Introduction
Highly structured microenvironments in secondary lymphoid organs (SLOs) maximize the efficacy of immune responses to viral infection1, 2, 3, 4, 5, 6. In the spleen, blood-borne antigens are trapped and processed in the marginal zone by dendritic cells (DCs), macrophages and marginal zone B cells7. The territory of T cell and B cell zones is defined by constitutive chemokines, whereby local expression of the chemokine CXCL13 forms the B cell zone8, 9 and expression of the chemokines CCL19 and CCL21 shapes the T cell zone6, 10. In addition, CCL19 and CCL21 contribute to the positioning of macrophages in the marginal zone11. In the T cell zone, mature DCs prime naive T cells, whereas in the B cell zone, antibody responses and germinal center reactions are initiated.
Nonhematopoietic stromal cells form the backbone of SLO microarchitecture by producing CXCL13, CCL19 and CCL21 (ref 12). Follicular DCs represent B cell zone stromal cells12, 13, whereas T cell zone stromal cells are fibroblastic reticular cells (FRCs), which form continuous sheaths around a meshwork of collagen fibers and function as a scaffold for the T cell zone13. FRCs are multifunctional cells that produce constitutive chemokines, generate support structures for migrating T cells and DCs14 and form a conduit system that distributes small soluble antigens throughout the SLO parenchyma15, 16, 17. Moreover, FRCs enhance the survival of naive T cells by producing interleukin 7 (IL-7)18, present peripheral antigen to T cells19 and support the differentiation of regulatory DC subsets20.
The cellular and molecular basis for the generation of SLO microstructures during embryonic development and the early postnatal period has been studied extensively. CD3-
CD4+CD45+ lymphoid tissue–inducer (LTi) cells are among the first hematopoietic cells to colonize lymphoid tissue anlagen during embryonic development, where they interact with mesenchymal lymphoid organizer cells21, 22. Ligation of the lymphotoxin 
-receptor (LTR; A001440) on mesenchymal lymphoid organizer cells by lymphotoxin 
12 (LT12), expressed on LTi cells, is a mandatory event for SLO formation23, 24, 25. The retinoic acid–related orphan receptors ROR
and RORt are believed to function as 'master transcription factors' for the generation of LTi cells26. Although LTi cells can still be found in adult SLOs25, 27, 28, their function in fully developed SLOs is not completely understood.
Infection with various pathogens can be associated with a profound loss of immunocompetence29, 30. Given the many functions of FRCs, interference of pathogens with the integrity and function of FRC networks may render the host particularly vulnerable to secondary infections. Indeed, infection with human and simian immunodeficiency virus31, 32 or the intracellular pathogen Leishmania donovani33 can lead to disruption of SLO microanatomy, and it is reasonable to assume that altered lymphoid microarchitecture is a chief factor underlying infection-associated immunodeficiency. It is thus important to identify the cellular participants and the molecular pathways involved in the successful restoration of lymphoid microenvironments after immunopathological infection.
Here we show that T cell zone FRCs were infected efficiently by lymphocytic choriomeningitis virus (LCMV) in vitro and in vivo. The LCMV-induced CD8+ T cell–mediated immunopathological process resulted in complete destruction of the FRC network and concomitant loss of immunocompetence. Reacquisition of immune responsiveness required rebuilding of the lymphoid microarchitecture. The integrity of lymphoid microenvironments was restored through the proliferation of LTi cells and productive, LTR-dependent interaction of LTi cells with lymphoid stromal cells. Our work shows that LTi cell–stromal cell crosstalk continues into adulthood and is an important mechanism for restoring SLO integrity.
Results
LCMV-mediated loss and restoration of the FRC network
LCMV is a noncytopathic virus with strong tropism for SLOs. LCMV infection is controlled by activated cytotoxic T lymphocytes (CTLs) that eliminate target cells through perforin-dependent and perforin-independent cytoxicity mechanisms34, 35. LCMV infection–associated immunopathology leads to the loss of infected macrophages and DCs in SLOs36, 37, whereas B cells secreting neutralizing antibodies38 are spared from CTL-mediated elimination. To assess the extent to which the stromal cell network is affected by CTL-mediated immunopathological damage, we infected C57BL/6 mice with a low dose of the LCMV strain WE, which shows intermediate replication kinetics39 and infects both macrophages and DCs. Other LCMV strains, such as Armstrong or clone 13, are characterized by considerable tropism to either splenic red pulp macrophages or CD11c+ DCs, respectively40. Immunofluorescence analysis showed that complete loss of the white pulp structure (Fig. 1a) coincided with the phase of maximum activation of virus-specific CD8+ T cells, which occurred around day 8 after infection1, 41 (Supplementary Fig. 1a,b online). During the contraction phase of the CTL response that followed viral clearance, lymphoid structures became partially reorganized (Fig. 1a, day 16), but marginal zones and distinct T cell and B cell areas were not fully rebuilt until almost 4 weeks after infection (Fig. 1a, day 25). Most notably, this cycle of destruction and restoration of lymphoid organization strongly affected the podoplanin (gp38)–positive FRC network of the T cell zone. The collapse of the gp38+ FRC network was mediated by CD8+ T cells, as the lymphoid microarchitecture, including the FRC scaffold, remained intact in mice depleted of CD8+ T cells. It seemed that both perforin-dependent and perforin-independent immunopathological mechanisms mediated FRC loss, as infection of perforin-deficient mice with LCMV led to disruption of the lymphoid architecture (Supplementary Fig. 2 online).
