Nkx2-3 Controls the Vascular Identity and Homing in the

This information is current as Tamás Czömpöly, Árpád Lábadi, Zoltán Kellermayer, of September 24, 2021. Katalin Olasz, Hans-Henning Arnold and Péter Balogh J Immunol 2011; 186:6981-6989; Prepublished online 18 May 2011; doi: 10.4049/jimmunol.1003770

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Transcription Factor Nkx2-3 Controls the Vascular Identity and Lymphocyte Homing in the Spleen

Tama´s Czo¨mpo¨ly,* A´ rpa´dLa´badi,* Zolta´n Kellermayer,* Katalin Olasz,* Hans-Henning Arnold,† and Pe´ter Balogh*

The vasculature in the spleen and peripheral lymph nodes (pLNs) is considerably different, which affects both homing of and antigenic access to these peripheral lymphoid organs. In this paper, we demonstrate that in mice lacking the homeodomain transcription factor Nkx2-3, the spleen develops a pLN-like mRNA expression signature, coupled with the appearance of high endothelial venules (HEVs) that mediate L-selectin–dependent homing of lymphocytes into the mutant spleen. These ectopic HEV-like vessels undergo postnatal maturation and progressively replace MAdCAM-1 by pLN together with the display of CCL21 arrest in a process that is reminiscent of HEV formation in pLNs. Similarly to pLNs, development of HEV-like vessels in the Nkx2-3–deficient spleen depends on -b -mediated signaling. The Downloaded from replacement of splenic vessels with a pLN-patterned vasculature impairs the recirculation of adoptively transferred lymphocytes and reduces the uptake of -borne pathogens. The Nkx2-3 mutation in BALB/c background causes a particularly disturbed splenic architecture, characterized by the near complete lack of the , without affecting lymph nodes. Thus, our obser- vations reveal that the organ-specific patterning of splenic vasculature is critically regulated by Nkx2-3, thereby profoundly affecting the lymphocyte homing mechanism and blood filtering capacity of the spleen in a tissue-specific manner. The Journal of Immunology, 2011, 186: 6981–6989. http://www.jimmunol.org/

he spleen in mammals is the largest single peripheral The development of the spleen is initiated by the formation of its lymphoid organ, contributing to both innate and adaptive mesenchymal anlage, controlled by the concerted action of mul- T immune responses against invading pathogens. Its de- tiple transcription factors, including Pbx1, Nkx3-2 (also known as velopment and tissue architecture are strikingly different from Bapx1), TLX1 (formerly known as HOX11), and Nkx2-5 (5, 6). In other secondary lymphoid organs, including the peripheral lymph the absence of any of these factors, development of the spleen is nodes (pLNs) and Peyer’s patches. Different lymphoid regions in arrested early, resulting in asplenia. TLX1 may be regulated in- the spleen are arranged around various vessels. The zone dependently by the homeodomain transcription factor Barx1, by guest on September 24, 2021 is located around central arterioles, and the is adja- which is also involved in stomach development (7). Importantly, cent to terminal arterioles in human or marginal sinus in rodents, loss of TLX1 does not prevent the formation of lymph nodes (8). whereas the red pulp is richly perfused by a sinus network lined by Embryonic lethality of Pbx1- and Nkx2-5–deficient mice and different endothelial subsets (1, 2). In contrast to the leukocyte neonatal death of Nkx3-2–null mutants precludes the analysis of homing in pLNs and Peyer’s patches, the spleen lacks high en- their potential roles in development (9–11). dothelial venules (HEVs) and requires no L-selectin binding for Following formation of the splenic primordium, spleen pro- subsequent leukocyte extravasation (3, 4). genitor cells undergo expansion and local hematopoiesis starts, controlled by the transcription factor Tal-1 (also known as Scl) (12). Subsequently, CD4+/CD32/CD45+/IL-7Ra+ lymphoid cells ac- cumulate around the developing blood vessels in close proximity *Department of Immunology and Biotechnology, University of Pe´cs, Faculty of with stromal cells that express MAdCAM-1 and VCAM-1 (13). Medicine, H-7624 Pe´cs, Hungary; and †Department of Cell and Molecular Biology, This close cellular association is thought to be necessary for ef- Institute of Biochemistry and Biotechnology, Technical University of Braunschweig, ficient interaction between members of the TNF/lymphotoxin (LT) 38106 Braunschweig, Germany family displayed by lymphoid cells (termed lymphoid tissue in- Received for publication November 12, 2010. Accepted for publication April 15, 2011. ducer) and their cognate receptors on stromal cell precursors (14– The work was supported by a research fund from the Faculty of Medicine of the 16). In contrast to the spleen-autonomous defect in TLX1- University of Pe´cs (to P.B.) as well as by “Science Please” Research Team Innovation deficient mutant mouse (8), loss of either LTa or the receptor Grant SROP-4.2.2/08/1/2008-0011 to the University of Pe´cs (to P.B.). T.C. is a re- for LTb (LTbR) prevents the formation of lymph nodes and cipient of the Bolyai Ja´nos Postdoctoral Fellowship of the Hungarian Academy of Sciences. P.B. is supported by the O¨ veges Jo´zsef Research Fund from the National Peyer’s patches, whereas the spleen develops, although with se- Office for Research and Technology of Hungary. verely disturbed architecture of and marginal zone Address correspondence and reprint requests to Dr. Pe´ter Balogh, Department of (17). Distinct lymph node and spleen phenotypes elicited in Immunology and Biotechnology, University of Pe´cs, Faculty of Medicine, Szigeti mutants lacking LT ligands or corresponding receptors and in u´t 12, H-7624 Pe´cs, Hungary. E-mail address: [email protected] TLX1 mutants suggest that development of the various peripheral Abbreviations used in this article: FDC, follicular ; HEV, high endothelial venule; KO, knockout; LT, lymphotoxin; LTbR, receptor for lymphotoxin b; mLN, lymphoid organs follows distinct regulatory programs. In this mesenteric lymph node; P0.5, ,1-d-old neonates; P10.5, postnatal day 10.5; pLN, process, the factors for cell lineage specification related to vas- peripheral lymph node; PNAd, peripheral lymph node addressin; qPCR, quantitative cular patterning in distinct lymphoid organs are mostly unknown. RT-PCR. More recently, the transcription factor Nkx2-3 has Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 been identified as an important regulator of spleen ontogeny (18– www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003770 6982 Nkx2-3 DETERMINES THE VASCULAR PATTERNING IN THE SPLEEN

