Defective T cell development and function in calcineurin A beta -deficient mice

Abstract

The calcium-dependent phosphatase calcineurin and its downstream transcriptional effector nuclear factor of activated T cells (NFAT) are important regulators of inducible gene expression in multiple cell types. In T cells, calcineurin-NFAT signaling represents a critical event for mediating cellular activation and the immune response. The widely used immunosuppressant agents cyclosporin and FK506 are thought to antagonize the immune response by directly inhibiting calcineurin-NFAT signal transduction in lymphocytes. To unequivocally establish the importance of calcineurin signaling as a mediator of the immune response, we deleted the gene encoding the predominant calcineurin isoform expressed in lymphocytes, calcineurin A beta (CnA beta). CnA beta(-/-) mice were viable as adults, but displayed defective T cell development characterized by fewer total CD3 cells and reduced CD4 and CD8 single positive cells. Total peripheral T cell numbers were significantly reduced in CnA beta(-/-) mice and were defective in proliferative capacity and IL-2 production in response to PMA/ionomycin and T cell receptor cross-linking. CnA beta(-/-) mice also were permissive to allogeneic tumor-cell transplantation in vivo, similar to cyclosporin-treated wild-type mice. A mechanism for the compromised immune response is suggested by the observation that CnA beta(-/-) T cells are defective in stimulation-induced NFATc1, NFATc2, and NFATc3 activation. These results establish a critical role for CnA beta signaling in regulating T cell development and activation in vivo.

Figure 1

Targeted disruption of the CnAβ gene. (A) Schematic representation of the CnAβ genomic locus, the targeting vector (TV), and the result of homologous recombination. (B) Western blot analysis of protein extracts from brain and spleen tissue with pan-calcineurin A catalytic subunit antisera, CnAβ-, or CnAα-specific antisera. (C) Calcineurin activity from splenocytes derived by measuring the dephosphorylation rate of a synthetic phosphopeptide substrate (RII peptide). *, P< 0.01 vs. CnAβ^+/+.

Figure 2

Defective NFAT activation in CnAβ^−/− thymocytes. Western blot analysis of protein extracts isolated from the cytosol of unstimulated or PMA/ionomycin-stimulated thymocytes taken from either wild-type or CnAβ^−/− mice. The arrows on the left illustrate the change in NFAT migration due to phosphorylation status of each NFAT isoform. Controls include Cos cell protein extracts from NFATc1 or NFATc3 transfections (lanes at right) as well as Western blotting for a nonmodulated cytosolic protein, Cbl, to monitor for extract integrity and equivalent loading.

Figure 3

Impaired T cell development in CnAβ^−/− mice. (A) Total number of CD3 positive cells (T cells) and CD19 positive cells (B cells) isolated from spleen and lymph nodes were quantified by flow cytometric analysis (four independent experiments). (B) A representative FACS experiment of thymocytes (Top) or splenocytes (Bottom) from wild-type, CnAβ^−/−, or wild-type mice treated with CsA were stained with FITC-CD4 and PE-CD8 antibodies. (C) Percentage of CD4- and CD8-positive cells summarized from the FACS data. (*, P < 0.01 vs. wild type).

Figure 4

Impaired lymphocyte proliferation in CnAβ^−/− mice. (A) Enriched splenic T cells stimulated with αCD3 antibodies or PMA and ionomycin for 48 h show reduced proliferation. All proliferation assays were performed in triplicate within a single experiment, and similar results were observed in multiple experiments (*, P < 0.01 vs. wild type). (B) IL-2 gene expression from enriched CnAβ^−/− or wild-type splenocyte T cells was assessed by RNase protection assay after stimulation with PMA/ionomycin for 16 h and expressed relative to GAPDH (n = 3; *, P < 0.01 vs. wild type). (C) Enriched T cell proliferation levels from wild-type (WT) and CnAβ^−/− (KO) mice stimulated with either αCD3 or PMA/ionomycin in the presence of increasing amounts of CsA or FK506. The assay background was approximately 300 dpm, whereas PMA/ionomycin stimulation induced approximately 4,000 dpm in CnAβ^−/− lymphocytes (because of the scale of the graph, lower levels of proliferation are not obvious). The results were derived from three wild-type and three CnAβ^−/− mice. Similar results were observed in three independent experiments.

Figure 5

Impaired allogeneic tumor cell rejection in CnAβ^−/− mice. (A) J558L mouse plasmacytoma cells (BALB/c background) were injected s.c. into BALB/c, wild-type, CnAβ^−/−, and CsA-treated wild-type mice so that allogeneic tumor growth could be scored 10–17 days later. (B) Model for T cell activation supports a critical role for the CnAβ gene as the major calcium-dependent transducer of lymphocyte activation, when compared with CnAα. Calcineurin activation then promotes NFATc1/c2/c3 dephosphorylation, which promotes their translocation into the nucleus, where NFAT factors interact with other response pathways to orchestrate the immune response.