Figure 1: Disruption and restoration of lymphoid organization during LCMV infection.
(a) In situ analysis of spleen sections from C57BL/6 mice infected with 200 plaque-forming units of LCMV WE. Top, B cell areas, T cell area and the marginal zone, identified with anti-B220 (red), anti-Thy1.2 (blue) and anti-MOMA-1 (green), respectively. Bottom, B cell and T cell zones distinguished by staining with anti-B220 and anti-gp38. Scale bars, 100 
m. Data are representative of three mice per group. (b,c) Analysis of LCMV infection–associated immunosuppression in C57BL/6 mice (n = 3 mice per group) infected with 200 plaque-forming units of LCMV and immunized (challenged) with 5 g recombinant VSV-G (time after LCMV infection, above graphs). (b) VSV-neutralization assay of neutralizing VSV-specific IgM and IgG. (c) Time-course analysis of maximum neutralizing VSV-specific IgM responses (top; day 4 after challenge) and IgG responses (bottom; day 14 after challenge). Data represent the mean values (
s.e.m.) of titers from three mice per group in two experiments.
The FRC network most likely facilitates T cell–DC interactions, as T cells have been found to 'crawl' along FRC strands14. To assess whether the T cell zone stroma depletion noted during LCMV infection resulted in impaired immune responsiveness, we immunized mice with recombinant glycoprotein from vesicular stomatitis virus (VSV-G). Immunization with VSV-G leads to T helper cell–dependent B cell activation with immunoglobulin class switching around day 6, at which time immunoglobulin M (IgM) is no longer detectable in the serum. If T helper cell activation and/or T cell–B cell 'collaboration' is impaired5, class switching is delayed and IgM is present for a longer period. Both IgM and IgG responses after immunization with VSV-G are dependent on productive DC–T helper cell interactions5. Challenge with VSV-G at various times after LCMV infection (Fig. 1b,c) showed that loss and gain of immunocompetence followed the same kinetics as those of disruption and reorganization of the FRC network; that is, antibody responses to VSV-G were absent around day 8 after infection and could be elicited only during the reconstruction phase after day 12 of LCMV infection. The lack of functional B cell responses after day 8 of LCMV infection was not due to lack of sufficient B cells, because adoptive transfer of B cells from naive C57BL/6 mice at the peak of the CTL response to LCMV (day 8) did not enhance the B cell response to the secondary antigen VSV-G (Supplementary Fig. 3 online). These data collectively suggest that cells of the FRC network suffered immunopathological damage and that integrity of the stromal network is an important factor for the maintenance of immunocompetence.
Elimination of T cell zone stromal cells by CTLs
To study the effect of LCMV infection on FRCs in more detail, we established short-term stromal cell cultures from adherent cells in the low-density fraction of splenocytes. The cultured FRCs had a homogeneous fibroblastic morphology (Fig. 2a), and the presence of gp38 together with expression of the integrin VCAM-1 and an absence of expression of the macrophage marker CD11b or the adhesion molecule PECAM-1 indicated high FRC purity and lack of macrophage and/or endothelial cell contamination (Fig. 2a and data not shown). After in vitro LCMV infection, stromal cells began to upregulate the integrins ICAM-1 and VCAM-1 (Fig. 2b), which indicated that LCMV replication in these cells led to their stimulation. In addition, we monitored productive infection of stromal cells in vitro by assessing secretion of viral progeny into the supernatant (data not shown) and staining for intracellular LCMV nucleoprotein (Fig. 2c). FRC cultures infected with LCMV in vitro were efficiently lysed by virus-specific CTLs (Fig. 2d), which further supported the idea that elimination of stromal cells can be mediated by LCMV-induced CTLs in vivo.
Figure 2: CTL-mediated lysis of T cell zone stromal cells in vitro.
(a) Fluorescence microscopy (left) and flow cytometry (right) of stromal cell cultures. Scale bar, 10 m. Numbers in quadrants indicate percent cells in each. Data are representative results from three independent experiments. (b) Flow cytometry (left) of gp38+ stromal cells left uninfected (–LCMV) or infected in vitro for 48 h with LCMV at a multiplicity of infection of 0.1 (+LCMV). Numbers in quadrants indicate percent cells in each. Right, flow cytometry quantification of ICAM-1 and VCAM-1 in gp38+ stromal cells. MFI, mean fluorescence intensity. **, P < 0.005; *, P < 0.05. Data are representative of three independent experiments. (c) Fluorescence microscopy of uninfected and LCMV-infected stromal cells with the LCMV nucleoprotein–specific antibody VL4. Scale bar, 10 m. Data are representative results from one of three independent experiments. (d) Standard 51Cr-release assay of the lysis of LCMV-infected stromal cells by splenocytes from C57BL/6 mice acutely infected with LCMV (day 8 after infection) as effectors (+LCMV). Uninfected stromal cells alone (No peptide) or loaded with 10 M LCMV peptide gp33 (+gp33) serve as negative or positive controls, respectively. E:T, effector cell/target cell. Data are from one experiment of two with similar results.