20) without affecting pLN development. The Nkx2-3 transcrip- Cambridge, MA) and was injected in newborn Nkx2-3 KO mice as described tion factor belongs to the structurally conserved Nk family of 18 previously (26). Pooled human IgG was used as control. members sharing a common DNA recognition sequence. This Flow cytometry group of transcription factors is involved in the cell type specifi- Lymphocytes were isolated from the spleen and lymph nodes by teasing cation of visceral mesoderm, including heart, lungs, pancreas, and apart the organs between the frosted ends of two microscopic slides and gastrointestinal tract, in a highly complex expression pattern (21). filtered through a 70-mm pore-size cell strainer. The cells were incubated Nkx2-32/2 mice lack the marginal sinus and have atrophic red with a mixture of fluorescein-labeled anti-B220 and biotinylated anti-CD3 pulp sinus network, two specific vascular structures in the spleen or anti-CD62L mAb in the presence of 2.4G2 anti-CD16/32 mAb. The biotinylated mAbs and the biotinylated cells in homing studies were that are absent in other peripheral lymphoid tissues (22). In this detected with PE-streptavidin. Dead cells were excluded based on their paper, we report that Nkx2-3 deficiency in the mouse transforms light scattering properties. At least 20,000 live cells were collected by the vasculature in a mutant spleen structurally and functionally a BD Biosciences FACSCalibur cytometer and analyzed using the Cell- toward the typical vascular pattern of pLNs. The disruption of the Quest software. Nkx2-3 results in the formation of splenic pLN addressin Immunohistochemistry and immunofluorescence (PNAd)-positive HEVs that display CCL21 arrest chemokine and The single and dual-label immunofluorescence and immunohistochemical mediate L-selectin–dependent homing. The development of ec- procedures are previously described (24, 26) for control staining normal rat topic HEV-like structures in a mutant spleen requires LTbR- IgG at 10 mg/ml concentration was used. After mounting, the sections were mediated signaling, similar to the HEVs in pLNs. Moreover, the viewed under an Olympus BX61 fluorescent microscope. The acquisition mutants show reduced marginal zone-associated capture of of digital pictures with a charge-coupled device camera was performed using the analySIS software. pathogens. Thus, our observations provide evidence that specifi- Downloaded from cation of the vascular pattern in the spleen and its consequences Adoptive cell transfer and bacterial injection for lymphocyte homing are critically dependent on the transcrip- Lymphocytes from pLNs or mesenteric lymph nodes (mLNs) were isolated tion factor Nkx2-3. and labeled with CFSE (Invitrogen) or sulfo-N-hydroxysuccinimide biotin ester (Sigma-Aldrich) as described previously (27). For lymphocyte Materials and Methods homing studies, 200 ml cell suspension at 5 3 107 CFSE-labeled cells was Mice injected i.v. in the tail vein, followed by the removal of the spleen at various intervals. The distribution of labeled cells was tested cells by http://www.jimmunol.org/ Nkx2-32/2 mice from a 129Sv 3 B6 mixed background were backcrossed immunofluorescence using anti-PNAd or IBL-11 mAb against white pulp with BALB/c mice (obtained from Charles River Laboratories, Budapest, fibroblasts (24) in conjunction with PE-labeled anti-rat IgG. For compet- Hungary) through 14 generations and genotyped as described previously. itive homing, two different cellular labeling procedures were used in which C57BL/6 and 129Sv Nkx2-3 mutants were maintained at the Technical the CFSE-labeled cells were subsequently incubated with MEL-14 IgG University of Braunschweig (Braunschweig, Germany). For homing (purified by G FPLC) or IBL-10 control rat mAb. After washing, studies, BALB/c mice from the faculty’s specific pathogen-free breeding the cells were counted and mixed at 1:1 ratio with biotinylated cells as unit were used as a lymphocyte donor for adoptive transfer. LTbR- reference population, followed by the i.v. injection of mixed cells. The deficient mice (23) were provided by Drs. K. Pfeffer (Heinrich Heine mice were sacrificed 30 min after the injection, and their distribution of University, Du¨sseldorf, Germany) and F. Weih (Leibniz Institute for Age CFSE-labeled and biotinylated lymphocytes in different lymphoid tissues Research, Fritz Lipmann Institute, Jena, Germany). All procedures in- was determined by flow cytometry or immunofluorescence. The CFSE: by guest on September 24, 2021 volving live animals were conducted in accordance with the guidelines set biotin ratio of the cells in lymphoid organs was calculated following the out by the Ethics Committee on Animal Experimentation of the University normalization of labeled cell frequencies with the preinjection CFSE:bi- of Pe´cs (Pe´cs, Germany). The double-knockout (KO) mice were identified otin ratio; thus, the CFSE:biotinylated cell ratio recovered from each organ in the F2 generation by simultaneous PCR amplification of the Nkx2-3, was divided by the CFSE:biotinylated cell ratio in the preinjection cell neomycin resistance cassette, and LTbR loci from genomic DNA by using mixture. For bacterial capture, mice were i.v. injected with 103 Escher- the following primer pairs: Nkx2-3,59-GCGGGAGACTGTAAGACGAG- ichia coli previously conjugated with FITC in 250 ml PBS. 9 9 9 3 (forward) and 5 -TTATCCTGCCGCTGTCTCTT-3 (reverse), amplicon Quantitative RT-PCR size: 238 bp; neomycin resistance cassette, 59-GTCGATCAGGATGATC- TGGAC-39 (forward) and 59-AAGGCGATAGAAGGCGATGC-39 (reverse), Total RNA was isolated with the RNeasy Plus Mini Kit (Qiagen) and was amplicon size: 321 bp; and LTbR, 59-GCATGTAGCCATGAAGACAG- treated with DNase I (Sigma-Aldrich). cDNA was prepared with the High GAT-39, (forward) and 59-CGCAAAGACAAACTCGCCTAT-39 (reverse), Capacity cDNA Archive Kit (Applied Biosystems). PCR primers used for amplicon size: 150 bp. real-time quantitative amplification of Glycam1, Madcam1, B3gnt3, Gcnt1, Abs and reagents Chst2, Chst4, and Fut7 were described previously (27). PCR primers for Cd34, endomucin, Cd300lg, podocalyxin-like protein, 18S rRNA, and RatmAbsagainstmousefibroblasticreticularcell markers(IBL-11)andThy- Hprt1 were designed by Primer Express Software (Applied Biosystems). 1 (IBL-1) were developed in our laboratory (24). For flow cytometry and dual PCRs were run in triplicates using the Power Sybr Green Master Mix immunohistochemistry, IBL-1 mAb was conjugated with sulfo-N-hydrox- (Applied Biosystems) on an ABI 7500 Real Time PCR System (Applied ysuccinimide biotin ester (Sigma-Aldrich, Hungary, Budapest). Rat hy- Biosystems). Standard curves were generated for each transcript, and ex- bridoma cell lines secreting anti-CD3 (KT-3), anti–MAdCAM-1 (MECA- pression levels were normalized to b-actin; the relative expressions were 367), L-selectin/CD62L (MEL-14), and B220 (RA3-6B2) were obtained calculated using b-actin normalized expression levels of wild-type from the American Type Culture Collection and used either as a hybridoma as reference samples. supernatant or a fluorescein-conjugated reagent. PE-conjugated anti-mouse Statistical analysis CD21/35 mAb (clone 7G6), MECA-79 mAb against PNAd, and FITC- labeled and PE-conjugated goat anti-rat IgG were purchased from BD Bio- Normal distributions of means were tested with a one-sample Kolmogorov- sciences (Soft Flow Kft, Pe´cs, Hungary). Goat Abs against CCL21 and Smirnov test. Means were compared with one-way ANOVA, followed by CXCL13 and NorthernLights 557-conjugated donkey Ab a Bonferroni test. A p value , 0.05 was considered statistically significant. against goat IgG were obtained from R&D Systems (Biomedica Hungary Statistical analyses were performed with SPSS 14.0 software. Kft, Budapest, Hungary). Biotinylated rat mAb against mouse IgM was produced in our laboratory from B7.6 clone. Goat anti-mouse IgM–HRP Results conjugate was produced by Invitrogen (Csertex Kft, Budapest, Hungary). Altered expression of marker associated with HEV Rabbit Abs against Prox1 were purchased from AngioBio (Del Mar, CA). Affinity-purified rabbit polyclonal Abs against HEC-GlcNAc6ST sulfo- development in the spleen and lymph nodes transferase (25) were provided by Dr. N. H. Ruddle (Yale University School Previous studies of Nkx2-32/2 mice in our laboratory and else- of Medicine, New Haven, CT) and were detected with FITC-conjugated goat anti-rabbit IgG (Sigma-Aldrich). Streptavidin-Alexa Fluor 350 and where already revealed a broad range of structural alterations of streptavidin-Alexa Fluor 488 conjugates were purchased from Invitrogen. the spleen (18, 22, 26), affecting the marginal sinus and red pulp LTbR–Ig fusion protein was provided by Dr. J. L. Browning (Biogen Idec, sinuses, two highly specific segments of the splenic vasculature The Journal of Immunology 6983 involved in splenic recirculation in mice (reviewed in Ref. 2). and pLNs of wild-type mice was reduced compared with wild- Despite the lack of these vessels in mutants, the accumulation type spleens. of lymphocytes in the mutant spleen indicates the existence of Both GlyCAM-1 and MAdCAM-1 may serve as backbone a selective homing process to that organ, prompting an analysis of for PNAd in a process depending on glycosylation and those endothelium-associated genes that have been implicated in sulfatation, mediated by the glycosyltransferases B3gnt3 and Gcnt1, tissue-specific homing. Microarray analysis of the Nkx2-3–mutant the fucosyltransferase Fut7, and the sulfotransferases Chst2 and spleen suggested that expression of several genes associated with Chst4, the latter also known as HEC-GlcNAc6ST, respectively endothelium-mediated tissue-specific homing (28–33) was altered (34). Quantitative RT-PCR (qPCR) analysis for these genes in (data not shown). In this study, we performed real-time PCR to wild-type and mutant spleens revealed that the majority of these quantify expression of some of these Nkx2-3–regulated candidate transcripts were upregulated in the Nkx2-32/2 mutant, although to genes in wild-type and mutant spleens as well as in pLNs and different degrees (Fig. 1B). B3gnt3 glycosyltransferase in Nkx2- mLNs. The analysis demonstrated profound reduction of 3–mutant spleen was expressed at a lower amount compared with MAdCAM-1 expression in the spleen of the homozygous mutant wild-type spleen; however, this reduced mRNA transcription was mouse in agreement with previous reports (19, 20). Importantly, also observed at various degrees in lymph nodes from both mutant expression of GlyCAM-1 in the mutant spleen was dramatically and wild-type samples. Gcnt1 glycosyltransferase enzyme mRNA upregulated (∼10, 000-fold), whereas transcript levels for other was the only transcript showing differences in expression in lymph marker genes, such as CD34, endomucin, CD300 Ag-like family nodes between Nkx2-3 mutants and wild-type samples in which, member G (Cd300lg, also known as nepmucin), and podocalyxin- in both pLNs and mLNs of Nkx2-3–mutant mice, mRNA for like protein, were also increased, although to a lesser extent (be- Gcnt1 glycosyltransferase was expressed at a robustly increased Downloaded from tween 4- and 16-fold) in comparison with wild type (Fig. 1A). amount, similarly to the mutant spleen. Chst4 sulfotransferase Particularly, the upregulation of the endothelial marker GlyCAM- mRNA showed a similar degree of increased expression in the 1 and the reduction of MAdCAM-1 expression in the mutant mutant spleen as well as in the pLNs and mLNs from both mutant spleen appeared more similar to pLNs than in wild-type spleens. and wild-type mice. Taken together, these observations suggest In comparison, the pLNs from Nkx2-3–deficient mice showed that, in the absence of the transcription factor Nkx2-3, endothelial

