Table 1.

Immunological characteristics of NFAT5 deficiency mouse models.

NFAT5 deficiency mouse model (References)	Loss of NFAT5 protein expression confirmed (References)	Cell types analyzed	Immunological effect of NFAT5 deficiency (References)
Genomic deletion of the first exon of the DBD. NFAT5-null mice suffer severe perinatal mortality but 30% of mice survive to adulthood in a 129 background, whereas much fewer or no adult mice are obtained in a C57BL/6 background (13).	Essentially complete loss of NFAT5 protein confirmed by Western blot in mature T cells (16) and BMDM (11).	T lymphocytes, BMDM, tissue macrophages	Poor T cell responsiveness and function under high salt stress in vivo and in vitro (16). Altered balance of naïve and memory CD4 and CD8 T cells in vivo under high salt stress (16). In vivo defective rejection of allogeneic tumors (16). T cell proliferative deficiency under high salt (which in vivo is associated with systemic hypernatremia in these mice) (16). Defective response of BMDM to TLR stimulation in vitro in the absence of osmostress (11). In vivo defective expression of iNOS and impaired clearance of pathogen Leishmania major (11). Alterations in cytokine and TLR-regulated M1 and M2 polarization of BMDM in vitro (18). Reduced expression of CIITA and MHCII in macrophages (BMDM) (12).
Transgenic mice expressing a dominant-negative NFAT5 DBD in thymocytes and mature T cells under the control of a CD2 promoter (19).	Not applied	Thymocytes and mature T lymphocytes	Reduced numbers of thymocytes and mature T cells in vivo. Poor T cell responsiveness and function under high salt stress in vitro. Reduced T cell survival to amino acid deprivation in the absence of osmostress (19).
Genomic deletion of the first two exons of NFAT5 DBD. Severely impaired viability of NFAT5-null mice without reaching adulthood in a C57BL/6 background. Experiments in adult mice limited to heterozygous animals (14).	Western blot confirmed 50% reduction in expression of NFAT5 protein in thymocytes (14), peritoneal macrophages and BMDM of heterozygous mice with one mutant NFAT5 allele (20).	Thymocytes, mature T lymphocytes and BMDMs	Reduced numbers of thymocytes and splenocytes in vivo in heterozygous mice. Reduced Ig production upon immunization with OVA in heterozygous mice (14). Reduced proliferation in response to mitogenic stimuli for T (anti-CD3 and anti-CD28 antibody) and B cells (LPS) under high salt stress in vitro (14). Reduced T cell survival to amino acid deprivation in the absence of osmostress (14). NFAT5-haploinsufficient BMDM show poorer migratory capacity in response to M-CSF than wild-type ones (21). NFAT5-haploinsufficient peritoneal macrophages and BMDM show enhanced IL-10 expression in response to LPS than wild-type ones (20).
Systemic NFAT5 deletion upon tamoxifen administration in mice that have the first DBD exon floxed and are transgenic for a ubiquitin C (UBC) promoter-driven fusion of Cre/ERT2 activated by tamoxifen (17). These Nfat5-floxed mice have also been used to obtain NFAT5-deficient macrophages upon crossing them with LysM-Cre transgenic animals (22–24).	Essentially complete loss of NFAT5 protein in renal medulla (17) and 60–80% reduction of NFAT5 protein in BMDM in tamoxifen-treated mice (22). 50–80% reduction in NFAT5 protein confirmed by Western blot in BMDM from LysM-Cre x Nfat5fl/fl mice (22).	BMDM, macrophages in footpad lesions by Leishmania major infection.	Enhanced susceptibility to infection with Leishmania major in vitro in NFAT5-deficient BMDM cultured from tamoxifen-treated UBC-Cre/ERT2 × Nfat5fl/fl mice (22). In vivo defective expression of iNOS and impaired clearance of pathogen Leishmania major in footpad macrophages from LysM-Cre Nfat5fl/fl mice kept in high salt diet (HSD) and infected with the parasite (22). Decreased local expression of VEGFC, and impaired lymphatic capillary density and chloride anion (Cl−) balance in skin of mice with a myeloid-specific deletion of NFAT5 (LysM-Cre Nfat5fl/fl) under HSD (23). In vivo reduced expression of iNOS and TNFα in peritoneal macrophages from LysM-Cre Nfat5fl/fl mice injected with LPS (24).
Mice with the first DBD exon floxed allow deletion of NFAT5 upon crosses with lineage-specific Cre drivers (thymocytes and T cells Lck-Cre and CD4-Cre, myeloid LysM-Cre, blood lineages Vav-Cre), and the type I IFN-responsive driver Mx1-Cre (12, 15, 16, 18, 25).	Essentially complete loss of NFAT5 protein confirmed by Western blot in mature T cells (CD4-Cre x Nfat5fl/fl) (15, 16), thymocyte subsets (from DP to SP stages in CD4-Cre and Lck-Cre x Nfat5fl/fl) (25) and BMDM (Vav-Cre and poly I:C-treated Mx-Cre x Nfat5fl/fl) (18). Essentially complete loss of NFAT5 mRNA in peritoneal macrophages of LysM-Cre and Vav-Cre x Nfat5fl/fl mice (18), and BMDM, BMDC, and lymph node and spleen T and B cells of poly I:C-treated Mx-Cre x Nfat5fl/fl (12).	Thymocytes, T lymphocytes, BMDM, BMDC, tissue DCs and macrophages	Impaired proliferation of T cells, cell cycle arrest and defective induction of cell cycle regulators under high salt stress in vitro (15). Altered balance of naïve and memory CD4 and CD8 T cells and reduced homeostatic survival in response to IL-7 in vitro under high salt stress (16). Defective induction of CD24 in response to high salt stress in vivo and in vitro (16). Thymocyte development arrest at the transition from DN3 to DN4 associated with imbalanced expression of prosurvival and proapoptotic regulators (25). Defective induction of Th17 features in activated CD4 T cells in response to high salt (26). In addition, and independently of osmotic stress, activated CD4 T cells in CD4-Cre Nfat5fl/fl conditional knockout mice show altered induction of Treg, Th1 and Th17 responses (26). Diverse osmostress-independent defects in macrophages: reduced activation by LPS, reduced phagocytic and bactericidal capacity against Escherichia coli, altered polarization responses to IFNγ plus LPS (M1) or IL-4 (M2), reduced capacity to stimulate Th1 T cell responses, and reduced anti-tumor activity in BMDM (18). Reduced expression of CIITA and MHCII in macrophages (BMDM, peritoneal, skin) due to lack of NFAT5 binding to a Ciita remote enhancer (12).

NFAT5 knockout mice described in López-Rodríguez et al. (13), Go et al. (14), Küper et al. (17), all show substantial or complete loss of NFAT5 protein in different cell types analyzed, including T cells and macrophages. However, it has been found that mouse embryonic fibroblasts (MEFs) of NFAT5-deficient mouse models express a spliced NFAT5 product that lacks a portion of its DBD and is expressed at comparable protein levels to the wild-type NFAT5 protein (14, 24).