Essential Roles for the Tec Family Kinases Tec and Btk in M-CSF Receptor Signaling Pathways That Regulate Macrophage Survival This information is current as of September 26, 2021. Martin Melcher, Bernd Unger, Uwe Schmidt, Iiro A. Rajantie, Kari Alitalo and Wilfried Ellmeier J Immunol 2008; 180:8048-8056; ; doi: 10.4049/jimmunol.180.12.8048 http://www.jimmunol.org/content/180/12/8048 Downloaded from

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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 © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Essential Roles for the Tec Family Kinases Tec and Btk in M-CSF Receptor Signaling Pathways That Regulate Macrophage Survival1

Martin Melcher,2* Bernd Unger,2† Uwe Schmidt,3* Iiro A. Rajantie,‡ Kari Alitalo,‡ and Wilfried Ellmeier4*

Tec family kinases have important roles in lymphocytes; however, little is known about their function in monocytes/macrophages. In this study we report that Tec family kinases are essential for M-CSF (M-CSF)-induced signaling pathways that regulate macrophage survival. Compared with wild-type bone marrow-derived macrophage (BMM) cultures, Tec؊/؊Btk؊/؊ BMM cultures displayed increased cell death that correlated with a severe drop in macrophage numbers. In addition, macrophages deficient in either Tec or Btk showed expression and activation of caspase-11. Elucidation of M-CSF receptor (M-CSFR) signaling pathways Downloaded from revealed that the total tyrosine phosphorylation pattern upon M-CSF stimulation was altered in Tec؊/؊Btk؊/؊ macrophages despite normal expression and phosphorylation of the M-CSFR. Further, Tec and Btk are required for proper expression of the GM-CSF receptor ␣ (GM-CSFR␣) chain in macrophages but not dendritic cells, implicating Tec family kinases in the lineage- specific regulation of GM-CSFR␣ expression. Taken together, our study shows that Tec and Btk regulate M-CSFR signaling- induced macrophage survival and provides a novel link between Tec family kinases and the regulation of caspase-11 and GM-

CSFR␣ expression. The Journal of Immunology, 2008, 180: 8048–8056. http://www.jimmunol.org/

acrophages are large phagocytic mononuclear cells review see Ref. 4 and references therein). Thus, a better under- that play important roles in innate and adaptive im- standing of M-CSFR signaling may also be of medical relevance. munity. Their progenitors, the monocytes, enter the Members of the Tec kinase family (Bmx, Btk, Itk, Rlk, and Tec) M 5 blood stream from the bone marrow (BM) and migrate to tissues constitute the second largest family of nonreceptor tyrosine ki- where they mature into resident tissue macrophages (1). The dif- nases and are preferentially expressed in the hematopoietic system. ferentiation, proliferation, and survival of macrophages are regu- A large number of studies have shown important roles for these lated by the M-CSF. In fact, M-CSF receptor (M-CSFR)-deficient kinases in the lymphoid system. Furthermore, mice with combi- by guest on September 26, 2021 mice or mice with an inactivating mutation of M-CSF have pleio- natorial deletions of Tec family kinases revealed both unique and tropic phenotypes including decreased macrophage numbers in redundant functions in B cells (Tec, Btk) and T cells (Rlk, Itk). vivo (2, 3). In mice, impaired M-CSF signaling has also been Although the Tec family kinase members Tec, Btk, and Bmx are implicated in the pathogenesis of several disorders (for a detailed expressed in monocytes/macrophages (5–7), little is known about their function in this lineage. Several studies implicated Tec family kinases in the LPS-induced signaling in macrophages leading to the induction of TNF-␣ production. Btk-defective X-linked immu- *Institute of Immunology, Center for Physiology, Pathophysiology and Immunology, † nodeficient (xid) macrophages have impaired secretion of the Medical University of Vienna, Vienna, Austria; Competence Center for Biomolec- ␣ ␤ ular Therapeutics, Vienna, Austria; and ‡Molecular/Cancer Biology Laboratory, Bio- proinflammatory cytokines TNF- and IL-1 after stimulation medicum Helsinki, University of Helsinki, Helsinki, Finland with LPS (8) and are also incapable of producing efficient bursts of Received for publication August 7, 2007. Accepted for publication April 10, 2008. reactive oxygen intermediates (9). In line with this, xid macro- The costs of publication of this article were defrayed in part by the payment of page phages show impaired p65 phosphorylation and transactivation charges. This article must therefore be hereby marked advertisement in accordance upon LPS stimulation, whereas IkB␣-degradation is normal (10). with 18 U.S.C. Section 1734 solely to indicate this fact. Meanwhile, another study could not find any differences in TNF-␣ 1 This work was supported by the START program (Grant Y-163) of the Fonds zur Fo¨rderung der Wissenschaftlichen Forschung and the Austrian Ministry of Education, expression between control and xid macrophages after LPS stim- Science and Culture (to W.E.), by the K-Plus Competence Center for Biomolecular ulation (5), which may reflect differences in the genetic back- Therapeutics (to W.E.), by the Sonderforschungsbereich project F2305-B13 of the Austrian Research Fund (to W.E.), and by a postdoctoral fellowship (to U.S.) from the grounds or different macrophage populations used in these studies. Deutsche Forschungsgemeinschaft (Schm 2128/1-1). However, the importance of Tec family kinases for monocyte 2 M.M. and B.U. contributed equally to this work. function has been confirmed through the analysis of Btk-deficient 3 Current address: Nabriva Therapeutics, Brunnerstrasse 59, 1235 Vienna, Austria. human monocytes. Blood monocytes isolated from X-linked 4 Address correspondence and reprint requests to Dr. Wilfried Ellmeier, Institute of agammaglobulinemia (XLA) patients who lack a functional Btk Immunology, Medical University Vienna, Center for Physiology, Pathophysiology have impaired phagocytic functions and altered chemotactic and Immunology, Lazarettgasse 19, A-1090 Vienna, Austria. E-mail address: ␣ [email protected] responses (11) and are impaired in the production of TNF- and IL-1␤ upon stimulation of TLR 2 or 4 (12), although another study 5 Abbreviations used in this paper: BM, bone marrow; BMDC, BM-derived dendritic cell; BMM, BM-derived macrophage; HPRT, hypoxanthine phosphoribosyltrans- reports that Btk is not essential for LPS/TLR4 signaling (13). ferase; LCM, L929 cell-conditioned medium; M-CSFR, M-CSF receptor; PI, pro- Overexpression of Btk in wild-type human monocytes leads to the pidium; xid, X-linked immunodeficient (gene); XLA, X-linked agammaglobulinemia. stabilization of TNF-␣ mRNA and therefore to an increase in Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 TNF-␣ production (5, 12). Interestingly, incubation of XLA www.jimmunol.org The Journal of Immunology 8049