a slightly elevated expression of MAdCAM-1 and an equal ex- MadCAM-1 expression in the spleen is essentially replaced by http://www.jimmunol.org/ pression of GlyCAM-1 compared with wild-type mice, whereas GlyCAM-1, and the modifying enzymes required to generate mLNs had less MAdCAM-1 and a higher level of GlyCAM-1 functional PNAd are also upregulated. Thus, this pattern of endo- mRNA expression than wild-type controls. The differences be- thelial marker in mutant spleens is reminiscent of tween the expression level of other core proteins genes was less HEVs in pLNs. dramatic, although the general tendency was an increased mRNA 2/2 production for all genes investigated. Pdxl, a lesser investigated Ectopic formation of HEV-like vessels in the Nkx2-3 PNAd core protein, showed an increased expression in all lym- mutant spleen phoid tissues of Nkx2-3 mutants, whereas its expression in mLNs The pLN-like signature of transcripts for HEV-related genes and the described alterations of the marginal zone and white pulp (19– by guest on September 24, 2021 22, 26) in the Nkx2-3–deficient spleen prompted us to investigate the vasculature in more detail by using MECA-79 anti-PNAd (35) and anti–MAdCAM-1 mAbs. In contrast to the wild-type spleen, which never contains HEVs, in the spleens of young adult Nkx2- 32/2 mice, PNAd-positive HEVs in close proximity of -rich foci were prominently present. Dual immunofluorescence for HEC-GlcNAc6ST sulfotransferase (25) and the PNAd epitope showed coexpression on HEV-like vessels in the mutant spleen, similarly to pLNs from either wild-type or mutant mice (Fig. 2A). To test whether ectopic HEVs develop only in BALB/c back- ground, we also tested Nkx2-3 mutants from both 129Sv and C57BL/6 strains. We found that spleen sections from these hap- lotypes also contained PNAd-positive HEVs (data not shown). These vessels were lined with plump endothelial cells that express CD31 endothelial Ag in which transmigrating lymphocytes could also readily be observed (Fig. 2A). It is interesting to note that elevated expression of PNAd on ectopic HEV-like vessels in the Nkx2-3–deficient mouse mutant appeared to be limited to the spleen, because HEVs in Peyer’s patches or in mLNs did not show any increased reactivity for this marker (data not shown). To test whether the PNAd epitope is accessible for recirculat- ing lymphocytes, MECA-79 mAb against PNAd was adminis- tered to Nkx2-32/2 mutants and wild-type mice i.v. We found a strong luminal binding in mutant spleens to a degree similar to pLNs from either mutant or wild-type mice, demonstrating that FIGURE 1. qPCR analysis of mRNAs for PNAd core proteins (A) and modifying enzymes (B) in the Nkx2-32/2 and wild-type (WT) spleens, this L-selectin ligand is accessible for recirculating lymphocytes pLNs, and mLNs. Values in different organs are normalized to the ratio of in the mutant spleen, similarly to pLNs, whereas wild-type spleens target mRNA to b-actin in the WT spleen, represented by the line drawn at had no discernible reactivity (Fig. 2B). y = 1, and are expressed on a log scale. Bar diagram shows the mean 6 SD We also found that, unlike Nkx2-3 mouse mutants on other of six parallel samples from three mice per organ; *p , 0.05, **p , 0.001. genetic backgrounds (129Sv 3 B6 [F1] or C57BL/6), ∼75% of 6984 Nkx2-3 DETERMINES THE VASCULAR PATTERNING IN THE SPLEEN

