Luther Sanjiv, Associate Professor

Secondary lymphoid tissues, such as lymph nodes and spleen, are the only sites where immune responses against pathogens are efficiently initiated. It is within the T cell rich zone of these organs that dendritic cells present the captured pathogens to recirculating T cells in order to activate the rare antigen-specific T cells. While we have made considerable progress in understanding the biology of dendritic cells and T lymphocytes, we know very little about the stromal cells that form the 'niches' within this unique microenvironment. Over the last four years we have developed the technology to isolate, culture and characterize stromal cells of the T zone at the phenotypic and functional level. We could demonstrate that these cells are indeed critical for efficient T cell homeostasis and activation. The interest of the lab is now focused on dissecting further the development, phenotype and function of these poorly characterized T zone stromal cells, both during homeostasis and disease.

Stromal cells of secondary lymphoid organs

Stromal cells in primary lymphoid organ, such as the bone marrow and thymus, have been extensively studied and shown to be important for the generation of niches allowing the correct localization, differentiation and proliferation of haematopoietic cells. Besides providing a structural framework, they do so by providing several factors such as cytokines, chemokines, adhesion molecules and extracellular matrix proteins.

Much less is known about stromal cells in secondary lymphoid organs. In the last few years it has become clear that the compartmentalization of these organs into B and T cell-rich zones is achieved by specialized resident stromal cells which constitutively produce chemotactic factors (chemokines). B zone stromal cells (also referred to as follicular dendritic cells or FDC) have been characterized phenotypically and functionally and attributed several important functions for B cells, including the production of the B cell attractant CXCL13.

However, their T zone counterpart, called fibroblastic reticular cells (FRC), has been mainly characterized in tissue sections. They resemble fibroblasts, associate with collagen fibres and can be selectively stained using the marker gp38. For a long time FRCs were thought to be mainly responsible for the structural stability of the T zone. In the year 2000 we proposed a more active function for gp38+ T zone stromal cells based on the evidence that a stromal cell type in the T zone represents the major constitutive source of the chemokines CCL19 (ELC) and CCL21 (SLC). These chemokines share the same receptor CCR7 and efficiently attract CCR7-bearing dendritic cells and T cells. The importance of T zone stromal cells and the CCR7 ligands most likely secreted by them is best illustrated in a natural mouse mutant (plt/plt) that we and others have described to lack both CCR7 ligands within lymph nodes and spleen. In plt/plt mice dendritic cells and T cells fail to accumulate in the T zone leading to an inefficient induction of T cell activation.

More recently, FRCs were shown to form a 3-dimensional network to which dendritic cells adhere. Interestingly, migrating T cells use this network as a road system for their continuous migration leading them past the antigen-presenting dendritic cells. Therefore, they are thought to improve the efficiency of immune response induction by bringing antigen-specific T cells together with the antigen-presenting cells. Despite this recent progress a detailed study of FRC was not possible due to the lack of appropriate cell isolation techniques.

Phenotype of T zone stromal cells in lymph nodes

Our histological analysis of lymph nodes revealed that gp38 expression is not restricted to FRC in T zones. Lymphatic endothelial cells in lymph nodes co-express gp38 and CD31 while B zone stromal cells upon activation coexpress gp38 and CD35.

When we tested various cell isolation techniques based on enzymatic digestion of lymph nodes we found that a one-hour-long collagenase digestion released best gp38+CD31-CD35-CD45- cells. These cells had a distinct surface phenotype, including the expression of LTbR and TNF-R1 which are thought to act upstream of CCL19/21 expression. Indeed, sorted FRC expressed very high levels of CCL19 and CCL21 transcripts when compared to 5 other cell populations in LN. Thus, we have developed the first protocol allowing the isolation and ex vivo characterization of CCL19/21+ FRCs. The coexpression of both PDGFR chains (αβ) suggested a mesenchymal origin for these cells. As we observed desmin and α-SMA expression within these cells, their contractile capacity was tested (in collaboration with Boris Hinz, EPFL). Indeed, these cells were able to contract efficiently a thin silicone substrate suggesting they are functional myofibroblasts.