To confirm that interpretation, we infected CD8-depleted mice with LCMV and monitored key markers of SLO morphology (Supplementary Fig. 2). Indeed, lymphoid structures remained intact in CD8-depleted mice and gp38+ FRCs showed very positive staining for LCMV nucleoprotein during infection (Fig. 3a). The susceptibility of FRCs to infection with LCMV WE is compatible with the presence of LCMV clone13 antigen in T cell zone FRCs expressing the marker ER-TR7 (ref. 42). To further substantiate the link between LCMV infection and loss of gp38+ FRCs, we analyzed the content of stromal cells in the CD45- fraction of splenocytes during the course of LCMV infection. Whereas CD45- cell populations in naive mice included about 60% gp38+VCAM-1+ stromal cells, these cells nearly completely disappeared on days 8–12 after infection (Fig. 3b). It is noteworthy that loss of FRCs was a feature exclusive to infection with LCMV WE; infection with mouse cytomegalovirus (MCMV) or VSV did not lead to disruption of lymphoid architecture (Supplementary Fig. 4a online), loss of FRCs (Supplementary Fig. 4b) or considerable impairment of immune responsiveness (Supplementary Fig. 4c). Overall, these data indicate that CTL-mediated effects on LCMV-infected stromal cells led to destruction of the FRC network.
Figure 3: Loss of T zone stromal cells in LCMV-infected mice.
(a) Immunofluorescence histology of the presence of infected T cell zone stromal cells in spleens from C57BL/6 mice depleted of CD8+ T cells and subsequently infected with LCMV, analyzed on day 4 after infection by costaining with anti-gp38 and anti–LCMV nucleoprotein (VL4). B, B cell area (dashed lines); T, T cell area (boxed). Scale bars, 50 m. Data are representative images from two independent experiments. (b) Percent VCAM-1+gp38+ cells in the CD45-
fraction of splenocytes at various times after LCMV infection (left), and total number of CD45-
VCAM-1+gp38+ cells in spleens from naive and LCMV-infected mice on day 8 after infection (right). Data represent mean values ( s.e.m.) of three mice per group. (c) RT-PCR analysis of the expression of constitutive cytokines in LCMV-infected spleens at various times after infection (n = 3–8 mice per time point). Values are relative to mRNA expression in naive C57BL/6 mice. Data are representative of two experiments.
Initiation of a 'transcriptional reorganization' program
As FRCs are the main producers of the constitutive chemokines CCL19 and CCL21 (ref. 43), we assumed that the destruction of FRC networks during LCMV infection would lead to a profound loss of these chemokines. In fact, quantitative PCR of whole-spleen RNA showed that, like infection with LCMV Armstrong44, infection with LCMV WE was also associated with a transient loss of expression of CCL19 and CCL21 (Fig. 3c). Notably, infection with other viruses such as with MCMV or VSV did not elicit such a substantial alteration in the expression of constitutive chemokines (Supplementary Fig. 4d). Overall, the loss and gain of expression of constitutive chemokines during LCMV infection followed the same pattern as that noted for FRC loss and immune responsiveness. To further explore the link between the profound alterations in the cellular constitution and altered gene expression, we did 'custom-made' gene array analyses to assess the expression pattern of 'candidate genes' involved in the development of lymphoid organs. In addition to the downmodulation of CCL19 mRNA and CCL21 mRNA, we found a substantially less IL-7 mRNA in spleens (Supplementary Fig. 5 online) and lymph nodes (Supplementary Fig. 6 online) affected by the LCMV-induced disruption of lymphoid architecture. The loss of stromal cell–associated genes was paralleled by a profound increase in the abundance of mRNA encoding members of the tumor necrosis factor (TNF) or TNF receptor 'superfamily', including LT, LT, LTR, LIGHT, TNFRI and TNFRII (Fig. 4a and Supplementary Fig. 5), as well as mRNA encoding adhesion molecules, such as VCAM-1, ICAM-1 and 2 integrins (Fig. 4b). All these gene products are essential during the development of lymphoid tissue and are critical for the crosstalk between stromal cells and LTi cells7. It therefore seems that the substantial population expansion of effector CTLs and the increase in their activity during acute LCMV infection in adults, reflected by the upregulation of genes encoding molecules involved in inflammation and cytotoxicity (Fig. 4c), are accompanied by a 'transcriptional reorganization' program reminiscent of the one operating during lymphoid organ ontogeny in the embryo.
Figure 4: Gene-expression patterns in LCMV-infected spleens.
Low-density array-based quantitative RT-PCR analysis of genes required for lymphoid organ development and organization (a), genes encoding adhesion molecules (b) and genes encoding inflammatory mediators (c) in spleens from C57BL/6 mice infected with LCMV (n = 3–8 mice per group). Each sample was analyzed in duplicate, and values are relative to mRNA expression in naive mice. Data are representative of two experiments.