FIGURE 2. Absence of the Nkx2-3 gene in the BALB/c mouse leads to HEV forma- tion and pLN-like lymphocyte composition in the spleen. A, Frozen sections of the spleen and pLNs from mutant and wild-type mice were stained with fluorescence-labeled Abs as indicated. The purple color represents the merge of complement receptor (CR) expres- sion by FDCs and IgM reactivity of adjacent B cells. Scale bar, 100 mm. H&E staining (upper right) shows plump endothelial cells (asterisks) and lymphocytes either closely associated with (arrowhead) or transmigra- ting between (arrow) these cells (scale bar, 25 mm). Endothelial cells in the mutant spleen coexpress CD31 and PNAd (lower right). Scale bar, 100 mm. B, The PNAd epitope is detected at the luminal surface of the endothelium in HEVs in the mutant spleen (upper left) and pLNs from mutant Downloaded from (upper right) or wild-type (lower right) mice following i.v. administration of MECA-79 anti-PNAd mAb without any detectable re- activity in the wild-type spleen (lower left). Dashed lines, Edges of tissue; inset, higher magnification of the marked rectangular area 3

in left panel (60 objective). Ellipses in the http://www.jimmunol.org/ pLN sections demarcate follicles. Scale bar, 100 mm. Lower panels, PNAd-positive HEVs that strongly coexpress HEC-GlcNAc6ST in the Nkx2-32/2 spleen (lower panel). Scale bar, 50 mm. The figures are representative from a cohort of three to five mice repeated three times.