monocytes with M-CSF leads to an increase in the expression of containing M-CSF (10 and 30 ng/ml), GM-CSF (500 and 1500 U/ml), or Tec and restores their ability to produce TNF-␣ upon LPS stimu- LCM was exchanged daily until the end of the culture. For the supernatant 7 Ϫ/Ϫ Ϫ/Ϫ lation (5). This suggests a compensatory role for Tec similar to the exchange experiments, 10 wild-type and Tec Btk cells were re- seeded onto 10-cm dishes on day 6, and the corresponding supernatants situation observed in murine B cells (14), which may also explain were added daily to the culture. For the M-CSF titration experiments, cells why XLA patients show normal innate immune responses (5). were reseeded in 6-well plates at 0.8 ϫ 106 cells per well on day 5 and In this study, we aimed to further dissect the role of Tec family different concentrations of M-CSF (15, 30, 60, and 90 ng/ml) were added. kinases in monocytes/macrophages. We used a genetic approach to Generation of BM-derived dendritic cells (BMDCs) study macrophages lacking various members of the Tec kinase family and generated combinatorial Tec family kinase knockout BM cells were isolated and RBCs were lysed as described above for the 6 mice. We could show that Tec and Btk regulate the survival of generation of BMM. To generate BMDCs, 2 ϫ 10 BM cells were cultured on 10-cm dishes in RPMI 1640 medium with 10% FCS, 100 U/ml peni- BM-derived macrophages (BMM) by controlling M-CSFR signal- ␮ L Ϫ/Ϫ Ϫ/Ϫ cillin, 10 g/ml streptomycin, 2 mM -glutamine, 10 mM HEPES (Sigma- ing. A severe drop in cell numbers in Tec Btk macrophage Aldrich), 50 ␮M 2-ME (Invitrogen) and 700 U/ml recombinant murine cultures was observed, and this correlated with increased cell death GM-CSF (Peprotech) for 10 days (19). of macrophages. Despite normal expression and M-CSF-induced autophosphorylation of the M-CSFR, M-CSF stimulation of RT-PCR Ϫ Ϫ Ϫ Ϫ Tec / Btk / macrophages resulted in an altered tyrosine phos- Total RNA from the various cell types was isolated with TRIzol reagent phorylation pattern. Because Btk was activated upon M-CSF stim- (Sigma-Aldrich), treated with DNase I (Boehringer Mannheim), and con- ulation of primary BMMs, our study thus shows that Tec family verted into cDNA by reverse transcription with oligo(dT) and random primer according to the manufacturer’s protocol (SuperScript II first- kinases play an important role in M-CSFR signaling pathways that strand synthesis for RT-PCR; Invitrogen). The following primers were Downloaded from Ϫ/Ϫ Ϫ/Ϫ lead to macrophage survival. Interestingly, Tec or Btk mac- used for expression analysis: Tec, 5Ј-TAACCATGGTGACTCGTGGC rophages showed constitutive expression of caspase-11, an induc- CA-3Ј (forward) and 5Ј-GGTATACATGGCTGGCACTCA-3Ј (re- ible member of the caspase family (15). Finally, we found that Tec verse): Btk, 5Ј-GAGTAACATTCTAGATGTGATGG-3Ј (forward) and 5Ј-CAGTCTGTTAGGAGTCTTGAA-3Ј (reverse); Bmx, 5Ј-gcagccctat and Btk are required for proper expression of the GM-CSF recep- gacttatatgat-3Ј (forward) and 5Ј-CAGATAAACAGCACATAGACC-3Ј tor ␣-chain (GM-CSFR␣) in macrophages but not dendritic cells, (reverse); Hprt, 5Ј-GATACAGGCCAGACTTTGGTTG-3Ј (forward)

therefore implicating Tec kinases also in the lineage-specific reg- and 5Ј-GGTAGGCTGGCCTATAGGCT-3Ј (reverse); and Csf2Ra, 5Ј- http://www.jimmunol.org/ ulation of GM-CSFR␣ expression. CCCCCACGGAGGTCACAAGGTCAA-3Ј and5Ј-CAGGGCAACAGG GGTCCAGTCACA-3Ј (reverse).

Materials and Methods BrdU incorporation Mice BM cells were differentiated into macrophages as described above. On day Tec-deficient mice (14), Btk-deficient mice (16) (purchased from The Jack- 5, cells were reseeded at 0.8 ϫ 106 cells per well of a 6-well plate. At day son Laboratory), and Bmx-deficient mice (17) were intercrossed and main- 6, BMM cultures were incubated with 0.1 mM BrdU (Sigma-Aldrich) for tained in the animal facility of the Medical University of Vienna, Vienna, a 1.5-h period. Cells were removed from the plate with citric saline as Austria. The mice used in this study were of mixed 129/Sv ϫ C57BL/6 described above, resuspended in 500 ml 0.15 M NaCl. Ninety-five percent

background. C57BL/6 Ly 5.1 mice were obtained from the European EtOH (Ϫ20°C) was added dropwise. After 30 min on ice, the cells were by guest on September 26, 2021 Mouse Mutant Archive (Strasbourg, France). All animal experiments were washed with PBS and resuspended in 1 ml of 1% paraformaldehyde with performed according to protocols approved by the Federal Austrian Min- 0.01% Tween 20 in PBS and incubated at 4°C overnight. The cells were istry for Education, Science and Art (Vienna, Austria). incubated in 1 ml DNase I solution (50 Kunitz units/ml DNase I, 0.15 M

