viral infections in vivo [17,18]. Together, these observations have strengthened the notion that TRC help in the induction of T cell responses by accelerating T cell priming and expansion. However, recent reports have suggested that TRC may also negatively regulate T cell responses. TRC were shown to express the inhibitory programmed death ligand 1 (PD-L1) thereby reducing CD8 T cell mediated pathology [19]. TRC also express self-antigens in the context of MHC class I thereby promoting CD8+ T cell tolerance [20,21] (reviewed in [22,23]). In addition, stromal cells isolated from neonatal or adult spleen were shown to induce over 1–2 weeks the development of DC that inhibit T cell proliferation in vitro. The spleen contains many stromal cell subsets and the precise identity of the cells used as well as their localization relative to DC and T cells has remained unclear [24,25]. Together, these observations indicate that lymphoid tissue stromal cells may also inhibit T cell responses. Currently, the exact role of LN TRC in T cell activation and differentiation is not known. This is in part due to the difficulty of isolating sufficient numbers of TRC for in vitro experiments and the lack of appropriate tools to investigate TRC in vivo. Here we used a combination of in vitro and in vivo approaches to study the effect of TRC on CD8+ T cell priming by antigen-pulsed DC. We demonstrate that TRC diminish T cell expansion by releasing NO. They share this property with a subset of DC. We show that NO production by TRC and DC is strongly dependent on cytokines from activated T cells suggesting a negative feedback loop once T cell priming has started. Our in vivo findings using Inos2/2 mice indicate that TRC and DC limit the speed of T cell expansion possibly to ensure organ functionality during the early phase of acute lymph node swelling. Results Lymph node TRC dampen T cell expansion To dissect the role of TRC in T cell activation and differentiation, we initially adopted a reductionist approach: T cells were co-cultured with antigen-pulsed bone-marrow derived DC (BM-DC), either in the presence or absence of TRC. Given the difficulties in isolating pure TRC populations, several independent TRC cell lines were established from peripheral lymph nodes (pLN = pool of inguinal, axillary and brachial LN) of C57BL/6 wild-type (WT) mice (referred to as ‘pLN1’ and ‘pLN2’) or GFP-expressing mice (‘GFP-pLN’). All these lines showed typical fibroblastic morphology and a uniform surface marker expression pattern comparable to ex vivo isolated TRC (Fig. S1 and data not shown). In contrast to ex vivo TRC [9] cell lines expressed only low levels of Ccl19, Ccl21 and Il7 transcripts. To circumvent this caveat, initial experiments included exogenously added CCL19, CCL21 and IL-7 protein with no difference in the outcome (data not shown). To study T cell priming CD45.1+ congenic ovalbumin (OVA)- specific OT-I T cell receptor (TCR) transgenic CD8+ T cells were labeled with the proliferation dye carboxyfluorescein succinimidyl ester (CFSE), mixed with unspecific WT T cells (CD45.2+) in a ratio of 1:50, and cultured together with antigen-pulsed BM-DC on top of an adherent layer of the TRC line. TRC were previously irradiated to limit their proliferation and nutrient consumption. Surprisingly, the total OT-I cell number was strongly decreased in presence of the TRC line pLN2 (Fig. 1A). Using CFSE dilution to measure T cell proliferation, both the percentage and number of dividing OT-I T cells were strongly reduced in the presence of pLN2 (Fig. 1B). The increase in cell size (FSC) and CD44 expression (Fig. 1C) as well as the loss of CD62L expression (Fig. 1D) occurred in presence of TRC but to a reduced extent. Lymph Node Fibroblasts Limit T Cell Expansion The co-cultures were supplemented with IL-7 and IL-2, so a lack of known pro-survival factors for na¨ıve and activated T cells is unlikely to be the cause. In line with that, the number of na¨ıve, undivided OT-I T cells was not affected by the TRC presence, nor was the up-regulation of the high-affinity receptor chain for IL-2, CD25, on dividing T cells (Fig. 1E). Importantly, several other fibroblast lines established independently from LN and spleen [26] not only shared the same surface phenotype (Fig. S1) but also the inhibitory effect on T cell expansion with a reduction in proliferating OT-I T cell numbers of 60–90% (Fig. 1F). Impor- tantly, primary TRC isolated from na¨ıve pLN limited T cell expansion at least as strongly as TRC lines (Fig. 1G). Even TRC isolated from pLN of mice immunized 3 days earlier with NP- CGG in Montanide adjuvant maintained these inhibitory properties (Fig. 1G). Next, we examined the effect of TRC on CD8+ T cell differentiation. OT-I T cells primed in presence of TRC expressed intracellular interferon gamma (IFNc) protein (Fig. 2A) and killed target cells (Fig. 2B), although with markedly reduced efficiency (Fig. 2B). Together these results demonstrate that the presence of TRC during T cell activation diminishes the expansion or survival of CD8+ T cells and to a lesser extent their differentiation into effector cells. Fibroblasts from non-lymphoid organs also attenuate T cell proliferation It has been reported that murine and human fibroblasts can have anti-proliferative effects on activated T cells, similar to mesenchymal stem cells (MSC) and certain tumor lines [27,28,29,30,31]. Therefore we tested in our system the inhibitory potential of several fibroblastic cell lines established de novo from different non-lymphoid organs, as well as their ex vivo equivalents (CD452CD352CD312EpCAM2gp38+; see Fig. S2), and com- pared them to our pLN2 line. In addition, epithelial-like cells from the epidermis and kidney, a MSC line and two tumor cell lines (MC38 colon carcinoma and B16-F10 melanoma) were tested. All cell lines inhibited OT-1 T cell proliferation to an extent comparable with pLN2 (Fig. 3 A–D), highlighting that inhibition of T cell expansion is a property common to many cell types, including fibroblasts from various non-lymphoid organs. TRC directly inhibit T cell proliferation and render DC less stimulatory MSC have been shown to inhibit T cell proliferation by intracellular expression of enzymes such as indoleamine 2,3- dioxygenase (IDO) and arginase-1, surface expression of PD-L1, or by secretion of molecules like NO, prostaglandin E2 (PGE2), IL- 10 and TGFb(reviewed in [30,31]). The proposed pathways included direct inhibition of T cell proliferation or conversion of DC to a non-stimulatory or suppressive phenotype (reviewed in [31,32]). To examine whether TRC can directly interfere with T cell proliferation pLN2 were co-cultured with anti-CD3/CD28 bead-stimulated T cells. Clearly, TRC diminished the number of dividing T cells but the inhibitory effect was only half of what we had observed in the BM-DC-induced T cell activation assay (Fig. 4A). Next, potential effects of TRC on the stimulatory capacity of antigen-pulsed BM-DC were investigated by co- culturing first pLN2 with BM-DC before incubating these ‘TRC- conditioned’ BM-DC alone with T cells. The capacity to stimulate T cell proliferation was reduced by half in conditioned relative to unconditioned BM-DC (Fig. 4B). Together these results indicate that TRC limit T cell expansion in two additive ways, by making DC less immunogenic and by directly inhibiting T cell proliferation or survival. PLoS ONE | www.plosone.org 2 November 2011 | Volume 6 | Issue 11 | e27618