2/2 homozygous BALB/c mice carrying the Nkx2-3 mutation Homing of lymphocytes in the Nkx2-3 spleen involves the by guest on September 24, 2021 exhibited spleens with nearly no red pulp or had spleens with ectopic HEV-like vessels producing CCL21 and depends on macroscopically detectable red pulp only in a segment of the organ L-selectin/CD62L + (Fig. 3A). The lack of MAdCAM-1 marginal sinus-lining cells or Next, we investigated whether the ectopic PNAd+ vessels formed aberrant distribution of topographic markers, including marginal in the Nkx2-3–mutant spleen function as lymphocyte exit ports. zone-associated sessile macrophages or white pulp-associated fi- CFSE-labeled lymphocytes isolated from wild-type pLNs or broblastic cells (20, 22), excluded the precise determination of the mLNs were injected i.v., and their localization was determined at distribution of various lymphocyte subpopulations. These “red- various times after injection using double immunofluorescence less” spleens or the spleen segments devoid of red pulp are entirely microscopy. Shortly after, injection-labeled lymphocytes were filled with T and B cells without forming discrete territories (Fig. found to be closely associated with PNAd+ HEVs in the spleen of 3B). Thus, the formation of red pulp sinuses seems to be partic- mutant animals, whereas in wild-type spleens, they were located ularly sensitive to the Nkx2-3 mutation in BALB/c mice; however, at the marginal zone (Fig. 4A). The mutant spleens appeared to re- HEV-like vessels develop in homozygous Nkx2-3 mutants irre- ceive significantly fewer lymphocytes than the wild-type organ; spective of the genetic background. however, their accumulation within the HEVs in the mutant spleen The lymphocyte composition of mutant spleens was tested by was almost indistinguishable from pLNs, and by 12 h, the lym- flow cytometry. Our analyses revealed a slightly increased pro- phocytes showed the same degree of tissue dispersion as in pe- portion of lymphoid cells expressing L-selectin/CD62L homing ripheral lymph nodes (data not shown). receptors together with a relative increase of T cells in a mutant The initial binding of lymphocytes to marginal sinus-lining spleen in BALB/c background, thus approaching a T/B cell ratio cells in the spleen occurs independently of L-selectin (2, 3), but that is more characteristic for pLNs than for the spleen (Fig. 3D). in pLNs, binding to HEVs critically requires L-selectin. To Taken together, these observations suggest that both the vascula- determine whether the presence of PNAd+ HEVs in the spleen is ture and lymphocyte composition in the Nkx2.3-deficient spleen coupled with an altered dependence on L-selectin during homing, resemble that of peripheral lymph nodes without, however, the we performed short-term competitive homing experiments in typical tissue compartmentalization of pLNs. To test whether the which wild-type lymphocytes were treated with the specific Ab absence of organized follicles is caused by the lack of CXCL13 MEL-14 to block L-selectin (36). This mAb prevents the homing homeostatic chemokine production by follicular dendritic cells of lymphocytes to pLNs in vivo but does not eliminate the cells (FDCs), we stained sections with anti-CXCL13 and anti-CD21/35 (37). Equal numbers of CFSE-labeled lymphocytes treated with Abs. We found that, although the FDC network in mutant spleens MEL-14 mAb and mock-treated biotinylated control cells (27) was considerably more compact compared with wild-type controls were then injected into Nkx2-32/2 mutant and wild-type recipi- (Fig. 2A), FDCs showed detectable CXCL13 production, similarly ents. Thirty minutes after the injection, spleen and pLN cells were to the wild-type samples (Fig. 3C). isolated and subjected to flow cytometry. We observed that the The Journal of Immunology 6985

FIGURE 3. Nkx2-3 mutation induces “redless” variant in BALB/c mice. A, Macroscopic appearance of “redless” mutant spleen (Spl) (top and middle samples) in comparison with wild type (bottom sam- ple). Arrow points to the splenic hilus. B, Frozen sections of young adult spleens from wild-type and Nkx2-32/2 mutant mice were immunostained with anti-CD3 and anti-IgM for T and B cells, respectively. Note the mixed cell distribution in the mutant spleen. C, Frozen sections of young adult spleens from wild- type and Nkx2-32/2 mutant mice were labeled with anti-CR1.2 (left panels) and anti-CXCL13 (middle panels) Abs to delineate FDCs producing CXCL13 Downloaded from (merge, right panels)(n = 5, in two independent experiments). Scale bar, 250 mm. D, Flow cytometric analysis of cellular composition in Nkx2.32/2 and the wild-type spleen and pLNs. Representative examples illustrate the T-to-B cell ratio and proportion of CD62L (L-sel) expressing lymphocytes in mutant and http://www.jimmunol.org/ wild-type control. Numbers indicate the percentages of cells in each quadrant of gated viable lymphocytes. The results are representative for three independent experiments with at least three mice each. Fo, follicle; Mz, marginal zone; PALS, periarteriolar lymphoid sheath; Rp, red pulp. by guest on September 24, 2021