NaCl, 4.2 mM MgCl2, and 10 mM HCl; Sigma-Aldrich) at 37°C for 30 Flow cytometry and Abs min. The samples were washed with PBS, stained with FITC anti-BrdU or isotype control Ab (BD Pharmingen), and analyzed by FACScan (BD The spleen was removed from euthanized mice and placed into 60-mm Biosciences). tissue culture dishes containing staining buffer (PBS, 2% FCS, and 0.1% sodium azide). Peritoneal cells were obtained by lavage of the peritoneum Propidium iodide (PI) stainings and cell death analysis with 10 ml of PBS. Single-cell suspensions were made by passing the tissue through a 70-␮m nylon cell strainer. BM cells were harvested from Macrophages were harvested with citric saline as described above and reconstituted mice by flushing femur and tibiae with PBS containing 2% resuspended in PBS. PI (4 ␮g/ml in PBS) was added and the percentage of FCS. After hypotonic lysis of RBCs with ACK lysis buffer (0.15 M NH4Cl, PI-positive cells was determined by flow cytometry (FACSCalibur; BD ϫ 5 1.0 mM KHCO3, and 0.1 mM Na2EDTA (pH 7.2)), 1–5 10 cells were Biosciences). incubated on ice for 5 min with Fc block (BD Pharmingen) and subse- quently stained with respective Abs for 30 min on ice in staining buffer. Bone marrow competition Afterward, the cells were washed once with staining buffer and analyzed. Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ The following mAbs were used: FITC-anti-mCD11b, PE-anti-Gr1, and BM cells from wild-type and Tec / Btk / Bmx / as well as from Ly allophycocyanin-anti-B220 from Caltag Laboratories. Flow cytometric 5.1-positive wild-type mice were isolated and RBCs were lysed with ACK analysis was performed on a FACSCalibur device (BD Biosciences) and (ammonium chloride-potassium carbonate) buffer. The cells were washed data were analyzed with CellQuest Pro software. three times with PBS and counted. Ly 5.1 BM cells were mixed in the ratio of 1:1 with either wild-type BM or knockout BM. Mixed BM cells (1 ϫ Generation of BMMs 106) were injected into the tail vein of lethally irradiated Ly 5.1 mice (2 ϫ ␮ 7 3500 mGy; Hille TH-150). Mice were treated with 25 g/ml neomycin BMMs were generated as described (18). Briefly, after RBC lysis, 10 BM (Invitrogen) and 25,000 U/ml polymyxin B sulfate (Sigma-Aldrich) in the cells were seeded onto 10-m bacterial dishes in 10 ml of DMEM (Sigma- acidified drinking water for 1 wk. After 6–8 wk the reconstituted mice Aldrich) supplemented with 10% FCS (Invitrogen), 100 U/ml penicillin, 10 were sacrificed and analyzed by flow cytometry (FACSCalibur; BD ␮ g/ml streptomycin, 2 mM L-glutamine, 10 mM HEPES (Sigma-Aldrich), Biosciences). and 50 ␮M 2-ME (Invitrogen) in the presence of 20% L929 cell condi- tioned medium (LCM). The culture medium was changed on days 3 and 6 RNase protection assay unless indicated otherwise. Cells were analyzed by flow cytometry and microscopy (Nikon Eclipse TS100). LCM was produced as described (18). RNA from wild-type and TecϪ/ϪBtkϪ/Ϫ BMMs was isolated with TRIzol For determining the effects of M-CSF and GM-CSF and those of wild-type reagent (Sigma-Aldrich) at day 10. The multiprobe RNase protection assay and TecϪ/ϪBtkϪ/Ϫ supernatant exchanges on macrophage numbers, culture was performed according the manufacturer’s protocol (BD Biosciences). medium changes were done on days 2 and 5. For the M-CSF and GM-CSF The hybridization products were separated on a 4.75% denaturating poly- rescue experiments, BMMs were removed on day 6 from the plate with acrylamide gel. The gel was dried and exposed to autoradiography films citric saline (0.135 M potassium chloride and 15 mM sodium citrate) and (Kodak) overnight at Ϫ80°C. Probes for the housekeeping gene probes reseeded in 6-well plates at 0.8 ϫ 106 cells per well. Medium (total of 2 ml) L32 and GAPDH were used as normalization controls. 8050 Tec AND Btk REGULATE M-CSF RECEPTOR SIGNALING

Preparation of BMM lysates and immunoblot analysis Cell lysates were prepared by washing the macrophages on the tissue cul- ture dish with ice-cold PBS followed by their lysis in 120 ␮l (for 107 cells) lysis buffer (1% Nonidet P-40, 20 mM Tris-HCl (pH 8.0), 138 mM NaCl, 10 mM EDTA, and 10% glycerol) supplemented with 1 mM orthovanadate and complete protease inhibitor mix (Roche). Cell lysates were cleared by centrifugation and concentrations were determined using the Brad- ford method (Bio-Rad). The cell lysates were analyzed by standard West- ern blotting techniques using the following Abs: anti-phospho-Tyr (PY99; catalog no. sc-7020, Santa Cruz Biotechnology), rabbit-anti- phospho-Tyr223-Btk (catalog no.3531, Cell Signaling Technology), rabbit anti-Btk (catalog no. 556365, BD Pharmingen), rabbit anti-M-CSFR (cat- alog no. sc-692, Santa Cruz Biotechnology), rabbit anti-phospho-M-CSFR (catalog no.3155, Cell Signaling Technology), rat anti-caspase-11 (catalog no.C1354, Sigma-Aldrich), rabbit anti-actin (catalog no. A2066, Sigma- Aldrich), hypoxanthine phosphoribosyltransferase (HPRT)-coupled anti- rabbit Ig (Jackson ImmunoResearch Laboratories), HPRT-coupled anti- goat Ig (Jackson ImmunoResearch), HPRT-coupled anti-rat Ig (catalog no. P0450, DakoCytomation) and rabbit anti-Tec (gift from Prof. H. Mano, Jichi Medical University, Tochigi, Japan). Immunoblot protein bands were visualized by ECL (Amersham Biosciences). Downloaded from Stimulation of BMM BMMs were incubated overnight in 10 ml of medium without LCM at a 7 density of 10 cells per 10-cm dish. The following day the adherent cells FIGURE 1. Myeloid cell development in TecϪ/ϪBtkϪ/ϪBmxϪ/Ϫ mice. were stimulated directly on the dish with 100 ng/ml M-CSF (Peprotech) in A, RT-PCR analysis of RNA isolated from peritoneal macrophages (PM), a total volume of 4 ml for the indicated time periods at 37°C. To terminate BMM, and BM showing expression of Btk, Bmx, and Tec. Expression data the stimulation, the plates with the adherent cells were put on ice and the are representative of two independent experiments. B, Immunoblot analysis cells were washed with ice-cold PBS. Protein lysates were harvested as http://www.jimmunol.org/ Ϫ/Ϫ Ϫ/Ϫ described above. showing expression of Tec and Btk in wild-type (wt), Tec , and Btk BMM. Expression data are representative of two independent experiments. Cell surface M-CSFR immunoprecipitation C, Histograms showing Ly5.1 expression in macrophages (CD11bhigh/ Gr1medium) or B cells (B220high) isolated from the spleen of irradiated mice The protocol for immunoprecipitation of cell surface M-CSFR was adapted ϩ 7 that were reconstituted with a 1:1 mixture of either Ly5.1 wild-type (wt) from Lee et al. (20). In brief, day 8 BMMs were reseeded at 10 cells per ϩ ϩ ϩ 10-cm dish and incubated overnight with medium without LCM. The next and Ly5.2 wild-type (upper panels) or Ly5.1 wild-type and Ly5.2 day, the adherent macrophages were stimulated with M-CSF as described (TBB; lower panels) BM cells. Numbers in the histograms indicate the above, washed three times with ice-cold PBS, and incubated with 3 ml of percentage of cells in the indicated regions. One representative mouse from PBS containing 6 ␮g/ml sheep anti-M-CSFR Ab (catalog no. AF3818, a total of three mice reconstituted with two different batches of BM cells