Full size image (76 KB)LTi cell–FRC interaction during LCMV infection
LTi cells are essential for the development of lymphoid organs and have been linked to maintenance of the T cell area in the spleen25. The continuous need for SLO integrity during infection and the transcriptional profile of LCMV-infected spleens suggested that LTi cells might be involved in the reconstruction of the lymphoid microarchitecture in adults. Indeed, LCMV infection was associated with considerable accumulation of CD45+CD4+IL-7R+ and lineage-negative (CD3-
B220-
CD11c-
) cells in both lymph nodes and spleen (Fig. 5a). Maximum LTi cell accumulation in SLOs was between days 8 and 12 after infection (Fig. 5b); that is, at the peak of immunopathological damage. To investigate whether the increase in LTi cells during LCMV infection was due to proliferation or recruitment of the cells, we pulsed naive and LCMV-infected mice with bromodeoxyuridine (BrdU) on day 8 and day 12 after infection. These assays showed enhanced proliferation of LTi cells during the course of LCMV infection (Fig. 5c). The DNA content in LTi cells on day 10 after infection indicated that LTi cell proliferation in these conditions occurred outside SLOs (data not shown). Nevertheless, the accumulation of LTi cells during the course of virus infection seemed to be specific for LCMV, as infection with MCMV or VSV did not lead to such considerably higher LTi cell numbers in SLOs (Supplementary Fig. 4e).
Figure 5: Accumulation and proliferation of LTi cells in SLOs after LCMV infection.
(a) Flow cytometry of CD45+CD4+IL7-R+ LTi cells. Gates were set on the lineage-negative (CD3-
CD11c-
B220-
) and CD4+ population of splenocytes (boxed area, left), and CD45+ cells expressing IL7-R were acquired as LTi cells. Data are representative results from three independent experiments (n = 3 mice per group). (b) Time-course analysis of CD45+CD4+IL7-R+ cells in the lineage-negative fraction of splenocytes (left) and cells from the inguinal lymph node (ILN; right). Data represent mean values ( s.e.m.) of two experiments with three to nine mice per group. (c) Flow cytometry of BrdU incorporation to assess LTi cell proliferation in spleens and inguinal lymph nodes of mice that received intraperitoneal injection of 2 mg BrdU on day 8 after LCMV infection and BrdU (0.8 mg/ml) in their drinking water on days 8–12, analyzed on day 12. -
LCMV, uninfected mice treated with BrdU as described above. Numbers above bracketed lines indicate percent proliferating cells. Right, pooled data. **, P < 0.005. Data are representative of three independent experiments with three mice per group.
To directly determine the function of LTi cells in the reconstruction of the stromal cell network in LCMV-infected mice, we created chimeric mice by using bone marrow from ROR-deficient (Rorc-
/-
) mice26 to reconstitute lethally irradiated wild-type C57BL/6 (B6) mice (Rorc-
/-
B6 chimeras). As expected, Rorc-
/-
B6 chimeras had normal numbers of DCs (CD11c+), T cells (CD4+ and CD8+) and B cells (B220+) in the spleen, but lacked LTi cells (Fig. 6a). Most notably, these chimeric mice had normal lymphoid organization in the spleen (Fig. 6b) and in lymph nodes (data not shown). Immunoreactivity in Rorc-
/-
B6 chimeras after LCMV infection was similar to that of control chimeras of lethally irradiated wild-type C57BL/6 mice reconstituted with wild-type C57BL/6 bone marrow (B6B6 chimeras), as shown by the population expansion of LCMV-specific CD8+ T cells (Supplementary Fig. 7 online). In spleens of both B6B6 and Rorc-
/-
B6 chimeras, LCMV infection caused substantial loss of gp38+ FRCs (Supplementary Fig. 8 online). However, in the spleens of B6B6 control chimeras, but not those of Rorc-
/-
B6 chimeras, rebuilding of the gp38+ FRC network had begun by day 16 after infection (Fig. 6b). Only by day 25 after infection could we detect large numbers of gp38+ FRCs in confined T cell areas of Rorc-
/-
B6 chimeras (Fig. 6b). Quantification of gp38+ signals in splenic T cell areas showed that restoration of the gp38+ FRC network was significantly delayed in the absence of LTi cells in Rorc-
/-
B6 chimeras (Fig. 6c). Furthermore, quantitative real-time RT-PCR analysis of CCL19 and CCL21 confirmed impaired rebuilding of the stromal cell compartment, as re-expression of these constitutive chemokines was delayed in Rorc-
/-
B6 chimeras relative to that in control chimeras at all times (Fig. 6d). Adoptive transfer of enriched LTi cell preparations into Rorc-
/-
B6 chimeras and B6B6 control chimeras accelerated restoration of the FRC network (Supplementary Fig. 9 online), which provided further support for the interpretation that LTi cells have a decisive function in restoration of the lymphoid architecture.
Figure 6: Lymphoid organ reorganization is supported by LTi cells.