Function of T zone stromal cells in lymph nodes

Our demonstration of CCL19 and CCL21 production by FRC is clear evidence for an important role of these cells in adaptive immunity given that plt/plt mice show a defect in T cell priming. When looking for novel functions of FRC we considered its strategic position in the T zone where T cells spend hours migrating along the FRC network. Recirculating T cells are known to depend on the cytokine IL-7 for survival; however, the organ and cells providing this factor were not known. In situ hybridization analysis revealed a reticular pattern of IL-7 expressing cells throughout the T zone of LNs and realtime PCR on cDNA of sorted LN cells showed the highest IL-7 expression within FRCs. In cell culture FRC were the only cell type able to keep naïve CD4+ and CD8+ T cells alive over several days. Surprisingly, this effect was only partially due to IL-7. The second signal proved to be pertussis-toxin sensitive and mainly due to CCL19. Recombinant CCL19 alone was sufficient to keep naïve T cells alive although to a lesser extent than recombinant IL-7. As an in vivo testfor the role of LNs and FRCs in naïve T cell homeostasis, we blocked LN access for the transferred naïve T cells by interfering with critical integrin or chemokine signals (collaboration with Hans Acha-Orbea, Lausanne). Indeed, such interference led to a similar decrease in peripheral T cell numbers as blocking the survival signal IL-7 itself. To assess the potential role of CCL19 in T cell survival in vivo, wildtype T cells were transferred into CCL19-/- mice which show a normal lymphoid tissue organization (collaboration with Jason Cyster, San Francisco). In a CCL19-deficient environment, naïve T cells disappeared more rapidly than in wildtype mice indicating its non-redundant role in naïve T cell homeostasis. In summary, recirculating T cells appear to 'recharge their batteries' during their several hour long stay within T zones by collecting survival factors such as IL-7 and CCL19 produced by TRCs. Secondary lymphoid organs and in particular FRCs within the LN T zone control the size of the peripheral T cell pool and thereby the available repertoire for an adaptive immune response. Therefore, FRCs are not just structurally important cells but play a central and very active role in adaptive immunity.

Figure 3: Model showing the central role FRC play for recirculating T cells. On the one hand they attract T cells by producing the chemoattractants CCL19 and CCL21. On the other hand they provide them with survival factors, such as IL-7 and possibly CCL19.

Function of T zone stromal cells in disease

In collaboration with the laboratory of B. Ludewig (St.Gallen) we have characterized lymphoid tissue stromal cells during LCMV-WE infection. GP38+ FRCs in spleen were destroyed 8-12 days after infection due to FRC infection by LCMV and their elimination by CD8+ T cells. This destruction led to the disappearance of distinct white pulp cords and was associated with a strong immunodeficiency towards a subsequent VSV infection. Surprisingly, the stromal cell network reappeared within few days leading to white pulp formation and return of immunocompetence. This reconstruction phase depended on lymphoid tissue inducer cells (LTi) that started to proliferate during the phase of acute immunosuppression. Therefore, LTi cells come into play during lymphoid tissue injury and recapitulate processes involved in lymphoid tissue genesis. A key target of LTi cells are presumably the stromal cells which act as tissue organizer cells.

Figure 4: Stromal cell networks of the splenic white pulp on day 0 (left), 12 (middle) and 25 (right) after LCMV-WE infection. CD35+ B zone stroma (in green) and gp38+ T zone stroma (in red) are destroyed completely on day 12 and reconstructed few days later.

Perspective

Given that we can now isolate, culture and characterize FRC at the single cell level opens up many avenues of future research. Our present and future studies aim at improving our understanding of the T zone stromal cells and their role for T cells and dendritic cells in homeostasis and immune response. Importantly, lymphoid infiltrates observed at sites of inflammation or cancer are associated with similar stromal cell networks. Therefore our studies should help in designing intervention strategies to enhance beneficial or suppress harmful immune responses within secondary or tertiary lymphoid tissues by targeting directly the organizer cells, such as the T zone stromal cells.