R&D Systems) specific for the extracellular domain of the M-CSFR for 15 is shown. by guest on September 26, 2021 min at 4°C. Unbound Ab was removed by washing the cells five times with ice-cold PBS. Macrophages were lysed as described above and protein lysate (ϳ300 ␮g of protein) was incubated with protein G-agarose beads (Roche) for1hat4°C. The beads were pelleted by centrifugation and washed five times with lysis buffer. were removed from the beads competitive BM reconstitution experiments were performed. Wild- by boiling in Laemmli buffer and the surface fraction of M-CSFR was type (Ly5.1ϩ) BM cells were mixed in a 1:1 ratio with either determined by immunoblotting. Internal M-CSFR levels were measured in Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ ϩ ϩ cell lysate aliquots taken after the incubation with protein G-agarose beads. Tec Btk Bmx (Ly5.2 ) or wild-type (Ly5.2 ) BM cells ϩ Total M-CSFR levels were determined in cell lysate aliquots after the stim- and transplanted into irradiated Ly 5.1 wild-type recipients. After ulation with M-CSF. 6–8 wk of reconstitution, the ratio of Ly 5.1ϩ and Ly 5.2ϩ pop- ulation of the various cell lineages in the BM chimeras was de- Results termined. There was equal reconstitution of Ly5.2ϩ to Ly5.1ϩ Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ Normal myeloid cell development in Tec Btk Bmx mice macrophages (defined as CD11bhighGr1medium) (Fig. 1C, left pan- Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Tec family kinases are broadly expressed in the hematopoietic sys- els). As expected, Tec / Btk / Bmx / BM cells were not able tem, and Tec, Btk, and Bmx have been detected in the murine and to reconstitute the B cell lineage (Fig. 1C, right panels) due to a human monocyte/macrophage lineage (5–7). RT-PCR analysis severe block of B cell development in the combined absence of showed that peritoneal macrophages expressed Btk, Tec, and Bmx Tec and Btk (14). (Fig. 1A), while BMMs expressed Btk and Tec (Fig. 1, A and B). Therefore, we focused our further studies on the analysis of Impaired survival of BM-derived macrophages in the absence TecϪ/ϪBtkϪ/Ϫ BMMs and TecϪ/ϪBtkϪ/ϪBmxϪ/Ϫ mice (Btk and of Tec and Btk Bmx map to the X and thus the genotype of male To generate BMMs, BM cells of the various genotypes were knockout mice is Y/Ϫ; however, for simplicity we refer to Btk- isolated and differentiated using LCM, an established source of deficient or Bmx-deficient mice as BtkϪ/Ϫ or BmxϪ/Ϫ mice, re- M-CSF (21). Wild-type, TecϪ/Ϫ, and BtkϪ/Ϫ BM cell cultures spectively, throughout the article regardless of whether they were resulted in a similarly confluent layer of macrophages after 10 male of female). days in culture (Fig. 2A). In contrast, TecϪ/ϪBtkϪ/Ϫ macro- FACS analysis of various organs indicated that myeloid cell phage cultures were less dense (Fig. 2A) and showed dramati- subsets are present in TecϪ/ϪBtkϪ/ϪBmxϪ/Ϫ mice under homeo- cally reduced cell numbers already at day 8 of culture (Fig. 2B). static conditions. However, because the lack of Tec and Btk leads The drop in cell numbers occurred after day 6, because until this to reduced peripheral B cell numbers (14), the relative percentage time point cell numbers were equal in the various macrophage of the various subpopulations was different compared with wild- cultures. TecϪ/ϪBtkϪ/ϪBmxϪ/Ϫ BMM cultures showed a simi- type mice (data not shown). To determine whether TecϪ/ϪBtkϪ/Ϫ lar reduction in cell numbers as TecϪ/ϪBtkϪ/Ϫ cultures (data BmxϪ/Ϫ myeloid cells display the same developmental kinetic, not shown). Although cell numbers were reduced in the absence The Journal of Immunology 8051