(a) Total lymphocytes (Lymph), DCs (CD11c+), T helper cells (CD4+), CTLs (CD8+) and B cells (B220+) in spleens of Rorc-
/-
B6 and B6B6 chimeras (top), presented as absolute cell numbers (mean and s.e.m.), and flow cytometry of LTi cells in the chimeras (bottom), with percent LTi cells among CD45+CD3-
CD11c-
B220-
cells adjacent to boxed areas. (b) In situ analysis of spleens from Rorc-
/-
B6 and B6B6 chimeras at various times (above images) after LCMV infection. Staining for B220 (blue) and Thy-1.2 (green) defines the T cell zone, and anti-gp38 (red) is used to visualize T cell zone stromal cells. Insets, staining with anti-gp38 alone. Scale bars, 100 m. (c) Density of gp38+ stromal cells in the T cell zone. Values (mean and s.e.m.) indicate pixel ratio of gp38 positivity of the total T cell area (T cell zone stroma/total T cell zone) of 30–60 white pulp areas for two to three mice per group. (d) Expression of mRNA at various times after LCMV infection in Rorc-
/-
B6 chimeras relative to that in B6B6 chimeras (Rorc-
/-
/B6). (e) Density of gp38+ stromal cells assessed as described in c in spleens from naive mice or LCMV-infected mice at day 12 after infection, treated for 9 d with LTR-hIg and analyzed on day 25 (n = 3 mice per group). Far right, LCMV-mediated FRC destruction analyzed on day 12 after infection (reference). **, P < 0.005; *, P < 0.05; NS, not significant. Data are from one representative experiment with three mice per group (a) or three independent experiments with three mice per group (d; mean values) or are representative of two experiments (b,c,e).
In the embryo, LTi cells are an important source of LT12, which is instrumental during interaction with their target cells for induction of the SLO microstructure21. We therefore tested whether blocking LTR signaling might alter reconstruction of the FRC network during recovery from LCMV infection. Indeed, treatment of C57BL/6 mice with a fusion protein of LTR and human immunoglobulin (LTR-hIg)45 between days 12 and 25 after LCMV infection led to a significant delay in the accumulation of gp38+ stromal cells (Fig. 6e), which suggested that LTi cell–mediated FRC stimulation through the LT12-LTR axis may represent an important mechanism for the reorganization and maintenance of the T cell zone stromal cell network in adult lymphoid tissues.
LTi cells in the adult continue to express LT12, and we noted more binding of recombinant LTR-hIg to LTi cells during LCMV infection (Supplementary Fig. 10 online). Furthermore, quantitative RT-PCR analysis of sorted LTi cells showed that expression of LT in particular was upregulated in LTi cells during LCMV infection, concomitant with the loss of expression of constitutive chemokines and IL-7 in the CD45-
cell fraction (Fig. 7a). To assess whether LTi cells could directly activate stromal cells, we did a series of in vitro experiments (Fig. 7b–d). Because primary stromal cells rapidly lose expression of constitutive chemokines in vitro46, upregulation of CCL19 or CCL21 serves as a biomarker for productive LTi cell–stromal cell interaction. In cocultures of gp38+ stromal cells and enriched CD4+CD3-
LTi cell preparations from adult donors deficient in recombination-activating gene 1, there was substantial upregulation of CCL19 transcription (Fig. 7b) and expression of ICAM-1 and VCAM-1 (Fig. 7d) after 4 d of culture. Notably, neither each cell population alone nor coculture with CD11c+ cells, used to assess the effect of contaminating DCs, led to new chemokine expression (Fig. 7b). Coculture of flow cytometry–sorted LTi cells with stromal cells cultured short-term confirmed that specific interaction of these two cell populations restored the expression of constitutive chemokines in stromal cells (Fig. 7c). These data collectively indicate that LTi cells from adult mice are able to productively interact with gp38+ stromal cells and induce a transcriptional program for the reorganization of the lymphoid structure.
Figure 7: Interaction of LTi cells with stromal cells.
(a) Quantitative RT-PCR analysis of gene expression in flow cytometry–sorted LTi cells (left) and CD45-
cells (right) from spleens of LCMV-infected mice on day 8 after infection. Values are relative to mRNA expression in naive C57BL/6 mice. ND, not detectable. Cells from ten mice were pooled and analyzed in triplicate. Data are representative of one experiment. (b) RT-PCR of the expression of CCL19, CCL21, LT, LT and TNF on stromal cells cultured for 4 d together with cell samples enriched for LTi cells or CD11c+ cells (DCs) in the presence (+ GM) or absence of recombinant granulocyte-macrophage colony-stimulating factor (left five lines) or cells cultured alone (right five lanes). Far right (cDNA spleen), positive control for CCL19 and CCL21. TBP (bottom), expression of mRNA encoding TATA-binding protein (control). Data are representative of three independent experiments. (c) Expression of mRNA assessed as described in b after 4 d of culture of flow cytometry–sorted LTi cells with stromal cells cultivated short-term. (d) Flow cytometry of the expression of ICAM-1 and VCAM-1 on stromal cells on day 4 of coculture of stromal cells and LTi cells as described in b (open histograms). Gray filled histograms, expression before coculture. Data are from one representative analysis of two.
Discussion
In this study, we have demonstrated that virus infection–associated immunopathological disruption of lymphoid organ integrity can be associated with a substantial loss of T cell zone stromal cells. After clearance of the virus, CD3-
CD4+ LTi cells were instrumental in efficiently re-establishing the formation of the FRC network, the proper segregation of T cell and B cell zones and recovery of the marginal zone. We also found that reorganization of the gp38+ stromal cell network was dependent on LTR signaling, which suggested that alterations in the homeostasis of adult SLOs reactivated LTi cell–stromal cell crosstalk through molecular pathways that had been operative during lymphoid organ development.