homing of mock-treated biotinylated lymphocytes in the spleen HEV-like vessels in the mutant spleen also produce CCL21 che- of Nkx2-32/2 mutants was reduced compared with that in wild- mokine, spleen sections were subjected to double immunofluores- type recipients, but more importantly, the homing of MEL-14 cence analysis by using anti-CCL21 and MECA-79 Abs. Similarly mAb-treated CFSE-labeled cells to the mutant spleen was sig- to pLNs, the majority of PNAdhi vessels in the mutant spleen nificantly blocked compared with wild-type recipients (Fig. 4B). coexpressed CCL21, which was additionally present on fibro- The impaired homing activity of lymphocytes with blocked L- blastic reticular cells. In a normal spleen with no PNAd reactivity, selectin function was indicated by a 3-fold decrease in the CFSE: the CCL21 expression was restricted to the central arteriole and biotin ratio in the Nkx2.3-deficient spleen but not in the wild-type fibroblastic reticular cells in the T cell zone (Fig. 4C). Thus, the spleen. The ratio in the mutant spleen was very similar to that in CCL21 chemokine is readily available in ectopic PNAd+ vessels pLNs of wild-type or mutant mice, suggesting that the ectopic in mutant spleens, and it is likely to play a similar role in homing PNAd+ HEV-like vessels in the mutant spleen use L-selectin for as for the HEVs in pLNs. lymphocyte homing. As expected, blocking L-selectin significantly Impaired capture of blood-borne pathogens in the spleens of reduces the ratio of CFSE:biotin-labeled cells in both wild-type 2/2 and mutant pLNs, confirming that lymph nodes use L-selectin– Nkx2-3 mice dependent homing mechanism, regardless of the Nkx2-3 genotype. In addition to providing a microenvironment for the initial lod- Interestingly, the biodistribution of variously labeled lymphocytes ging of mobile lymphocytes during their splenic recirculation, without MEL-14 blockade also revealed a striking parallel between the marginal zone also plays an important role in capturing blood- the mutant spleen and pLNs, because the biotinylation of lympho- borne corpuscular Ags, including both encapsulated Gram- cytes favored their splenic homing in wild-type mice, whereas positive bacteria and Gram-negative E. coli (1). The abnormal in pLNs, the frequency of CFSE cells was higher, resulting in a vascular arrangement and the reported loss of marginal zone CFSE:biotin ratio , 1 in the wild-type spleen, and CFSE:biotin macrophages and a subset of B cells in Nkx2-3–mutant spleens ratio . 1 in pLNs as well as in the mutant spleens, respectively. (18–22, 26) prompted us to examine the clearance of bacteria by Subsequent to the binding of L-selectin by PNAd, extravasation i.v. injection of fluoresceinated E. coli (39). Because of the lack of of lymphocytes in pLNs involves the arrest chemokine CCL21 marginal zone macrophages and MAdCAM-1–positive sinus- that is produced by HEV endothelium (38). To test whether the lining cells as reference markers in the spleens of mice with 6986 Nkx2-3 DETERMINES THE VASCULAR PATTERNING IN THE SPLEEN

FIGURE 4. HEVs in Nkx2-32/2 mice function as L-selectin–dependent exit ports to the spleen and express CCL21. A, CFSE-labeled lymphocytes (green) are localized to HEVs identified with MECA-79 anti-PNAd mAb (red) in the Nkx2-32/2 spleen (left) and pLN 30 min after i.v. injection. In the wild- type spleen (right), the lymphocytes are associated with the marginal zone visualized by IBL-11 mAb. Scale bar, 200 mm; the results are representative for three independent experiments with at least three mice each. B, MEL-14 mAb against L-selectin reduces short-term homing to the spleen and pLN in Nkx2-32/2 mice. CFSE-labeled cells were treated with MEL-14 mAb or control mAb and injected to- Downloaded from gether with biotinylated cells into wild-type or Nkx2- 32/2 mutant recipients as described in Materials and Methods. Representative examples are shown. Num- bers indicate the percentages of cells in the four quadrants. The lower diagram summarizes the results of three independent experiments performed with three to five mice for each genotype. The percentage http://www.jimmunol.org/ data of retrieved cells were corrected by the ratio of CFSE:biotinylated cell percentages in the preinjec- tion mixture. Shaded bars, MEL-14 mAb; open bars, control mAb; **p , 0.001. C, The majority of PNAdhi HEVs (arrows) in both the mutant spleen (left) and control pLNs (middle) express CCL21 (red and green fluorescence, merged as yellow) in addi- tion to fibroblastic cells (arrowhead), whereas in the

normal spleen (right), CCL21 expression is over- by guest on September 24, 2021 whelmingly restricted to the T zone fibroblastic reticular cells and central arteriole (asterisk) without detectable PNAd reactivity. Dashed lines demarcate follicles (scale bar, 200 mm; the results are repre- sentative for three independent experiments with at least three mice each).

disrupted Nkx2-3, IBL-11 mAb against white pulp fibroblastic it is still detectable in mLNs of newborn mutants (19, 20). Under reticular cells was used, and IBL-11 mAb detects circumferential physiological conditions, MAdCAM-1 is similarly downregulated reticulum in wild-type mice and perivascular stromal cells in during postnatal maturation of HEVs in pLNs (40). In contrast, mutants (22, 26). Although efficient retention of bacteria in the PNAd+ HEVs can also be induced to occur at an ectopic location marginal zone of wild-type animals was observed, capture of upon chronic infection coupled with accumulation of lymphoid bacteria in the mutant spleens was markedly reduced (Fig. 5). cells (41). To investigate whether the appearance of ectopic HEVs These data indicate that the replacement of splenic marginal sinus in a mutant spleen follows the same postnatal developmental ki- and other splenic vessels with ectopic HEVs and the attenuated netics characterized by upregulated PNAd expression in place of recirculation of lymphocytes in the Nkx2-3–deficient spleen are MAdCAM-1 as in pLNs, we compared MAdCAM-1 and PNAd accompanied by a considerably reduced capacity to collect blood- expression in mutant mice after birth using immunofluorescence borne pathogens, presumably because of the severe reduction of analysis. At P0.5 (,1-d-old neonates), spleens of both Nkx2-3– marginal zone macrophages in this mutation (20), which cells are closely associated with the marginal sinus in wild-type mice (1). deficient and wild-type mice contained cell clusters expressing MAdCAM-1 but no PNAd (Fig. 6A). Significantly, the spleen of 2/2 Ectopic HEVs in the spleen of the Nkx2-3 mouse develop the Nkx2-32/2 mutant at postnatal day 10.5 (P10.5) exhibited less b postnatally and require LT R activity MAdCAM-1, which was limited to a few dispersed vessels, si- It has been previously reported that in the secondary lymphoid multaneously expressing PNAd (Fig. 6B). This was in marked organs of adult Nkx2-32/2 mouse MAdCAM-1 is absent, whereas contrast to the wild-type spleen at P10.5 in which a considerable The Journal of Immunology 6987

FIGURE 5. Impaired capture of blood-borne pathogens in the Nkx2-3– mutant spleen. A small number of i.v. injected fluoresceinated E. coli (green) are retained in the vessels (arrows) in the mutant spleen (left), whereas in control mice, a substantial accumulation can be observed in the marginal zone (arrowhead) outlined by IBL-11 mAb (red; scale bar, 500 mm; the results are representative for three independent experiments with at least three mice each).