FIGURE 2. Impaired survival of TecϪ/ϪBtkϪ/Ϫ BM- derived macrophages. A, Pictures depicting day 10 BMM cultures. The data are representative of 10 inde- pendent experiments. Original magnification was ϫ100. wt, Wild type. B, Diagram showing BMM numbers in day 6 and day 8 cultures. The summary of five inde- pendent experiments is shown. Error bars show SD. The p values were calculated using an unpaired Student’s t and ;0.0011 ,ءء ;0.0104 ,ء :test. The p values shown are Ͻ0.0001. wt, Wild type. C, Diagram showing the ,ءءء appearance of F4/80ϩCD11bϩ macrophages (as deter- mined by flow cytometry) in BM cultures. Numbers at the y-axis indicate the percentage of the F4/ ϩ ϩ 80 CD11b population. Data are representative of Downloaded from three independent experiments. wt, Wild type. http://www.jimmunol.org/

of Tec and Btk, the differentiation kinetics of macrophages ac- PI-positive TecϪ/ϪBtkϪ/Ϫ macrophages compared with PI-positive cording to the expression of F4/80 and CD11b were similar in wild-type macrophages on day 8 (Fig. 3B). all genotypes analyzed (Fig. 2C). Constitutive expression of caspase-11 in the absence of Tec The reduced cell numbers could be caused by increased cell family kinases death or reduced proliferation. BrdU labeling experiments revealed that there was no difference in the proliferation of TecϪ/ϪBtkϪ/Ϫ The increase in the percentage of PI-positive macrophages indi- by guest on September 26, 2021 macrophages on day 6 (Fig. 3A), whereas on days 8 and 10 there cated increased cell death in the absence of Tec and Btk. To test was no detectable proliferation of wild-type and TecϪ/ϪBtkϪ/Ϫ whether the expression of pro- or anti-apoptotic is altered in Ϫ Ϫ Ϫ Ϫ macrophages (data not shown). To determine whether increased Tec / Btk / macrophages, RNase protection assays were per- cell death caused the reduction in cell numbers, the percentage of formed. The expression of most of the genes analyzed was similar Ϫ Ϫ Ϫ Ϫ PI-positive TecϪ/ϪBtkϪ/Ϫ and PI-positive wild-type macrophages between wild-type and Tec / Btk / macrophages (Fig. 4A); was determined on days 6, 8, and 10 in BMM cultures. Although however, we observed that caspase-11 expression was induced in Ϫ Ϫ Ϫ Ϫ there was no difference in the percentage of PI-positive cells on Tec / Btk / macrophages. Two isoforms of 43 and 38 kDa exist days 6 and 10, there was an ϳ60% increase in the percentage of that can be processed to an active form of 30 kDa (15). Expressed and activated forms of caspase-11 could be detected by immuno- blot analysis not only in TecϪ/ϪBtkϪ/Ϫ but also in TecϪ/Ϫ and BtkϪ/Ϫ single knockout macrophages. This indicates a link be- tween Tec family kinases and the regulation of caspase-11 expres- sion (Fig. 4B). TecϪ/ϪBtkϪ/Ϫ BMM survival can be rescued by M-CSF The reduced survival of TecϪ/ϪBtkϪ/Ϫ BMM indicates that culture conditions that allow the survival of wild-type BMM are not suf- ficient for TecϪ/ϪBtkϪ/Ϫ BMM. This could be the result of a cell- intrinsic alteration. However, it is also possible that TecϪ/ϪBtkϪ/Ϫ BMMs secret a toxic factor, a survival factor is missing, or a survival factor is depleted faster compared with wild-type cells. Ϫ Ϫ FIGURE 3. Normal proliferation but increased cell death in Tec / To distinguish between these possibilities, medium exchange Ϫ/Ϫ Btk macrophage cultures. A, Diagram showing the percentage of BrdU- experiments were performed. Daily replacement of macrophage Ϫ/Ϫ Ϫ/Ϫ positive cells in wild-type (wt) and Tec Btk macrophages cultures culture medium (supplemented with LCM) led to a dense layer (day 6) after 90 min of incubation with BrdU. Data are representative of Ϫ Ϫ Ϫ Ϫ of Tec / Btk / macrophages (Fig. 5A, left panels) with nor- four different experiments. Error bars show SD. B, Diagram showing the TecϪ/ϪBtkϪ/Ϫ mal macrophage numbers (Fig. 5B), indicating that the survival percentage of wild-type (Wt) and PI-positive macrophages Ϫ/Ϫ Ϫ/Ϫ in day (d) 6, 8, and 10 cultures. The summary of four (day 6 and 8) and defect of Tec Btk macrophages is not caused by a cell Ϫ/Ϫ Ϫ/Ϫ three (day 10) independent experiments each performed in triplicates is autonomous process. Daily exchange of Tec Btk macro- shown. Error bars show SD. The p value was calculated using a paired phage culture supernatant to wild-type cultures did not affect ,is 0.0299. the survival of wild-type macrophages (Fig. 5, A, middle panels (ء) Student’s t test. The p value 8052 Tec AND Btk REGULATE M-CSF RECEPTOR SIGNALING

FIGURE 4. Expression of caspase-11 in the absence of Tec or Btk. A, RNase protection assay using RNA isolated from day 10 cultures of wild-type (wt) and TecϪ/ϪBtkϪ/Ϫ macrophages. One representative of two independent experiments is shown. B, Immuno- blot analysis of wild-type (wt), TecϪ/Ϫ, BtkϪ/Ϫ and TecϪ/ϪBtkϪ/Ϫ macrophages showing caspase-11 ex- pression. Actin was used as loading control. Expres- sion data are representative of two independent ex- periments. ␣-, Anti- (antibody). Downloaded from http://www.jimmunol.org/ and B). This argues against a toxic factor that is produced by The supernatant exchange experiments indicated that daily ad- TecϪ/ϪBtkϪ/Ϫ macrophages or a faster depletion of a survival dition of new culture medium that is supplemented with LCM factor. Adding wild-type macrophage culture supernatant (i.e., rescues the survival defect of TecϪ/ϪBtkϪ/Ϫ BMM. LCM is without additional LCM) to TecϪ/ϪBtkϪ/Ϫ cultures did not rescue viewed mainly as a source of M-CSF. Therefore, we tested the cell numbers of TecϪ/ϪBtkϪ/Ϫ macrophages (Fig. 5, A, middle whether daily addition of M-CSF can rescue macrophage numbers panels, and B), indicating that wild-type cells do not produce a in TecϪ/ϪBtkϪ/Ϫ BM cultures. Macrophage numbers were re- survival factor that is missing in TecϪ/ϪBtkϪ/Ϫ macrophages. As stored in a dose-dependent manner to a similar extent in wild-type expected, daily supernatant replacement with a culture medium not and TecϪ/ϪBtkϪ/Ϫ macrophage cultures (Fig. 5C). Next, we tested

supplemented with LCM led to a severe reduction in cell numbers whether a sufficiently high concentration of M-CSF can be iden- by guest on September 26, 2021 for both wild-type and TecϪ/ϪBtkϪ/Ϫ macrophages (Fig. 5A, right tified that, if added at day 5, would yield similar numbers of wild- panels). type and TecϪ/ϪBtkϪ/Ϫ macrophages. Thus, increasing amounts of