Current collaborators

Hans Acha-Orbea (Univ. of Lausanne), Jeff Browning (Biogen, Boston), Chris Buckley (Univ. of Birmingham, UK), Jason Cyster (UCSF, San Francisco), Daniela Finke (Univ. of Basel), Matthias Heikenwaelder (Univ. Hospital, Zuerich), Boris Hinz (EPF Lausanne), Lukas Kuehn (ISREC, Lausanne), Dan Littman (NYU, New York), Burkhard Ludewig and Elke Scandella (Kantonal Hospital of St.Gallen), Rob MacDonald (LICR, Lausanne), Ian MacLennan and Kai Toellner (University of Birmingham, UK), Michael Sixt (MPI Munich), Charles Surh (Scripps Clinics, San Diego), Melody Swartz (EPF Lausanne) and Carl Ware (LIAI, San Diego).

Recent publications:

• Tomei, A.A., Siegert, S., Britschgi, M.R., Luther, S.A.1, Swartz, M.A.1 (2009). Fluid flow regulates stromal cell organization and CCL21 expression in a tissue-engineered lymph node microenvironment. J Immunol., 183, 4273-83,  PubMed
• Vogt, T., Link., A., Perrin, J. Finke, D., Luther, S.A. (2009). Novel function for interleukin-7 in dendritic cell development. Blood 113, 3961-3968. PubMed
• Fiorini, E., Ferrero, I., Merck, E., Favre, S., Pierres, M., Luther S.A., MacDonald, H.R. (2008), Cutting edge: Thymic crosstalk regulates delta-like 4 expresion on cortical epithelial cells. J. Immunol. 181, 8199-8203. PubMed
• Britschgi, M., A. Link, T. Lissandrin and S. Luther (2008a) Dynamic modulation of CCR7 expression and function on naive T lymphocytes in vivo. J Immunol. 181, 7681-8. PubMed
• Scandella, E., Bolinger, B., Lattmann, E., Miller, S., Favre, S., Littman, D., Finke, D., Luther, S.A., Junt, T., Ludewig, B. (2008). Restoration of lymphoid organ integrity through interaction of lymphoid tissue inducer cells with the T cell zone stroma. Nature Immunol. 9, 667-675 (cover story). Commentary on this article in Nature Reviews in Immunology 8, 400. PubMed
• Link, A., Vogt, T.K., Favre, S., Britschgi, M.R., Acha-Orbea, H., Hinz, B., Cyster, J.G., Luther, S.A. (2007). Fibroblastic reticular cells in lymph nodes regulate naïve T cell homeostasis. Nature Immunol. 8 (11), 1255-65. Commentary on this article in Nature Reviews in Immunology 7 (11), 839. PubMed
• Luther, S.A., Serre, K., Cunningham, A.F., Khan, M., Acha-Orbea, H., MacLennan, I.C.M., Toellner, K.M. (2007). Recirculating CD4 memory T cells mount rapid secondary responses without major contributions from follicular CD4 effectors and B cells. Eur. J. Immunol. 37, 1476-84. PubMed
• Lang, K.S., Recher, M., Junt, T., Navarini, A.A., Harris, N.L., Freigang, S., Odermatt, B., Conrad, C., Ittner, L.M., Bauer, S., Luther, S.A., Uematsu, S., Akira, S., Hengartner, H., Zinkernagel, R.M. (2005). Toll-like receptor engagement converts T-cell autoreactivity into overt autoimmune disease. Nat. Med. 11, 138-145. PubMed
• Bistrup, A., Tsay, D., Shenoy, P., Singer, M.S., Bangia, N., Luther, S.A., Cyster, J.C., Ruddle, N.H., Rosen, S.D. (2004). Detection of a sulfotransferase (HEC-GlcNAc6ST) in high endothelial venules of lymph nodes and in high endothelial venule-like vessels within ectopic lymphoid aggregates: Relationship to the MECA-79 epitope. Am. J. Pathol. 164, 1635-1644. PubMed
• Luther, S.A., Ansel, K.M., Cyster, J.G. (2003). Overlapping function of CXCR5, IL-7 receptor a and CCR7 in lymph node development. J. Exp. Med. 197, 1191-1198. PubMed
• Finke, D., Luther, S.A., Acha-Orbea, H. (2003). The role of neutralizing antibodies for mouse mammary tumor virus transmission and mammary cancer development. Proc. Natl. Acad. Sci. USA 100, 199-204. PubMed