MAdCAM-1 reactivity was observed in the developing marginal sinus, but absolutely no PNAd-positive cells could be detected at

any period. After the second postnatal week, no MAdCAM-1 Downloaded from expression was observed in the mutant spleen, similarly to young adults (19, 20) (data not shown). Comparison of MAdCAM-1 and PNAd expression in the mutant spleen and in normal age-matched pLNs revealed that the kinetics of early postnatal shift of addressin expression were similar in both lym- phoid organs (Fig. 6B). http://www.jimmunol.org/ The appearance of pLN-like HEVs in Nkx2-3–mutant spleens in a fashion similar to the development of HEVs in pLNs prompted

FIGURE 7. The formation of ectopic HEVs requires the activity of LTbR. A, Frozen spleen sections from various genotypes from 4-wk-old

mice were labeled with the Abs indicated at the top (representative result by guest on September 24, 2021 from staining performed on three separate occasions on three to five mice). Scale bar, 250 mm; n =5.B, Postnatal treatment of Nkx2-3–deficient mice with the LTbR-Ig decoy receptor disrupts ectopic HEV formation and abolishes MAdCAM-1 expression (top row). HEVs in mice treated with control human IgG (bottom row) express both MAdCAM-1 and PNAd (arrowheads) in addition to stroma-associated MAdCAM-1 (asterisk; scale bars, 200 mm; n = 4; nuclei counterstained with DAPI).

us to investigate whether this shift of programming is restricted to the vasculature, or other elements in the mesenchymal anlage in mutant spleens also switch to pLN-like features, characterized by their dependence on signaling mediated by LTbR (41, 42). To test whether LTbR signaling influences the development of a spleen in Nkx2-3–mutant mice, we generated double-mutant mice lacking both LTbR and Nkx2-3. These double-KO mice at 4 wk of age entirely failed to display PNAd-positive HEVs in their spleens in addition to lacking IBL-9/2 red pulp vessels (a trait shared with Nkx2-3–deficient mice) and MAdCAM-1 marginal sinus (a feature observed in both parent strains) (Fig. 7A). This result suggests that the formation of ectopic HEVs in a mutant FIGURE 6. pLN-like postnatal switch of addressin expression in splenic spleen is dependent on LTbR function similarly to pLNs; how- HEVs in Nkx2-3–deficient mice. A, Frozen newborn (P0.5) mutant and ever, as the spleen as a separate organ in these double mutants wild-type spleen sections were immunostained as indicated. Nuclei were was present, we conclude that the nonvascular mesenchymal stained with DAPI (blue). B, By postnatal day 10.5 (P10.5), the expression elements of splenic anlage can still develop, in contrast to the pLN of MAdCAM-1 in the mutant spleen is limited to a few vessels that formation blocked in the absence of LTbR. coexpress PNAd (top row, the quadrangle indicates the same area). In the b wild-type spleen (middle row), MAdCAM-1 outlines the boundary be- We and others have shown that administration of the LT R–Ig tween white and red pulp; PNAd is not expressed. In wild-type pLNs fusion protein to neonates effectively blocks postnatal vascular (bottom row), most of the HEVs coexpress MAdCAM-1 and PNAd (left development in the murine spleen (26, 43). To test whether the and middle panels). The pictures are representative of three independent pLN-like programming of splenic vasculature in Nkx2-3 mutants experiments performed on three to five mice. Scale bars, 50 mm. can be modulated postnatally, we applied this decoy receptor to 6988 Nkx2-3 DETERMINES THE VASCULAR PATTERNING IN THE SPLEEN

Nkx2-3–deficient mice on P0.5 and P3.5 and harvested spleens on CXCL13 production needs to be complemented with Nkx2-3– P10.5. We found that postnatal interference with LTbR activity dependent processes to promote follicle development in the spleen. also resulted in a significant reduction of PNAd expression in- According to this observation, Nkx2-3 does not seem to act up- dicative for less ectopic HEVs in the spleen (Fig. 7B). Thus, the stream of lymphotoxin signaling, and it may indeed function in transformation of splenic vasculature to HEV-like vessels in an a parallel regulatory pathway. However, both regulators are dis- Nkx2.3-deficient mouse seems to follow a similar program that pensable for embryonic development of the mesenchymal spleen underlies vessel formation in pLNs. anlage, because in a double-mutant mouse the spleen is present. It has been suggested that MAdCAM-1 glycoprotein in wild-type Discussion mice may be instrumental for proper clustering of endothelial cells A common theme in the postnatal organization of structured (43). Because HEV-associated MAdCAM-1 is still present in the secondary lymphoid organs is their compartmentalization into Nkx2.32/2 mutant spleen during the first two postnatal weeks when discrete regions of T or B cell dominance as a consequence of the white pulp and marginal zone architecture is established (25, 44, lymphocyte extravasation from specialized vascular segments. In 45, 50), it is reasonable to assume that loss of additional, yet un- this study, we provide structural, molecular, and functional evi- identified, proteins contributes to the adult spleen phenotype in this dence that the transcription factor Nkx2-3 plays a key role in mutation. This view is in line with the observation that MAdCAM- determining the vascular identity of the spleen. The spleen in 1–deficient mice exhibit apparently no major alterations of the Nkx2-3–deficient mouse acquires pLN-like vessels, which struc- splenic architecture (51). Importantly, the regulation of MAdCAM- tures mediate L-selectin–dependent homing. These observations 1 expression by Nkx2-3 appears to be different for endothelial cells suggest an unexpected plasticity among the developmental pro- and other cells, such as FDCs, because FDC-associated MAdCAM- Downloaded from grams that control the vasculature of peripheral lymphoid organs. 1 expression is preserved in mutant Peyer’s patches (20), whereas The importance of the homeodomain transcription factor Nkx2-3 the HEV-associated MAdCAM-1 production is abolished, similarly in the correct splenic vessel development is most strikingly il- to HEVs in mLNs. Thus, the differential effects of the absence of lustrated by the redless spleen lacking nearly all of the red pulp. Nkx2-3 on the expression of MAdCAM-1 mRNA in pLN of wild- In contrast to vessel formation in pLNs, little is known about type and Nkx2-3–mutant mice (where mutants had a higher level)