FIGURE 5. Effects of M-CSF and GM-CSF on mac- rophage survival. A, Pictures depicting day 10 wild-type (wt; upper panels) and TecϪ/ϪBtkϪ/Ϫ (lower panels) BMM cultures. The culture medium was replaced daily with new LCM-containing medium (daily ϩ LCM; left panels), supernatant (SN) from TecϪ/ϪBtkϪ/Ϫ (daily TecϪ/ϪBtkϪ/Ϫ SN; upper middle panel) or wild-type (daily wt SN; lower middle panel) cultures, or medium not supplemented with LCM (daily Ϫ LCM; right pan- els). Original magnification was ϫ100. B, Diagram showing macrophage numbers in day 10 cultures after medium exchange experiments shown in A. Error bars show SD. Data are representative of two independent experiments. SN ex, SN exchange between wt and TecϪ/ϪBtkϪ/Ϫ cultures; ϩ LCM, culture conditions in which LCM was added on days 1, 3, and 6. C, Diagram showing macrophage numbers in day 10 cultures after daily addition (from day 6 on) of either LCM or M-CSF (10 and 30 ng/ml). Error bars show SD. Data shown are representative of two independent experiments. D, Di- agram showing macrophage numbers in day 10 cultures after addition (day 5) of increasing amounts of M-CSF (0, 15, 30, 60, and 90 ng/ml). Error bars show SD. Data show summary of two independent batches each per- formed in triplicates. The p values shown are as follows: .(for 60 ng/ml M-CSF) 0.029 ,ءء ;Ͻ0.0001 ,ءءء The Journal of Immunology 8053

FIGURE 6. M-CSF receptor signaling in wild-type and TecϪ/ϪBtkϪ/Ϫ macrophages. A, Immunoblot analy- sis of wild-type BMM (day 9) showing Btk activation upon M-CSF stimulation for various time points. pY223, Phosphotyrosine at position 223; ␣-, anti- (an- tibody). B, Immunoblot analysis showing the tyrosine phosphorylation of the M-CSFR (P-Y-M-CSFR) in wild-type (wt) and TecϪ/ϪBtkϪ/Ϫ BMM (day 9) upon M-CSF stimulation (upper panel). Total M-CSFR levels are shown as control (lower panel). ␣-, Antibody. C, Immunoblot analysis showing surface (top row), inter- Downloaded from nal (second row from top) and total (fourth row from top) expression levels of M-CSFR in wild-type (wt) and TecϪ/ϪBtkϪ/Ϫ BMM (day 9) upon M-CSF stimulation. Actin was used as a loading control (third and fifth rows from top). ␣-, Anti- (antibody). D, Immunoblot analysis showing the tyrosine phosphorylation (P-Y) pattern of Ϫ/Ϫ Ϫ/Ϫ

M-CSF stimulated wild-type (wt), Tec , Btk , http://www.jimmunol.org/ TecϪ/ϪBtkϪ/Ϫ, and TecϪ/ϪBtkϪ/ϪBmxϪ/Ϫ BMM (day 9). (A, B, and D) Actin was used as loading control. (A–D) The cell lysate equivalent of 2 ϫ 106 cells was used for each immunoblot. Data are representative of two independent experiments. ␣, Anti- (antibody). by guest on September 26, 2021

M-CSF were added at day 5 to wild-type and TecϪ/ϪBtkϪ/Ϫ cul- the TecϪ/ϪBtkϪ/Ϫ BMM cultures was caused by lower levels of tures. This led to a dose-dependent increase both in wild-type and M-CSF due to a more rapid endocytosis of the M-CSFR and, there- TecϪ/ϪBtkϪ/Ϫ macrophage numbers. Although at lower M-CSF fore, an increased use of M-CSF in the absence of Tec and Btk. concentrations the wild-type macrophage numbers were higher However, surface M-CSFR disappeared with a similar kinetic in compared with TecϪ/ϪBtkϪ/Ϫ cultures, at the highest M-CSF wild-type and TecϪ/ϪBtkϪ/Ϫ macrophages (Fig. 6C), indicating a concentration the numbers were equal between wild-type and similar turnover of the M-CSFR upon M-CSF stimulation. To in- TecϪ/ϪBtkϪ/Ϫ cultures (Fig. 5D). This indicated that similar vestigate the activation of signaling pathways of TecϪ/Ϫ, BtkϪ/Ϫ, numbers of TecϪ/ϪBtkϪ/Ϫ macrophages compared with wild- TecϪ/ϪBtkϪ/Ϫ, and TecϪ/ϪBtkϪ/ϪBmxϪ/Ϫ macrophages to type cells can be generated if the cells are generated under a M-CSF in more detail, the tyrosine phosphorylation pattern sufficiently high concentration of M-CSF. upon M-CSF stimulation was determined. TecϪ/ϪBtkϪ/Ϫ and TecϪ/ϪBtkϪ/ϪBmxϪ/Ϫ macrophages displayed a changed ty- Ϫ/Ϫ Ϫ/Ϫ Impaired M-CSFR signaling in Tec Btk macrophages rosine phosphorylation pattern as compared with wild-type, Ϫ Ϫ Ϫ Ϫ Our data suggested that the M-CSFR signaling pathway is altered Tec / or Btk / cells (Fig. 6D). Tyrosine phosphorylation of in TecϪ/ϪBtkϪ/Ϫ macrophages. To test whether Tec family kinases proteins of ϳ110–130 kDa was reduced or absent in macro- are activated by M-CSFR stimulation, wild-type BMMs were stim- phages lacking both Btk and Tec. ulated with M-CSF for 1, 5, 10, and 30 min. M-CSF stimulation ␣ resulted in the activation of Btk, indicated by the phosphorylation Myeloid subset-specific regulation of GM-CSFR expression by of Y223 (Fig. 6A), a known autophosphorylation site of Btk (22). Tec and Btk The M-CSFR showed normal tyrosine phosphorylation upon M- By performing RNase protection assays, we also noted that Csf2ra CSF stimulation in TecϪ/ϪBtkϪ/Ϫ macrophages (Fig. 6B), and M- (the gene encoding GM-CSFR␣) expression was reduced in CSFR levels were similar in wild-type and the various knockout TecϪ/ϪBtkϪ/Ϫ macrophages as compared with wild-type cells (Fig. macrophages (Fig. 6C and data not shown). Daily addition of M- 7A). In contrast, TecϪ/ϪBtkϪ/Ϫ and TecϪ/ϪBtkϪ/ϪBmxϪ/Ϫ BMDCs CSF rescued the cell numbers in TecϪ/ϪBtkϪ/Ϫ BMM cultures showed normal expression levels of GM-CSFR␣ as compared with (Fig. 5C). Thus, it is possible that the decrease in cell numbers in wild-type cells (Fig. 7B), indicating Tec/Btk-dependent expression 8054 Tec AND Btk REGULATE M-CSF RECEPTOR SIGNALING Downloaded from http://www.jimmunol.org/