The importance of LTi cells in the development of lymphoid organs has been shown before21, 22. LTi cells contribute to the organization of splenic architecture47, and it has been shown that LTi cells in adult SLOs have a phenotype similar to that of LTi cells found in neonatal tissues48. In LT-deficient mice, adoptive transfer of adult CD3-
CD4+ LTi cells promotes the segregation of B cell and T cell areas and leads to an increase in CCL21 production25. Furthermore, published work has suggested that LTi cells, through expression of the ligand for the cell survival factor OX40 and the CD30 ligand, are able to provide accessory function for the generation and sustenance of B cell memory27. Our data presented here have indicated that adult LTi cells are endowed with additional important functions; that is, to swiftly restore lymphoid organization and full immune responsiveness during immunopathological destruction of SLO structures. Proliferative accumulation of LTi cells was greatest in conditions of maximum stromal cell loss (that is, during LCMV infection). As we have shown, infection with VSV or MCMV precipitated neither a substantial loss of stromal FRCs nor a substantial increase in the LTi cell population. It is thus likely that as-yet-unidentified feedback mechanisms provide signals that lead to LTi cell proliferation and/or their homing to damaged SLOs. Clearly, infection-associated inflammation and destruction of stromal FRCs, as noted during LCMV infection, were required for substantial LTi cell mobilization.
Loss of expression of constitutive chemokines has been reported for the slowly replicating LCMV Armstrong strain and in other infections33, 44, 49. LCMV Armstrong–induced downmodulation of CCR7 ligands results in altered migration of T cells and DCs into the white pulp of the spleen and impairment of T cell responsiveness after secondary infection44. Likewise, chronic infection with L. donovani can precipitate loss of constitutive chemokine production, leading to altered DC migration. In this infection model, CCR7 ligand downmodulation is associated with TNF-mediated loss of T cell zone stromal cells33. Here we have identified direct infection of T cell zone stromal cells and subsequent damage by effector CTLs as the underlying reason for the downmodulation of constitutive chemokines, disruption of SLO integrity and loss of immunocompetence. Stromal cell destruction was mediated exclusively by the action of virus-specific CTLs, as depletion of CD8+ T cells abrogated the downregulation of constitutive chemokines and preserved the FRC network. It seems that, as in infection with L. donovani33, effector T cell–mediated eradication of stromal cells during LCMV infection is mediated mainly by cytokines, as infection of perforin-deficient mice with LCMV resulted in destruction of the FRC network. Extrapolating from the results of our study here and those of previous studies, it is reasonable to postulate that potent counter-regulatory mechanisms must exist that restore the integrity of the stromal cell network and thereby maintain immunocompetence.
Our results here have indicated that adult LTi cells essentially contribute to the restoration of the FRC network. However, it is possible that other cells that express molecules involved in SLO organization are involved in the effects noted. T cells and B cells, for example, represent potential sources of LT12, as conditionally LT-deficient mice lacking LT12 in T cells or B cells show defects in SLO integrity and impairment in mounting virus-neutralizing antibody responses4. It is thus conceivable that in addition to LTi cells, other cells in SLOs provide redundant signals that secure the restoration of SLOs after immunopathological damage. Furthermore, the presence of LTi cells is not mandatory for the maintenance of SLO homeostasis in noninflammatory conditions. That idea was confirmed by the finding that Rorc-
/-
B6 chimeras had a normal SLO architecture, including expression of constitutive chemokines similar to that of wild-type controls. Thus, reactivation of the LTi cell–stromal cell crosstalk seems to be a 'fail-safe' mechanism that is called into action only in conditions of severe impairment of lymphoid organ integrity. IL-7 is one of the likely candidates that contributes to the regulation of adult LTi cell–stromal cell interactions, because it promotes the survival and proliferation of adult LTi cells as well as their LT12 production50, 51. IL-7-driven proliferation of LTi cells may represent the key mechanism that initiates the restoration process. However, as IL-7-producing FRCs are lost during LCMV infection, other sources of IL-7, including DCs52, might represent accessory mechanisms that help to restore lymphoid architecture in the adult.
In conclusion, the results of our study have emphasized the importance of LTi cell–stromal cell interactions in adult SLOs. As infection-associated disruption of SLO microarchitecture is a hallmark of chronic infection31, 32, 33, and resistance to infection correlates with the maintenance of follicular architecture53, the identification of mechanisms that favor the restoration of lymphoid organ structure will help to delineate new approaches for clinical intervention. Molecularly targeted stimulation of the mobilization and activation of adult LTi cells might thus be a useful therapeutic strategy for counteracting the loss of immunocompetence in diseases such as AIDS.
Methods
Mice.
C57BL/6 mice and mice deficient in recombination-activating gene 1 were obtained from Charles River Laboratories. Rorc-
/-
(ROR-deficient) mice were created as described54. Mice were kept in individually ventilated cages in conventional conditions at the Research Department of the Kantonal Hospital of St. Gallen. All animal experiments were done in accordance with Swiss Federal legislation on animal protection.