vascular patterning in the spleen (1, 2, 4). A characteristic vascular compared with mLNs (where wild-type mice showed a higher level http://www.jimmunol.org/ segment formed during the early postnatal period in the rodent of expression) is probably caused by the enlarged germinal centers spleen is the marginal sinus composed of MAdCAM-1–positive containing MAdCAM-1–positive FDCs in pLNs of Nkx2-3 mutants cells (2, 44, 45). Allocation of MAdCAM-1+ cells to the marginal and the presence of highly MAdCAM-1–positive HEVs in the wild- sinus in the mouse spleen (43) is controlled by several signals and type mLNs, respectively. regulators, including Nkx2-3, which can probably activate the The transformation of splenic vasculature into pLN-like vessels transcription of MAdCAM-1 directly (19). Inactivation of Nkx2-3 as a result of the Nkx2-3 gene disruption emphasizes the importance function affects the expression of MAdCAM-1 and also the for- of correct vessel formation for spleen ontogeny in mammals. The mation of the spleen and Peyer’s patches. In contrast, loss of LTa red pulp is formed during embryonic development when Nkx2-3 is or LTbR blocks embryonic development of virtually all peripheral expressed (15, 20), whereas expansion and compartmentalization by guest on September 24, 2021 lymphoid organs, including the white pulp stroma of the spleen; of white pulp occur postnatally when Nkx2-3 expression is already however, segregation of vasculature into red pulp and white pulp downregulated. This scenario suggests that in the embryonic spleen territories takes place independently from LTbR ligands (14, 15, Nkx2-3 may be directly responsible for the development of the red 17). The Nkx2.3-null mutation causes a particularly severe defect pulp sinus network that ascertains blood supply essential for tissue in splenic vasculature affecting all vascular segments in the red growth. Nkx2-3 also seems to be required to set up early progenitor pulp, marginal zone, and white pulp as well (26). It results in cells for the correct vasculature in white pulp. Whether or not the nearly complete loss of red pulp vascular beds and disturbed white function of Nkx2-3 in this process is to actively promote the ap- pulp architecture, thus offering the opportunity to analyze the propriate cell fate for white pulp endothelium or to suppress HEV- putative role of red pulp on morphogenesis of the white pulp. fated endothelial progenitors is currently unclear. Alternatively, The Nkx2-32/2 mutant spleen contains vessels that fulfill the Nkx2-3 may play a role that affects postnatal development of the morphological and functional criteria of HEVs that are normally splenic vasculature in a non–cell-autonomous fashion, for instance, present only in pLNs. According to our qPCR data, these unusual by changing stromal signals. However, the transformation of splenic splenic HEVs use predominantly GlyCAM-1 besides some other vasculature to contain HEV-like vessels in the mutant spleen is not PNAd core proteins but no or very little MAdCAM-1 as homing simply the consequence of defective red pulp development, as it is receptor (28–33). Replacement of MAdCAM-1 by PNAd in the observed in BALB/c mutants that retain some red pulp and in Nkx2- spleen of the Nkx2-32/2 mouse occurs postnatally, suggesting that 3 mutants on other genetic backgrounds where red pulp is also early MAdCAM-1 expression may either not be controlled by present. In addition to the altered vascular organization, other cel- Nkx2-3 or, alternatively, may be regulated by coexpressed proteins lular compartments including marginal zone macrophage subsets such as Nkx2-5, providing redundant functions. Besides Nkx2-3, are also affected, leading to major functional deficits in lymphocyte expression of MAdCAM-1 is influenced by other regulators, such as recirculation and pathogenic uptake, and may explain defective relB/NF-kB (46), because NF-kB loss of function mutations in mice immune responses in Nkx2-3–mutant mice (49). Taken together, also lead to loss of marginal sinus-associated MAdCAM-1 ex- our observations clearly demonstrate that Nkx2-3 is an important pression (46, 47). In this context, NF-kB appears to act downstream factor for the distinct vasculature in spleens well beyond the regu- of LTbR-mediated signals (41, 48). Preliminary gene expression lation of MAdCAM-1 by possibly determining the embryonic analysis in Nkx2-32/2 mice failed to provide evidence for loss of vascular patterning and recirculation activity in this secondary LT-related ligands or receptors in agreement with the presence lymphoid organ. of splenic FDCs (20, 49), which depend on LTbR activity (23). Similarly to previous findings, we also confirm that although in Acknowledgments Nkx2-3–mutant mice FDCs are present and produce CXCL13 We thank Drs. Nancy H. Ruddle and Ann Ager for helpful critical com- chemokine (20), yet no follicles are formed, indicating that ments. We also thank Drs. Klaus Pfeffer and Falk Weih for providing the The Journal of Immunology 6989

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