FIGURE 7. Reduced expression of GM-CSFR␣ in TecϪ/ϪBtkϪ/Ϫ BMM. A, RNase protection assay using RNA isolated from day 10 cultures of wild-type (wt) and TecϪ/ϪBtkϪ/Ϫ macrophages. One representative of two independent experiments is shown. B, Semiquantitative RT-PCR showing expression of Csf2Ra in BMDCs of the indicated genotype. Hprt expression was used as input control. Data are representative of two independent experiments. wt, Wild type. C, Diagram showing macrophage numbers in day 10 cultures after daily addition (from day 6 on) of GM-CSF (500 and 1500 U/ml). daily ϩ LCM, Culture conditions in which LCM was added daily; ϪLCM, control cultures without LCM. Error bars show SD. Data shown are Wt, wild type. D, Pictures depicting adherent .0.0017 ,ءء ;0.0168 ,ء :representative of two independent experiments. The p values shown are as follows GM-CSF-generated wild-type (wt, upper panel) and TecϪ/ϪBtkϪ/ϪBmxϪ/Ϫ (lower panel) BMMs in day 10 GM-CSF cultures (after removal of nonadherent DC). E, Diagram showing the relative number of wild-type (wt) and TecϪ/ϪBtkϪ/ϪBmxϪ/Ϫ BMDCs. BM cells were differentiated with GM-CSF and nonadherant BMDCs were counted at day 10. The summary of three different experiments, each performed in duplicate, is shown. The percentages of by guest on September 26, 2021 TecϪ/ϪBtkϪ/ϪBmxϪ/Ϫ BMDC cell numbers compared with wild-type (wt) BMDC were 147, 108, and 75% for experiments 1, 2, and 3, respectively. The absolute cell numbers (ϫ105) were 4.6 Ϯ 1.2 (wild type) and 6.8 Ϯ 0.9 (TecϪ/ϪBtkϪ/ϪBmxϪ/Ϫ, TecϪ/ϪBtkϪ/ϪBmxϪ/Ϫ) for experiment 1, 8.6 Ϯ 1.0 (wild type) and 9.3 Ϯ 2.6 (TecϪ/ϪBtkϪ/ϪBmxϪ/Ϫ) for experiment 2, and 6.3 Ϯ 1.5 (wt) and 4.7 Ϯ 0.9 (TecϪ/ϪBtkϪ/ϪBmxϪ/Ϫ) for experiment 3.

of the Csf2ra gene in BMMs but not in BMDCs. The reduced sence of Tec and Btk. Elucidation of M-CSFR signaling pathways expression levels of Csf2ra were only observed in TecϪ/ϪBtkϪ/Ϫ revealed an impaired total tyrosine phosphorylation pattern in BMMs but not in TecϪ/Ϫ or BtkϪ/Ϫ BMMs (data not shown). TecϪ/ϪBtkϪ/Ϫ macrophages upon M-CSF stimulation despite nor- Unlike M-CSF, which upon addition restored cell numbers in mal expression and phosphorylation of the M-CSFR. Thus, our TecϪ/ϪBtkϪ/Ϫ BMM cultures to wild-type levels (Fig. 5D), the data provide a novel link between Tec family kinases and M- addition of GM-CSF restored cell numbers in TecϪ/ϪBtkϪ/Ϫ CSF receptor signaling pathways that regulate macrophage sur- BMM cultures only partially (Fig. 7C). The reduced expression of vival. Finally, Tec and Btk are required for the proper expres- GM-CSFR␣ in the absence of Tec and Btk, however, may explain sion of GM-CSFR␣ in macrophages but not in dendritic cells, why the cell numbers upon GM-CSF addition were always lower implicating Tec kinases in the lineage-specific regulation of in TecϪ/ϪBtkϪ/Ϫ cultures compared with wild-type cultures (Fig. GM-CSFR␣ expression. 7C). Culture of BM cells with GM-CSF instead of M-CSF leads to The generation of BMMs as assessed by surface marker expres- the generation of a confluent layer of adherent macrophages and sion was not influenced by the absence of Tec and Btk. This was the cogeneration of loosely attached CD11cϩ BMDCs (23). The also reflected by similar macrophage numbers on day 6 in wild- macrophage layer was observed in GM-CSF cultures (day 10) of type and TecϪ/ϪBtkϪ/Ϫ cultures. Therefore, Tec and Btk are not Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ wild-type but not Tec / Btk / Bmx / BM cells (Fig. 7D). How- required for the differentiation of precursor cells into macrophages. ever, the generation of BMDCs was not affected, because similar The drop in cell numbers starting after day 6 could either be caused Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ numbers of wild-type and Tec / Btk / Bmx / BMDCs devel- by reduced proliferation or reduced survival of differentiated mac- oped in the presence of GM-CSF (Fig. 7E). rophages. Because BrdU incorporation was the same in wild-type and TecϪ/ϪBtkϪ/Ϫ macrophages, it is likely that Tec and Btk reg- Discussion ulate macrophage numbers by promoting macrophage survival. In this study we performed a genetic approach to investigate in This is supported by the observation that the drop in cell numbers detail the role of Tec family kinases in murine macrophages. We correlated with an increase in PI-positive cells and also by the Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ observed reduced survival rates of Tec Btk bone marrow- occurrence of a sub-N2 population in Tec Btk macrophages derived macrophages. A severe drop in macrophage numbers cor- as revealed by DNA content analysis (data not shown). Tec family relating with increased numbers of dead cells occurred in the ab- kinases have already been implicated in regulating cell survival The Journal of Immunology 8055 and/or apoptosis in other cell lineages (24–26) and in the apoptosis pression of caspase-11 in macrophages depends on NF-␬B and of macrophages after stimulation (9). Our findings indicate that STAT1 (34), p38 MAPK (35), and the transcription factor CHOP, Tec and Btk also regulate cell survival of BMM. Neither Tec nor a C/EBP family transcription factor (36). CHOP is implicated in Btk single-deficient BMM displayed a survival defect. Thus, our endoplasmatic reticulum stress-mediated apoptosis, providing a data also indicate redundant activities of Tec and Btk during mac- link between stress response and caspase-11 expression. However, rophage generation, similar to the murine B cell lineage where Tec we consider it unlikely that the up-regulation of caspase-11 ex- and Btk are required for proper B cell development (14). pression in Tec family kinase-deficient macrophages is linked with M-CSF is a crucial cytokine required for the differentiation, pro- the increase in cell death, because caspase-11 is expressed in liferation, and survival of macrophages and is often provided in TecϪ/ϪBtkϪ/Ϫmacrophages on day 10 when there are no differences cultures of BM-derived macrophages with LCM (18). M-CSF star- in the percentage of PI-positive cells between wild-type and Tec/Btk- vation in macrophage cultures induces apoptosis (27). The obser- deficient cells. Furthermore, the observation that TecϪ/Ϫ or BtkϪ/Ϫ vation that high levels of M-CSF (by daily addition of new LCM) macrophages show caspase-11 expression also argues against a can rescue TecϪ/ϪBtkϪ/Ϫ macrophage numbers suggested that direct link between caspase-11 and the drop in macrophage num- there are differences in the use of M-CSF due to an increased bers. However, caspase-11 expression might indicate a stress re- internalization of the M-CSFR or an impairment of M-CSFR sig- sponse in Tec- or Btk-deficient macrophages. Finally, we noted naling or both. However, because the kinetic of the internalization that Tec and Btk are required for the expression of GM-CSFR␣ in of the M-CSFR upon M-CSF stimulation was similar in wild-type BMMs but not in BMDCs, implicating Tec family kinases in the and TecϪ/ϪBtkϪ/Ϫ macrophages, these data point toward a M- lineage-specific regulation of GM-CSFR␣ expression. This may