Infection and immunization.
LCMV WE (obtained from R.M. Zinkernagel) was propagated on mouse L929 fibroblast cells at a low multiplicity of infection and was quantified as described55. Mice were infected intravenously with 200 plaque-forming units of LCMV WE and were analyzed at various times after infection. Recombinant VSV-G was obtained from a culture of Spodoptera frugiperda 9 cells after infection with a recombinant baculovirus56. Mice were immunized intravenously with 5 g VSV-G and then blood was obtained from the tail vein. Titers of VSV-neutralizing antibodies in serum were determined as described57. Sera were prediluted (40 
) in MEM plus 2% (vol/vol) FCS and were 'titrated' in twofold dilution steps; the dilution that resulted in a 50% decrease in virus plaques was considered the neutralizing titer. For IgG titers, serum was incubated for 1 h at 25 °C with an equal volume of 0.1 M 2-mercaptoethanol in PBS before dilution. All mouse serum was heated for 30 min at 56 °C for complement inactivation.
Antibodies.
Fluorescein isothiocyanate–conjugated antibody to CD45 (anti-CD45; 30-F11), peridinin chlorophyll protein–conjugated anti-CD4 (RM4-5), and phycoerythrin-conjugated anti-B220 (RA3-6B2), anti-CD11c (HL3), anti-CD3 (145-2C11), anti-CD8 (53-6.7), anti-ICAM-1 (YN1/1.7.4) and anti-CD11b (M1/70) were all from BD Pharmingen. Alexa Fluor 488–conjugated anti-VCAM-1 (429), anti-B220 (RA3-6B2) and anti-Thy-1.2 (30-H12) were from Biolegend. Alexa Fluor 647–conjugated anti-IL7-R was from eBiosciences. Anti-CD8 (YTS169.4; ECACC; Sigma), for depletion experiments, anti–LCMV nucleoprotein (VL4; provided by R.M. Zinkernagel) and anti-gp38 (8.1.1; Hybridoma Bank) were purified from supernatants of hybridoma cell cultures. As secondary reagents, biotin-conjugated goat anti–Syrian hamster (107-065-142), biotin-conjugated goat anti-human (109-065-098), streptavidin–tetramethyl rhodamine isothiocyanate (016-020-084) and streptavidin-allophycocyanin (016-130-084) were from Jackson Immuno-Research Laboratories.
In vivo antibody treatment.
For depletion of CD8+ T cells, mice were injected intraperitoneally with 500 g purified anti-CD8 on days -
1, +1, +3 and +5 relative to LCMV infection. For blockade of LTR signaling, naive mice or LCMV-infected mice at day 12 were injected with 100 g soluble LTR-hIg (provided by X.Y. Fu) on days 0, 3, 6 and 9 after infection. Mice were killed and spleens were collected for immunohistochemistry on day 13 after treatment with LTR-hIg, which corresponded to day 25 after LCMV infection.
Stromal cell culture.
Spleens from C57BL/6 mice were cut into small pieces and were digested for 45 min at 37 °C with collagenase type II (0.5 mg/ml) in RPMI 1640 medium (Invitrogen). After being homogenized by passage through a 10-ml syringe with an 18-gauge needle, single-cell suspensions were washed twice with RPMI 1640 medium by centrifugation for 15 min at 50g. Cells in the pellet were resuspended at a density of 1 106 cells per ml in RPMI 1640 medium plus 10% FCS, were seeded into 75-cm2 cell culture flasks and were incubated overnight at 37 °C. The next day, nonadherent cells were removed and fresh medium was added. Cultures were continued for 4–7 d until fibroblast-like cells formed monolayers. Stromal cells were passaged at least two to over three times with trypsinization. As fibroblast-like stromal cells detached easily from the culture dish, macrophages were 'diluted out' after the second passage.
Enrichment of LTi cells and stromal cells.
For LTi cells, single-cell suspensions were generated from the spleens of mice deficient in recombination-activating gene 1 and were depleted of CD11c+ cells with MACS anti–mouse CD11c microbeads followed by negative selection with magnetic cell sorting according to the manufacturer's instructions (Miltenyi Biotec). CD4+ cells from the CD11c+ cell–depleted cell fraction were enriched with MACS anti–mouse CD4 microbeads and were positively selected. Samples were enriched 15–25% for LTi cells in the CD4+CD11c- cell fraction, as confirmed by flow cytometry. For the preparation of stromal cells, single-cell suspensions were generated from the spleens of C57BL/6 mice by collagenase treatment as described above and were depleted of lymphocytes with MACS anti–mouse CD45 microbeads (Miltenyi Biotec) followed by negative selection with magnetic cell sorting.
Flow cytometry and sorting.
