A Novel Bacterial Artificial Chromosome-Transgenic Podoplanin–Cre Mouse Targets Lymphoid Organ Stromal Cells in vivo

Stromal cells provide the structural foundation of secondary lymphoid organs (SLOs), and regulate leukocyte access and cell migration within the different compartments of spleen and lymph nodes (LNs). Furthermore, several stromal cell subsets have been implied in shaping of T cell responses through direct presentation of antigen. Despite significant gain of knowledge on the biology of different SLO-resident stromal cell subsets, their molecular and functional characterization has remained incomplete. To address this need, we have generated a bacterial artificial chromosome-transgenic mouse model that utilizes the podoplanin (pdpn) promoter to express the Cre-recombinase exclusively in stromal cells of SLOs. The characterization of the Pdpn–Cre mouse revealed transgene expression in subsets of fibroblastic reticular cells and lymphatic endothelial cells in LNs. Furthermore, the transgene facilitated the identification of a novel splenic perivascular stromal cell subpopulation that forms web-like structures around central arterioles. Assessment of the in vivo antigen expression in the genetically tagged stromal cells in Pdpn–Cre mice revealed activation of both MHC I and II-restricted TCR transgenic T cells. Taken together, stromal pdpn–Cre expression is well-suited to characterize the phenotype and to dissect the function of lymphoid organ stromal cells.


INTRODUCTION
Secondary lymphoid organs (SLOs) are characterized by a complex architecture with specialized compartments that facilitate the efficient interaction between immune cells. The structural foundation of this compartmentalization is formed by elaborate frameworks of stromal cell elements (Junt et al., 2008;Mueller and Germain, 2009). These non-hematopoietic cells can be divided in subpopulations that are distinguished by their properties, functions, and localization. Endothelial cells are located at the interface between liquid-transporting compartments and the parenchyma. For example, the highly plastic system of lymphatic vessels that is formed by lymphatic endothelial cells (LECs), distributes lymph fluid through the lymphoid tissues and regulates lymphocyte trafficking (Pham et al., 2010). Likewise, blood endothelial cells (BECs) facilitate controlled access of leukocytes to SLOs by building the network of high endothelial venules (HEVs; Miyasaka and Tanaka, 2004;Kumar et al., 2010). A bridging function, in terms of junction formation between the lymphatic vasculature and the conduit network, is fulfilled by marginal reticular cells (MRCs) that form a dense web of stromal cells underlying the subcapsular Abbreviations: BAC, bacterial artificial chromosome; EYFP, enhanced yellow fluorescent protein; FRC, fibroblastic reticular cell; LEC, lymphatic endothelial cell; LN, lymph node; pdpn, podoplanin; SLO, secondary lymphoid organ. endothelium (Katakai et al., 2008). Other stromal cells are critical for the formation and maintenance of distinct lymphocyte compartments. These include the CXCL13-producing follicular dendritic cells (FDCs) of the B cell zone (Gunn et al., 1998) and fibroblastic reticular cells (FRC) of the T cell zone. FRCs support T cell survival through expression of IL-7, and guide the migration of T cells and dendritic cells (DCs) via production of the constitutive chemokines CCL19 and CCL21 (Luther et al., 2000;Bajenoff et al., 2006;Link et al., 2007). Furthermore, FRCs form the microvascular conduit system that distributes small lymph-borne antigens within the lymphoid tissue (Sixt et al., 2005). Although significant insight into stromal cell biology has been generated over the recent years, the molecular and functional characterization of the above-described subsets has remained incomplete.
Phenotypical and functional characterization of cells in vivo can be achieved through the generation of transgenic mouse models expressing the bacteriophage P1 Cre-recombinase (Schmidt-Supprian and Rajewsky, 2007). Cell type-specific promoters can be utilized to express the Cre-recombinase in distinct cell subsets, and to activate markers for lineage tracing, to conditionally switchoff genes, or to express antigens in a cell type-restricted manner. The choice of a particular promoter for such studies depends on the pre-existing knowledge on the phenotypical diversification of the cell populations under investigation. The current distinction www.frontiersin.org of SLO stromal cells largely relies on the differential expression of CD31 (Pecam-1) and podoplanin (pdpn, also known as glycoprotein 38). CD31 is a marker for endothelial cells and therefore is expressed on BECs and LECs. Pdpn is a mesenchymally expressed glycoprotein with distinct functions in the development of the lymphatic system such as the separation between the lymphatic and blood vascular system (Schacht et al., 2003). FRCs express pdpn, whereas LECs express both pdpn and CD31. Since pdpn is a major marker for stromal cells, we used a bacterial artificial chromosome (BAC)-encoded pdpn promoter to generate a Cre-recombinase-transgenic mouse model that could serve as a tool to facilitate in vivo characterization of SLO stromal subsets. We found that pdpn promoter-driven Cre-recombinase expression labels subsets of FRCs and LECs in lymph nodes (LNs). In addition, the analysis of Pdpn-Cre mice uncovered a novel splenic stromal cell subpopulation that forms web-like structures around central arterioles. Further immunological characterization revealed that antigen expression directed to pdpn-Cre-expressing cells led to activation of both MHC I and II-restricted TCR transgenic T cells, underscoring the potential of certain stromal cell subsets in antigen presentation and T cell activation.