CSFR signaling defect in the absence of Tec and Btk. This is also explain why the addition of GM-CSF does not rescue mac- Downloaded from supported by the observation that the tyrosine phosphorylation pat- rophage numbers to the same extent in TecϪ/ϪBtkϪ/Ϫ cultures as tern upon M-CSF stimulation was changed in TecϪ/ϪBtkϪ/Ϫ mac- compared with wild-type cultures. However, it remains possible rophages despite normal expression and phosphorylation of the that Tec and Btk are, in addition, also required for proper GM- M-CSFR. These data indicate that Tec family kinases are required CSFR signaling. for the proper transmission of M-CSF signals in macrophages. Taken together, our analysis showed that Tec and Btk are cru-

Thus, suboptimal concentrations of M-CSF that still allow macro- cially involved in macrophage survival by M-CSFR signaling. Our http://www.jimmunol.org/ phage survival in wild-type cells do not induce a sufficient signal study provides a novel link between Tec family kinases and M- to allow survival of TecϪ/ϪBtkϪ/Ϫ macrophages. However, higher CSFR signaling as well as with the regulation of caspase-11 and M-CSF concentrations (due to daily replacement of M-CSF) pre- GM-CSFR␣ expression. Future in vivo studies addressing the roles sumable provide a stronger (i.e., above “threshold”) signal to of Tec and Btk in inflammation and infection will be of interest TecϪ/ϪBtkϪ/Ϫ macrophages that rescues the survival defect. Thus, because both caspase-11 and GM-CSF are already implicated in macrophages use Tec family kinases for proper “sensing” of M- these processes. CSF levels. In support of this hypothesis, we also observed a dose- dependent rescue of TecϪ/ϪBtkϪ/Ϫ macrophage numbers that can Acknowledgments reach wild-type cell numbers if sufficiently high amounts of M- We thank Dr. Thomas Decker for providing L929 cells and Drs. Mathias by guest on September 26, 2021 CSF are added at day 5. Activation of Tec family kinases is a Mu¨ller and Herbert Strobl for critical reading of the manuscript. process involving their localization to the plasma membrane fol- lowed by a -mediated phosphorylation of a ty- Disclosures rosine residue in the activation loop of the kinase domain. Full The authors have no financial conflict of interest. activation of Tec family kinases is achieved after a subsequent autophosphorylation of a tyrosine residue in the Src homology 3 References (SH3) domain of Tec kinases (28, 29). Membrane recruitment is 1. Gordon, S., and P. R. Taylor. 2005. Monocyte and macrophage heterogeneity. Nat. Rev. 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J. Immunol. 168: 2914–2921. 9. Mangla, A., A. Khare, V. Vineeth, N. N. Panday, A. Mukhopadhyay, apoptosis (32). In contrast to other caspases, caspase-11 is gener- B. Ravindran, V. Bal, A. George, and S. Rath. 2004. Pleiotropic consequences of ally not expressed in cells and tissues under homeostatic condi- Bruton tyrosine kinase deficiency in myeloid lineages lead to poor inflammatory responses. Blood 104: 1191–1197. tions (15). However, expression can be induced by stimuli such as 10. Doyle, S. L., C. A. Jefferies, and L. A. O’Neill. 2005. Bruton’s tyrosine kinase is LPS, systemic inflammation, or ischemic brain injury (33). Ex- involved in p65-mediated transactivation and phosphorylation of p65 on serine 8056 Tec AND Btk REGULATE M-CSF RECEPTOR SIGNALING

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515–525. popolysaccharide-induced inflammation. J. Immunol. 176: 6245–6253. by guest on September 26, 2021