Single-cell suspensions were generated from various organs, and 1 106 cells were incubated for 20 min at 4 °C with a specific monoclonal antibody. Cells were analyzed with a FACSCalibur and CellQuest software (BD Pharmingen). A FACSAria (BD Biosciences) was used for cell sorting. Splenocyte samples depleted of erythrocytes were stained with saturating concentrations of anti-CD45 (30-F11), anti-CD3 (145-2C11), anti-CD11c (HL3), anti-B220 (RA3-6B2), anti-CD127 (B12-1) and anti-CD4 (RM4-5) in Iscove's modified Dulbecco's medium plus 2% (vol/vol) FBS. After 30 min of incubation at 4 °C, cells were washed in PBS plus 2% (vol/vol) FBS and resuspended in PBS, were filtered through nylon mesh with a pore size of 20 m and were resuspended at a density of about 20 106 cells per ml in filtered PBS plus 2% (vol/vol) FBS before being sorted. Reanalysis of sorted cells indicated a purity of over 95%.
LCMV-specific cytotoxic T cell responses.
Specific ex vivo cytotoxicity was determined by a standard 51Cr-release assay. The radioactivity of supernatants of cytotoxicity assay cultures was measured with a Cobra II -counter (Canberra Packard). Percent specific lysis was calculated as follows: (experimental release – spontaneous release) / (total release – spontaneous release) 100. Spontaneous release was always less than 20%.
RNA isolation and quantitative RT-PCR.
Spleens or lymph nodes were disrupted in TRIzol and RNA was isolated according to the manufacturer's instructions (Invitrogen). Then, cDNA was prepared with a High Capacity cDNA Reverse Transcription kit (Applied Biosystems). Custom-designed low-density arrays (Applied Biosystems) for quantitative RT-PCR included 24 different genes (Supplementary Table 1 online). All RNA samples were analyzed in duplicate and three mice per group were analyzed. RNA concentrations were adjusted and data were normalized to -actin and 18S RNA. Quantitative RT-PCR for CCL19, CCL21 and CXCL13 was also done with a LightCycler FastStart DNA master SYBR Green I kit (Roche Diagnostics) on a LightCycler 1.5 (Roche Diagnostics). For LightCycler analysis, expression of TATA-binding protein was used for normalization. Relative expression of samples from naive and LCMV-infected mice was calculated by the comparative cycling threshold method (
CT). HotStar Taq DNA polymerase (Qiagen) was used to amplify cDNA by conventional RT-PCR. Primer sequences and amplification conditions for RT-PCR are in Supplementary Table 2 online.
Immunofluorescence microscopy.
Freshly removed organs were immersed in Hank's balanced-salt solution and were 'snap-frozen' in liquid nitrogen. Tissue sections 5 m in thickness were air-dried, were fixed for 10 min with acetone and were stored at -
70 °C. Cryosections were blocked for 30 min with 1 g Fc-blocking antibody 2.4G2 per sample, were washed in PBS and were incubated for 1 h at 4 °C with the appropriate fluorescent antibodies. Where needed, streptavidin–tetramethyl rhodamine isothiocyanate was added in a second step. After sections were washed with PBS, nuclei were counterstained with DAPI (4',6'-diamino-2-phenylindoledihydrochloride; Sigma) and sections were mounted with fluorescence mounting solution (Dako).
Generation of bone marrow chimeras.
Recipient mice were lethally irradiated with 900 rads from a linear accelerator (Clinics of Radio-Oncology, Kantonal Hospital of St. Gallen) and were injected intravenously 1 d later with 2 107 donor bone marrow cells. For the ensuing 3 weeks, chimeric mice were provided 'antibiotic water' containing sulfadoxin and trimethoprim (Borgal; Veterinaria AG). Mice were used for experiments 8–10 weeks after bone marrow reconstitution.
Quantification of gp38+ stromal cells.
Images of white pulp regions were acquired from distinct splenic regions of tissue sections. T cell area boundaries in the white pulp were identified according to structural characteristics defined by staining of Thy-1.2 and B220. T cell zone area sizes were determined with Adobe Photosphop CS software. The amount of T zone stromal cells in the T cell zone was calculated as the area of gp38 positivity. The T cell zone stroma/total T cell zone ratio was calculated as a 'read-out' of stromal cell regeneration. Overall, three to four tissue sections per mouse (each containing five to ten white pulp regions) were analyzed for two to three mice per group.
BrdU incorparation assay.
BrdU (2 mg diluted in PBS) was injected intraperitoneally into naive or LCMV-infected mice on day 8 after infection. In addition, BrdU was added to their drinking water at a concentration of 0.8 mg/ml. Then, 4 d later, mice were killed, splenocytes were collected and cell surfaces were stained as described above. Cells were fixed and made permeable and then were stained with the fluorescein isothiocyanate BrdU Flow kit (5559619) according to the manufacturer's instructions (BD Pharmingen).
Statistical analysis.
An unpaired two-tailed Student's t-test was used to determine statistically significant differences. P values of less than 0.05 were considered statistically significant. GraphPad Prism version 4.03 for Windows (GraphPad Software) was used for statistical data analysis.
Accession code.
UCSD-Nature Signaling Gateway (http://www.signaling-gateway.org): A001440.
Note: Supplementary information is available on the Nature Immunology website.
Author contributions
B.L. directed the study and wrote the manuscript; E.S. designed the study, did experiments and wrote the manuscript; T.J. did research and contributed to manuscript writing; E.L. contributed to low-density array analysis and real-time PCR; E.L., B.B., S.M., D.F. and S.F. did research; S.A.L. contributed to data analysis and provided reagents; and D.R.L. provided Rorc- /- mice.