ETHICS STATEMENT
Experiments were performed in accordance with federal and cantonal guidelines under permission numbers SG09/83 and SG09/87 following review and approval by the Cantonal Veterinary Office (St. Gallen, Switzerland).

PREPARATION OF STROMAL CELLS
Lymph nodes and spleens were dissected into small pieces and transferred into a well of a 24-well dish filled with RPMI 1640 medium containing 2% FCS, 20 mM Hepes (all from Lonza), 1 mg/ml Collagenase Type IV (Sigma), and 25 μg/ml DNaseI (Applichem). LN and spleen pieces were incubated at 37˚C for 30 min. After enzymatic digestion, cell suspensions were passed through a 45-μm gauze filter and washed with phosphate-buffered saline (PBS) containing 0.5% FCS and 10 mM EDTA (Sigma). The cell pellet was resuspended in 5 ml MACS-buffer and cells were counted. To further enrich the stromal cell fraction, lymphocytes were depleted by incubating the cell suspension with MACS anti-CD45 microbeads and passing over a MACS LS column (Miltenyi Biotec). The cells in the flow-through were collected and analyzed by flow cytometry.

CFSE-LABELING OF TCR TRANSGENIC T CELLS AND ADOPTIVE TRANSFER
Single-cell suspensions from the spleens of Bg1-Thy1.1 or Bg2-CD45.1 mice were subjected to hypotonic red blood cell lysis and stained with CFSE (Molecular Probes). A maximum concentration of 2.5 × 10 7 cells/ml were incubated in 5 μM CFSE in PBS for 10 min at 37˚C. Cells were washed twice with ice-cold balanced salt solution (BSS) and resuspended in BSS. Pdpn-Cre, R26-LacZ or control mice were injected intravenously with 10 7 Bg1-Thy1.1 and/or Bg2-CD45.1 splenocytes in 200 μl BSS.

