been shown to play a key role in the inhibition of mycobacterial growth in vitro [39] and in granuloma formation in vivo [40,41,42]. However, although the role of TNF in the pulmonary inflamma- tory response to MTb infection has been extensively demonstrated in murine models, the cellular source of TNF and the relative contribution of T cell- versus macrophage- or DC-derived TNF in protection against MTb and in clinical TB infection have not been defined. The transcription factor NFATp (also known as NFAT1 and NFATc2) is a major regulator of T cell-derived cytokine gene expression [43,44,45,46]. While NFATp plays a critical role in the transcription of the IFN-c gene [45] and the TNF gene [47,48] in T cells, it is not required for TNF transcription in the monocyte/ macrophage lineage [33,48]. This was strongly corroborated in vivo in mice by experiments that showed that NFATp2/2 mice were less susceptible to T cell superantigen-induced toxic shock, but were equally susceptible to lipopolysaccharide (LPS)-induced toxic shock [48]. Thus, the role of NFATp in the immune response to MTb infection, particularly in distinct cell types, presents a question of considerable interest that is tractable in the mouse model. Here, we show that NFATp2/2 mice have a dramatic increase in mortality after MTb infection relative to wild-type mice (mean survival of 119 days versus 205 days, p = 0.0042), and that they had an MTb burden in their lungs approximately three orders of magnitude higher at the symptomatic endpoint relative to the lungs of wild-type mice at the time of sacrifice (8.66108 CFU/lung versus 86105 CFU/lung). We show that in the lungs of MTb- infected NFATp2/2 mice, there is a significantly decreased level of IFN-c mRNA, while there in an initial increase of TNF mRNA expression that correlates with increased serum levels of TNF protein. Using in vitro analysis, we show that transcription and protein expression of TNF or IFN-c from na¨ıve or total CD4+ T cells was significantly impaired in NFATp2/2 mice, while TNF transcription and protein expression from DCs stimulated by TB was identical in NFATp2/2 and in wild-type mice. Furthemore, we show that blocking TNF with the pharmacological neutralizing agent Enbrel has a deleterious impact on survival of wild-type and NFATp2/2 mice when administered 6 weeks rather than 12 weeks following MTb infection, with a greater impact upon NFATp2/2 mice, indicating an early role for TNF derived from DC and monocytic cells in susceptibility to TB. Taken together, these results indicate that through its key role in regulating TNF and IFN-c, NFATp controls a major checkpoint in T cell cytokine expression required for control of MTb infection in the mouse. Materials and Methods Mice BALB/c NFATp+/2 mice (a generous gift from L.H. Glimcher, Harvard School of Public Health, Boston, MA) were bred and NFATp2/2 and wild-type (WT) littermates were genotyped by PCR as previously described [43] until a colony of NFATp2/2 was established. NFATp2/2 and WT (BALB/c) mice were harem- bred and maintained in pathogen-free conditions. Mice were used for experiments including infection at the age of 6–7 weeks. Their care was in strict accordance with NIAID and NIMR institutional guidelines, and all efforts were made to minimize suffering. Bacteria and infection MTb (HN878 clinical strain) was grown in 7H9 broth supplemented as recommended by the manufacturer (Becton Dickinson, Sparks MD). All animal infection with aerosolized MTb was performed as previously described [49,50] under BSL3 NFATp and TB Infection conditions in the animal facilities at NIAID. Enbrel (Wyeth Pharmaceuticals) and human isotype control antibody IgG1 (R&D Systems) were diluted in PBS and given intraperitoneally twice a week at 0.01 mg/mouse/dose, mimicking the physiological human dose (0.4 mg/kg) administered for rheumatoid arthritis. Experiments to determine differences in survival time were repeated at least twice. Necropsy and Histology Mice were sacrificed at described time points and the lungs, spleen, and any observed lymph nodes were removed aseptically and divided into portions for bacterial load determination, leukocyte cell typing and enumeration, RNA preparation, and histology. Histology tissues were fixed in 10% neutral buffered Formalin. Tissues were embedded in paraffin and 5–10 mm sections were cut and stained with H&E and Ziehl-Neelsen acid- fast stain to localize the acid fast MTb. FACS analysis of cell surface markers Age-matched NFATp2/2 and BALB/c mice (12 each) were infected with 30.3612 (SD) colony forming units of MTb strain HN878 per mouse. Lung cells were prepared from naive and infected mice at 2, 4, and 6 weeks post infection by crushing and pressing though a cell strainer (Becton Dickinson Labware, Lincoln Park NJ), lysing the erythrocytes with NH4CL-tris, and washing twice with DMEM without phenol red and resuspended to 26106 per mL in PBS containing 0.1% bovine serum albumin and 20% mouse serum. Cells from 2–4 mice were pooled if the number of viable cells recovered per mouse was less than 16106 and microspheres (Caltag laboratories, Burlingame CA) were added in order to determine the absolute cell number. Pooling was required for na¨ıve mice at all time points and for the 2 and 4 week time points. The cells were stained for 30 min with antibodies (0.2 mg/106 cells) against CD3 (clone 17A2, cy chrome) and CD11b (M1/70, FITC), and CD45 (clone 30-F11, R-PE), or CD8a (53-6.7, FITC), CD4 (clone RM4-5, cy chrome), and CD45 (clone 30-F11, R-PE) (BD Biosciences Pharmingen) in PBS containing 0.1% bovine serum albumin and 20% mouse serum. The cells were fixed with fixation medium (Caltag Laboratories) for 12 to 16 hours and analyzed by Fluorescence-activated cytometry using software provided by with the instrument (BD Immunocytometry Systems, San Jose, CA). The experiment was repeated with an additional set of infected mice since more mice were required per experiment than anticipated and the results were pooled. Isolation of murine primary T cells For T cell purification, spleen cell suspensions from na¨ıve (uninfected) mice were first depleted of APCs and CD8+ T cells using anti-B220, anti-CD8, and anti-CD11b antibodies. Total CD4+ T cells were purified by sorting for CD4+ and na¨ıve T cells were purified by sorting for CD4+CD62L+CD45RBhigh (98%) using a MoFlo flow cytometer (DakoCytomation). Cells were stimulated in vitro with anti-CD3/CD28 as described [51,52]. Cytokine measurement by intracellular cytokine staining (ICS) Th1 and Th2 CD4+ T cell subsets were prepared by polarizing FACS-purified naive CD4+ T cells from NFATp2/2 or WT (BALB/c) mice in the presence of IL-12 and anti-IL4 (Th1 conditions) or IL-4 and anti-IFN-c (Th2 conditions) for six days as previously described [51,52]. Cells were then stimulated with a combination of anti-CD3 and anti-CD28 antibodies for 4 h in the PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e41427