Figure 3. Infiltration of leukocytes into the lungs after MTb infection is similar in NFATp2/2 and wild-type mice. Age- matched NFATp2/2 and BALB/c (WT) mice were infected with Lung aerosolized cell samples HN878 from MTb MTb-infected strain at ,30 WT CFU/mouse (N) and NFATp2/2 as described (&) mice [50]. were obtained at 2, 4, and 6 weeks post-infection, and from uninfected WT (#) and NFATp2/2 (%) mice at times corresponding to 1, 2, 4, and 6 weeks post-infection, and the number of cells per lung for each indicated cell population (mean of 3 to 4 samples 6SD) was determined by FACS for samples at the indicated time points (A–F). The number of cells recruited to the lung during the course of MTb infection was determined for total leukocytes (A), CD11b+ cells, i.e., monocytes (B), total lymphocytes (C), CD3+ T cells (D), CD4+ T cells (E) and CD8+ T cells (F). doi:10.1371/journal.pone.0041427.g003 Loss of NFATp does not decrease TNF production by BM- DC but does decrease TNF and IFN-c production by CD4+ T cells in vitro. Since TNF produced by T cells is dependent upon NFATp, the finding that NFATp2/2 mice were able to produce high levels of TNF during acute TB infection indicated that cells other than T cells were able to vigorously produce TNF in response to TB in an NFATp-independent manner. Given the NFATp and TB Infection central role that DC play in phagocytosis and antigen presentation during acute TB infection in lungs [60,61] and the high levels of circulating TNF in these mice, we next investigated whether DC production of TNF was impacted by NFATp deletion. We derived BM-DC in vitro from WT and NFATp2/2 mice, stimulated them in vitro with a TB sonicate, and analyzed TNF mRNA and protein levels. Strikingly, the level and kinetics of TNF transcription (up to 6 hours) (Figure 5A) and protein production (up to 24 hours) (Figure 5B) are not impacted by NFATp deficiency in BM-DC. By contrast, when we examined TNF gene expression in primary na¨ıve and total CD4+ T cells from WT and NFATp2/2 mice activated with anti-CD3/CD28 antibodies, TNF mRNA levels were significantly lower in cells from NFATp2/2 animals in response to both first and second cellular stimulations as compared to their WT counterparts (Figure 5C). Consistent with our previous findings with bulk CD3+ T cells [48], these transcrip- tional differences in TNF were more pronounced at early time points (within 3 hours) post stimulation (Figure 5C). TNF protein levels were also lower in na¨ıve and total CD4+ T cells at all time points measured, consistent with the RNA results (Figure 5C). In the case of IFN-c, undifferentiated populations of T cells, na¨ıve and total CD4+ cells from both WT and NFATp2/2 mice, produced very low IFN-c mRNA in response to anti-CD3/CD28 stimulation (Figure 5D) consistent with the fact that the major source of IFN-c is a differentiated population of T cells (Th1). When these same T cell populations were stimulated again with the same antibodies after 6 days of culture in neutral conditions, IFN-c production was observed in the cells from WT animals consistent with their spontaneous differentiation into Th1 cells. In these T cell populations we observed a pronounced difference in IFN-c gene expression and protein production between WT and NFATp2/2 mice at all time points up to 72 and 96 hours, with the NFATp2/2 T cells producing significantly less IFN-c mRNA and protein at all time points (Figure 5D). For comparison, a previous study showed that at day 14 post-BCG infection, mediastinal lymph node cells from NFATp2/2 mice stimulated with purified protein derivative (PPD) from MTb or with a combination of anti-CD3 and IL-2 secreted less IFN-c, but not less IL-5, than WT cells, consistent with a reduced Th1 response [62]. As noted previously, while T cells from NFATp2/2 mice have an early defect in IL-4 gene expression following activation, they display a strong tendency to differentiate into Th2 cells [43,44,45,63]. This can be ascribed to prolonged maintenance of IL-4 mRNA expression in NFATp2/2 T cells; indeed, T cells from NFATp2/2IL-42/2 mice display wild-type levels of Th2 differentiation [44,45]. We previously demonstrated, using intra- cellular cytokine staining and FACS analysis, that expression of TNF protein upon activation by anti-CD3/CD28 of both Th1 and Th2 cells is reduced in NFATp2/2 mice [34]. Using this approach, we thus next analyzed Th1 and Th2 cells from wild- type and NFATp2/2 mice for expression of TNF, IFN-c, and IL- 4 following activation with anti-CD3/CD28 (Figure 6). IFN-c, as a canonical Th1 cytokine, is expressed almost exclusively in Th1 cells, and this expression is markedly reduced in Th1 cells from NFATp2/2 mice (Figures 6A and 6B). Similarly, IL-4 is predominantly expressed in WT Th2 cells, and its expression is strongly attenuated in NFATp2/2 Th2 cells (Figure 6A). In agreement with our previous results [34], TNF is expressed in Th1 and, to a lesser extent, Th2 cells, and the reduction of TNF expression in NFATp2/2 cells is much greater in Th2 cells relative to Th1 cells (Figure 6B). Thus, the decrease in IFN-c and TNF mRNA and protein expression in activated NFATp2/2 CD4+ T cells is consistent with reduced IFN-c expression in Th1 cells and PLoS ONE | www.plosone.org 5 July 2012 | Volume 7 | Issue 7 | e41427