GENERATION OF THE BAC TRANSGENIC PDPN-CRE MOUSE
In order to target stromal cells in vivo, we used the BAC homologous recombination technology (Sparwasser et al., 2004;Sparwasser and Eberl, 2007). Here, a BAC containing approximately 190 kb of the mouse chromosome 4 with the pdpn locus was used to replace exon 1 of the pdpn gene with a codon-optimized Crerecombinase sequence and a polyadenylization site ( Figure 1A). The recombination resulted in the expected integration of the Crerecombinase gene downstream of the pdpn start codon as shown by PCR reactions detecting the Cre-recombinase, the 5 ATG, and the 3 polyA regions ( Figure 1B). Primer pairs of the 5 -and 3 -PCRs contained one primer within the transgene and one primer within the adjacent region, so that only properly modified BAC clones could be detected. Positive BAC clones were sequenceverified and the presence of the transgene in the correct location on the BAC was analyzed using a digoxigenin-labeled DNA probe in a southern blot ( Figure 1C). Successfully recombined Pdpn-Cre BACs were linearized, purified, and injected into the pronucleus of C57BL/6 zygotes. The microinjection gave rise to 17 offsprings with three animals being PCR-positive for Cre-recombinase and the 5 ATG region ( Figure 1D).
The founders C57BL/6N-Tg (Pdpn-Cre) 406-408Biat , subsequently designated as line 6, 12, and 14 were crossed to R26-EYFP reporter mice in which a transcriptional stop-cassette can be excised in a Cre-recombinase-dependent manner (Srinivas et al., 2001). Initial examination of the resulting Pdpn-Cre,R26-EYFP mouse lines revealed that transgene expression could not be detected in mice derived from founder 12, suggesting that pdpn promoter-driven Cre expression was not sufficient for successful recombination. In mice derived from founder 6, some EYFP expression could be detected in the CD45 + non-stromal cell compartment (not shown). Founder line 6 was therefore excluded from further analysis. Examination of transgene activity in Pdpn-Cre,R26-EYFP offsprings from founder line 14 by flow cytometry revealed substantial transgene activity in CD45 − LN cells and a complete absence of transgene activity in non-stromal cells (Figure 2A and Figure A1 in Appendix) with coexpression of the stromal cell markers ICAM-1 and VCAM-1 (Figure 2B). Further analysis of LN stromal cells revealed transgene activity in approximately 15% of cells in the LEC and FRC fractions (Figure 2C,D). Transgene expression in BECs was very low (Figure 2D, left histogram) or completely absent in the double-negative fraction (not shown). Assessment of transgene activity in splenic stromal cells showed that the Pdpn-Cre transgene is almost exclusively active in the pdpn + CD31 − FRC fraction (Figure 2E,F). Taken together, these data indicate that the Pdpn-Cre transgene is well-suited to track particular stromal cell subsets within SLOs.

PDPN-CRE EXPRESSION TARGETS FRCs AND LECs IN VIVO
In order to localize transgene-expressing cells in spleen and LNs of Pdpn-Cre,R26-EYFP mice, we performed extensive in situ analysis using confocal laser scanning microscopy. LN sections were www.frontiersin.org stained for Pdpn and B220 to visualize the Pdpn + FRC network of the T cell zone, the Pdpn + lymphatic endothelium, and B220 + B cell areas ( Figure 3A). More detailed examination of transgene-expressing EYFP + cells in the T cell zone showed that transgene-expressing cells were indeed part of the Pdpn + FRC network ( Figure 3B). In addition, pronounced transgene activity could be detected in lymphatic vessel endothelial hyaluron receptor I (LyveI)-positive cells with a particularly high accumulation of these cells in the subcapsular sinus of the LN (Figure 3C). Thus, the Pdpn-Cre transgene facilitates selective tagging of FRCs and LECs in LNs.
Since the flow cytometric analysis of transgene activity in spleen revealed a more FRC-specific transgene expression pattern (Figure 2F), we wanted to confirm this finding using in situ analysis. Applying the identical immunofluorescent stainings as in the LN sections, we could detect transgene activity only within the Pdpn + stromal cell network in the white pulp (Figure 4A). High resolution analysis revealed that EYFP + cells accumulate in the central areas of the white pulp ( Figure 4B). Co-staining with anti-CD31 showed that the Pdpn-Cre + cells form a web-like structure around the central arteriole ( Figure 4C). The EYFP + perivascular stromal cells co-expressed the Pdpn protein, but were negative for CD31 ( Figure 4D). It is noteworthy that Pdpn-expressing EYFP + cells formed tube-like structures extending into the T cell zone ( Figure 4D). In summary, transgene expression in spleens of Pdpn-Cre,R26-EYFP mice identified a novel stromal cell subset within the splenic white pulp that displays a FRC-signature (Pdpn + CD31 − ), but shows an exclusive location in the center of the white pulp.

