Dendritic Cells in Granulomas Figure 5. CD4+ T cells maintain contact with CD11c-EYFP+ cells in both acute and chronic granulomas. A, Quantitative analysis of CD4+ T cell contact with CD11c-EYFP+ cells in granulomas from three- and ten-week liver sections. All CD4+ T cells per granuloma section were determined to be in contact (green bars), or not in contact (white bars) with CD11c-EYFP+ cells. For each time point, liver sections from 3 independent mice were used, and 10–15 lesions per section were analyzed. Data are represented as mean +/2 SEM, P,0.0001. B, Fluorescent images demonstrating CD11c- EYFP contact with CD4+ cells. Granulomas are outlined with white dashed lines at 10006 magnification and colors shown depict CD11c-EYFP (green cells), anti-CD4 Alexa 647 (red cells), and DAPI nuclear stain (blue). doi:10.1371/journal.pone.0011453.g005 lymphoid cuff of the granuloma and have high expression of MHCII and T cell stimulatory molecules CD40, CD80 and CD86. However, those from chronic granulomas have low expression of MHCII and co-stimulatory molecules, high expression of T cell inhibitory molecules PD-L1 and PD-L2, and also migrate toward the granuloma center. Most importantly, CD11c+ cells from acute granulomas have a greater capacity to induce IFNc production from CD4+ T-cells compared to those from chronic granulomas. When PD-L expression on CD11c+ cells was blocked ex vivo, their ability to restimulate allogenic CD4+ T cells to produce IFNc was restored to the level observed during acute infection. Although these data demonstrate a direct effect of granuloma DCs on CD4+ T cell IFNc production ex vivo, they do not rule out the possibility that other factors within the granuloma may also influence IFNc production. This CD11c+ shield may induce local tolerance within chronic granulomas, despite the high systemic level of CD4+IFNc+ anti-mycobacterial T cells. The ability of CD11c+ cells to shield mycobacteria from local IFNc T cell responses may be one explanation for the ability of mycobacteria to survive indefinitely in granulomas. IFNc is critical for activation of mechanisms in macrophages that kill engulfed bacteria –these include production of reactive nitrogen and oxygen species, as well as stimulation of phagosome- lysosome fusion [2]. The necessity of mycobacterium-specific IFNc-producing CD4+ T cells for controlling mycobacterial growth is demonstrated in mouse models with ablated CD4+ T- cell responses, HIV infected individuals, and patients lacking functional IFNc receptors, all of which are unable to control Mtb infection [4,42–47]. While it seems rational that improved vaccine strategies have aimed to increase CD4+ T-cell responses, the results have unfortunately been ineffective at achieving bacterial clearance [48,49]. Our work here detailing the presence of an inhibitory DC-like population in chronic granulomas provides a possible explanation for these failures. PD-Ls have been shown to negatively regulate T cells by inhibiting proliferation and cytokine production and are exploited by several microorganisms to evade anti-microbial immunity [50,51]. PD-L expression on antigen presenting cells is also key for the generation of peripheral tolerance [52]. What drives the transition of a population of DCs that stimulate T cells to one that PLoS ONE | www.plosone.org 7 July 2010 | Volume 5 | Issue 7 | e11453