysis of thymocyte precursors, single-cell suspensions of thymocytes from NF-ATc12/2 chimeric, NF-ATc11/2 chimeric, and 129/J control mice were stained with allophycocyanin-conjugated anti-CD4, anti-CD8, anti-TcRab, anti-B220, and anti-Mac-1; PE-conjugated anti-CD44; biotin-conjugated anti-CD25; and FITC-conjugated anti-Ly-9.1, followed by staining with streptavidin-conjugated RED670. Ly-9.11 lineage marker–negative populations were analyzed for their expression of CD44 and CD25. Single-cell suspensions of lymph node cells, spleen cells, and bone marrow cells were stained with anti-CD4 and anti-CD8, antiB220 and anti-IgM, or anti-B220 and anti-CD43, respectively. MNC

obtained from small intestine or liver were likewise stained with antiTcRb and anti-TcRd or anti-CD8a and anti-CD8b for IEL or antiTcRb and anti-IL-2Rb for liver MNC. Experiments were repeated three times with similar results. Cell Cycle Analysis For cell cycle analysis, Ly-9.11 lineage marker–negative thymocytes were cell-sorted. Cells were suspended in 1 ml of FCS followed by the addition of 1 ml of Iscove’s modified Dulbecco’s medium. Icecold 70% ethanol (6 ml) was added dropwise to a final concentration of 50% to permeabilize the cell membrane. After 30 min of incubation at 48C, cells were centrifuged and stained for 30 min at 48C with propidium iodide (50 mg/ml) in the presence of RNase (500 mg/ml) to visualize cellular DNA. Cell cycle analysis was performed with ModFit LT software (Becton Dickinson). Reverse Transcriptase–PCR CD251CD442 and CD252CD442 populations from 129/J mice were isolated from lineage marker–negative thymocytes by cell-sorting. Total RNA was extracted with Trizol solution (Gibco–BRL), reversetranscribed with a first-strand cDNA synthesis kit (Pharmacia Biotech, Uppsala, Sweden) using random primers, and amplified for NF-ATc1 gene expression using PCR. Primers used for amplification were: 59 primer, 59-GCA AGC ATC ACG GAG GAG AGC-39; and 39 primer, 59-CGG GTG GGA AGT CAG AAG TGG-39. As a positive control, the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene was amplified with a specific primer pair. As a negative control, the reaction was performed in the absence of reverse transcriptase. Cell Proliferation Assay For the T cell proliferation assay, lymph node cells (1 3 10 5 cells/ well) were placed into round-bottom 96-well plates and activated with PMA (12.5 ng/ml, Sigma, St. Louis, MO) plus Ca21 ionophore A23617 (100 ng/ml), soluble anti-CD3e (0.2 mg/ml), soluble anti-CD28 (0.1 mg/ml), or ConA (1.25 mg/ml) in the presence or absence of IL-2 (50 U/ml). Cells were harvested at 48 or 72 hr after an 8 hr pulse with [ 3H]thymidine (1 mCi/well). The incorporation of 3H-thymidine was measured with a b-counter. T cell culture supernatants were assayed in triplicate for IL-2 by enzyme-linked immunosorbent assay (ELISA) (Genzyme Diagnostics, Cambridge, MA). For the B cell proliferation assay, spleen cells were depleted of Thy1.21 T cells with anti-Thy1.2 antibodies (clone AT83) and rabbit complement (Cedarlane, Ontario, Canada). Viable B cells were isolated with Lympholite M (Cedarlane), and cells (1 3 10 5 cells/well) were placed into round-bottom 96-well plates and activated with PMA plus Ca21 ionophore, anti-IgM (20 mg/ml), anti-CD40 (1 mg/ml), or LPS (20 mg/ml), in the presence or absence of IL-4 (400 U/ml). [3H]Thymidine incorporation was measured as described above. ELISA for Immunoglobulin Subclasses ELISA for immunoglobulin subclasses were performed on serially diluted serum samples using anti-mouse immunoglobulin (IgG 1 IgA 1 IgM) antibodies and alkaline phosphatase–conjugated antimouse immunoglobulin isotype antibodies (Southern Biotechnology Associates, Birmingham, AL) according to the manufacturer’s directions. ELISA for IgE was performed using anti-IgE antibody (PharMingen) and alkaline phosphatase–conjugated anti-IgE antibody (Southern Biotechnology Associates). Western Blot Analysis Stimulated and unstimulated spleen cells from homozygous or heterozygous chimeric mice were lysed in a buffer containing 50 mM Tris (pH 7.5); 1% Triton-X; 150 mM EDTA; 150 mM NaCl; and 1 mg/ ml each of aprotinin, leupeptin, phenylmethylsulfoxidefluoride, and sodium orthovanadate. After a standard protein assay (Pierce, Rockford, IL), 20 mg of total protein from each sample was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane, and Western blotted for the presence of STAT5a and STAT5b, IRF-4, and actin as a control to ensure equal protein loading. Rabbit polyclonal antibodies against STAT5a and STAT5b (Zymed Laboratories, San Francisco, CA) and actin (Sigma) were used according to the manufacturer’s guidelines, and anti-IRF-4 was used at a dilution of 1:500. Donkey anti-rabbit

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horseradish peroxidase antibody (Amersham, Ontario, Canada) and enhanced chemiluminescence (Amersham) were used for visualization. In Vitro T Cell Differentiation Assay For in vitro T cell differentiation assays, spleen cells (1 3 106 cells/ ml) depleted of red blood cells were cultured in duplicate in Iscove’s modified Dulbecco’s medium and stimulated with 10 mg/ml platebound anti-CD3e in the presence of 1 ng/ml IL-12 for Th1 differentiation or 1000 U/ml IL-4 for Th2 differentiation. Cultured cells were fed with fresh medium after 4 days of culture. Cells were collected after 7 days and washed, and an equal number of viable cells (0.5 3 106 cells/ml) was restimulated with plate-bound anti-CD3e in the absence of any additional cytokines. Supernatants were collected 24 hr later, and the production of IL-4 and IL-6 in the Th2 differentiation culture or IFNg and TNFa in the Th1 differentiation culture was measured in duplicate by ELISA (Genzyme and Endogen, Woburn, MA). Acknowledgments The authors thank J. L. de la Pompa for critical reading of the manuscript; R. Yoshida, W. Khoo, and J. Lam for technical help and animal husbandry; and M. Saunders for scientific editing. Received October 28, 1997. References Beals, C.R., Clipstone, N.A., Ho, S.N., and Crabtree, G.R. (1997). Nuclear localization of NF-ATc by a calcineurin-dependent, cyclosporin-sensitive intramolecular interaction. Genes Dev. 11, 824–834. Carding, S.R., Hayday, A.C., and Bottomly, K. (1991). Cytokines in T-cell development. Immunol. Today 12, 239–245. Chen, J., Lansford, R., Stewart, V., Young, F., and Alt, F.W. (1993). RAG-2-deficient blastocyst complementation: an assay of gene function in lymphocyte development. Proc. Natl. Acad. Sci. USA 90, 4528–4532. Chen, J., Shinkai, Y., Young, F., and Alt, F.W. (1994a). Probing immune functions in RAG-deficient mice. Curr. Opin. Immunol. 6, 313–319. Chen, J., Stewart, V., Spyrou, G., Hilberg, F., Wagner, E.F., and Alt, F.W. (1994b). Generation of normal T and B lymphocytes by c-jun–deficient embryonic stem cells. Immunity 1, 65–72.

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