ACTIVATION OF TCR TRANSGENIC T CELLS IN ANTIGEN-EXPRESSING PDPN-CRE MICE
The organized structures of SLOs are critical for the activation of innate and adaptive immune responses (Junt et al., 2008). It is therefore intriguing that stromal cells can, at the same time, contribute to the tolerization of T cells. For example, it has been shown that direct presentation of self antigen by non-hematopoietic cells to autoreactive T cells results in activation, proliferation, and subsequent deletion of CD8 + T cells (Lee et al., 2007). Furthermore, a subpopulation of stromal cells in LNs and spleen was shown to express the transcription factor autoimmune regulator (AIRE) and was able to mediate the deletion of autoreactive CD8 + T cells (Gardner et al., 2008). Interestingly, it appears that different stromal cell subsets expressing peripheral tissue antigens may contribute to the maintenance of tolerance under distinct conditions (Cohen et al., 2010;Fletcher et al., 2010). To assess the impact of antigen expression by stromal cells in the Pdpn-Cre model, we crossed Pdpn-Cre to R26-LacZ mice that express the beta-galactosidase (βgal) antigen following Cre-recombinasemediated removal of a transcriptional stop-cassette (Soriano, 1999). We have shown previously that βgal expression in nonhematopoietic stromal cells can be used to dissect mechanisms Frontiers in Immunology | Antigen Presenting Cell Biology of peripheral tolerance (Bolinger et al., 2008(Bolinger et al., , 2010. Histological analysis of LNs from Pdpn-Cre,R26-LacZ mice revealed an antigen expression pattern ( Figure 5A) that was comparable to the EYFP expression in LNs of Pdpn-Cre,R26-EYFP mice ( Figure 3A). To assess whether βgal expression in Pdpn-Cre + stromal cells would suffice for activation of βgal-specific CD8 + and CD4 + T cells, we adoptively transferred Bg1 (Bolinger et al., 2008) and Bg2 (Tewalt et al., 2009) TCR transgenic cells into Pdpn-Cre,R26-LacZ mice. Single transfer of MHC I-restricted T cells did not result in significant activation (Figure 5B, left column), whereas transfer of Bg2 cells alone led to strong T cell proliferation as shown by substantial dilution of the intracellular dye CFSE (Figure 5B, right column). Co-transfer of Bg1 and Bg2 cells was accompanied by proliferation of both T cell populations ( Figure 5B, middle columns) indicating that under these conditions, stromal cell-specific CD8 + T cells received help from CD4 + T cells and could therefore enter into proliferative cycles. Taken together, these data underscore the high versatility of the Pdpn-Cre model with successful and comparable Cre-mediated recombination in different reporter strains, and secondly indicate that Pdpn-Cre mice can be utilized to investigate the interplay of stromal cells during the activation and/or tolerization of T cells that encounter antigens exclusively expressed in these cells.

DISCUSSION
In this study, we describe the generation and first phenotypical analysis of a stromal cell-specific Pdpn-Cre BAC transgenic mouse. The importance of stromal cell-immune cell interaction has received increased attention over the recent years (Mebius, 2007;Mueller and Germain, 2009;Turley et al., 2010). However, the characterization of stromal cells within SLOs has relied largely on particular cell surface marker combinations. The present study has addressed the need to generate models that facilitate (i) genetic definition of distinct LN and splenic stromal cell populations, (ii) in vivo labeling and tracking of particular stromal cell subsets, and (iii) functional assessment of immune activation/tolerization processes through selective expression of model antigens. Furthermore, it will be possible to utilize this model to selectively delete molecularly defined target structures through crossing with conditionally gene-targeted mouse strains such as the LTβR fl/fl strain (Wang et al., 2010), or to transiently deplete stromal cells in the inducible diphtheria toxin receptor system (Buch et al., 2005).
Genomic integration of modified BACs is a widely used technology to generate transgenic mouse models. Comparable to other BAC transgenic mouse lines expressing the Cre-recombinase (Nishikawa et al., 2010), we observed varying levels of transgene expression and recombination frequencies in different founder lines. Phenotypic variability among several founder lines most likely resulted from different chromosomal integration sites and varying numbers of integrated BAC copies thereby determining the level of Cre-recombinase expression. Here, one of the three Pdpn-Cre founder lines displayed a suitable transgene expression pattern that facilitated the identification of novel stromal cell subpopulations in SLOs such as the splenic perivascular stromal cell. This finding implies that the currently used FRC phenotype description (Pdpn + CD31 − ) may obscure the true diversity of

FIGURE 5 | βgal expression in FRCs and LECs in Pdpn-Cre,R26-LacZ mice leads to activation of βgal-specific TCR transgenic T cells. (A)
Pdpn-Cre mice were crossed to R26-LacZ reporter mice and βgal expression in LNs was detected by immunofluorescence analysis. Section of inguinal LN from Pdpn-Cre,R26-LacZ mouse stained for βgal (green), Pdpn (blue), and CD4 (red). Scale bar equals 100 μm. (B) Stimulation of βgal-specific TCR transgenic T cells by stromal βgal expression was determined by flow cytometry using CFSE-labeled congenic Bg1 and Bg2 cells. CFSE dilution in blood, spleen, and LNs was measured on day 5 after adoptive transfer in Pdpn-Cre,R26-LacZ mice (black lines) or Cre-negative littermates (gray shaded). Bg1 or Bg2 cells were adoptively transferred either alone or in combination. Values in the histograms indicate percentage of proliferated TCR transgenic cells; proliferation in Cre-negative littermates was always below 5%. stromal cells in SLOs. This interpretation has been confirmed in a recent study that defines a particular FRC subpopulation through JAM-C controlled chemokine secretion (Frontera et al., 2011). Using genetic tools such as BAC transgenic mouse lines with stromal cell-specific promoters will help to define both function and phenotype of SLO stromal cells in future studies.
Antigen presentation and activation of T cells is a wellcontrolled process that relies on a division of labor between different myeloid cell subsets, mainly DCs . It is possible that a similar specialization in the display of self-antigens for the tolerization of autoreactive T cells is distributed between different stromal cell subsets. Indeed, recent studies suggest that diverse stromal cell subsets, in particular FRCs and endothelial cells, participate in presenting antigen via MHC class I and thereby critically contribute to the shaping of a T cell response (Cohen et al., 2010;Fletcher et al., 2010). The Pdpn-Cre mouse will facilitate future studies on the exact nature of direct or indirect stromal cell-T cell interaction. It will be important to determine whether the observed lack of βgal-specific CD8 + T cell activation in Pdpn-Cre,R26-LacZ mice is simply the lack of interaction, www.frontiersin.org i.e., immunological ignorance (Bolinger et al., 2008) or an active process with down-tuning of T cell reactivity. Clearly, CD4 + T cells specific for a stromal cell-specific antigen were activated efficiently in the βgal system as presented here. Whether this activation was due to direct MHC II-mediated contact between stromal cells and CD4 + T cells or whether indirect activation through DCs had occurred, will require further investigations that exceed the scope of this initial characterization of this novel mouse strain.
In summary, stromal cell-specific Cre-recombinase expression is well-suited to better characterize SLO stromal cells through definition of functionally and phenotypically distinct subpopulations. In particular, the Pdpn-Cre mouse model can serve as a tool to dissect antigen presentation by FRCs and LECs in LNs and by the novel perivascular stromal cell population in spleen.

AUTHORSHIP STATEMENT
Burkhard Ludewig designed the study and wrote the paper; Lucas Onder and Elke Scandella performed research and wrote the paper; Qian Chai, Sonja Firner, Christian Mayer, and Thomas Rülicke performed research; Tim Sparwasser provided technical supervision and discussed data; Volker Thiel analyzed and discussed data.