Cutting Edge: FcR-Like 5 on Innate B Cells Is Targeted by a Poxvirus MHC Class I-Like Immunoevasin †,‡,x ‡ { Jessica A. Campbell,* Randall S. Davis, Lauren M. Lilly,‖ Daved H. Fremont, Anthony R. French,*,1 and Leonidas N. Carayannopoulos ,1,2 Under selective pressure from host immunity, evasins) must specifically bind one or more host-encoded have retained encoding immunoevasins, molecules molecules. In this regard, immunoevasins can be viewed as interfering with host viral recognition and clearance. probes with which the complex network of host-encoded Due to their binding specificities, immunoevasins can can be searched to identify those that are important be exploited as affinity labels to identify host-encoded to defense against the respective class of viruses. molecules of previously unsuspected importance in We previously described an orthopoxvirus MHC class I-like defense against the relevant class of . We previously (OMCP) encoded by monkeypoxvirus and cowpox- described an orthopoxvirus MHC class I-like protein virus (CPXV) (1). OMCP is secreted from infected cells (OMCP) that binds with high affinity to the activating and binds with high affinity to human and mouse NKG2D, receptor NKG2D on NK and subsets, implicating an activating receptor expressed by NK and T cell subsets. NKG2D in antiorthopoxvirus immunity. In this study, As a soluble NKG2D antagonist, OMCP potently inhibits NKG2D-dependent killing of target cells. These findings we report that OMCP also binds in an NKG2D- strongly implicated NKG2D in host defense against zoonotic independent manner to B cells and monocytes/macro- orthopoxviruses, and this conclusion was recently verified by phages. We identify murine FcR-like 5 (FCRL5), an Sigal and colleagues (2) using experimental mousepox. orphan immunoregulatory protein highly expressed Interestingly, OMCP also binds to B cells and monocytes/ by innate B lymphocytes, as a specific receptor for macrophages. The lack of NKG2D on these cell types (3–5) OMCP. The three N-terminal Ig domains of FCRL5 suggested the existence of a separate, non-NKG2D receptor are required for OMCP binding. The targeting of targeted by OMCP. In this study, we report that OMCP FCRL5 by an orthopoxvirus immunoevasin strongly binds to the protein FcR-like 5 (FCRL5), an immunoregu- implicates it in contributing to host defense against zoo- latory receptor of unknown specificity that is highly ex- notic orthopoxviruses. The Journal of Immunology, pressed by murine marginal zone (MZ) and B1 B cells. In 2010, 185: 28–32. addition to describing the first known ligand for FCRL5, these data implicate FCRL5 and innate B cells in defense against poxviruses. ffective immune responses depend on functional communication within and between host cells. At the Materials and Methods E molecular level, communication is controlled by Retroviral library receptor–ligand interactions. Every stage of an immune re- WEHI231 cell-derived mRNA was reverse-transcribed with Superscript II sponse to foreign Ag ultimately relies on such interactions, be (Invitrogen, Carlsbad, CA) and amplified and digested with the SMART they host–host or host–microbe in nature. Following receptor cDNA Library Construction Kit (BD Clontech, Palo Alto, CA). SfiI- digested cDNA was ligated into pMXs-IRES-EGFP (courtesy of Dr. T. Kita- ligation, downstream signaling events regulate cellular acti- mura, University of Tokyo, Tokyo, Japan) in which SfiIA and SfiIB sites had vation and effector functions. The latter impose great selective been incorporated, then transformed into XL-10 Gold Ultracompetent cells pressure on invading microbes. Therefore, microbial coun- (Stratagene, La Jolla, CA) for a final library complexity of 1.4 3 106. Retro- termeasures aimed at preventing or avoiding host immunity viruses were produced and used to infect C1498 cells as described previously (6). C1498 transductants were stained with OMCP tetramers and sorted for often disrupt receptor–ligand interactions. To accomplish the most brightly staining enhanced GFP (EGFP)+ cells. After four successive this, microbial immunomodulatory proteins (immuno- sorts, retroviral inserts from individual clones were amplified with primers

{ *Division of Pediatric Rheumatology, Department of Pediatrics, Department of Path- Address correspondence and reprint requests to Dr. Anthony R. French, Department ‖ ology and Immunology, and Division of Pulmonary and Critical Care Medicine, of Pediatrics, Washington University, 660 South Euclid Avenue, St. Louis, MO Department of Internal Medicine, Washington University School of Medicine, St Louis, 63110. E-mail address: [email protected] x MO 63110; and †Department of Medicine, ‡Department of Microbiology, and Depart- The online version of this article contains supplemental material. ment of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294 Abbreviations used in this paper: CPXV, cowpoxvirus; DIII, domain III; EGFP, enhanced GFP; FCRL5, FcR-like 5; FO, follicular; IRES, internal ribsosome entry site; 1A.R.F. and L.N.C. contributed equally to this work. MZ, marginal zone; OMCP, orthopoxvirus MHC class I-like protein. 2Current address: Merck Research Laboratories, Rahway, NJ. Ó Received for publication January 28, 2010. Accepted for publication May 4, 2010. Copyright 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 This work was supported by National Institute of Allergy and Infectious Diseases Grants R01 AI073552 and R01 AI067467 and National Heart, Lung, and Blood Institute Grant T32-HL073171. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000240 The Journal of Immunology 29 specific for the retroviral 59 long terminal repeat (59-tagactgccggatctagct-39) and internal ribosome entry site (IRES) (59-aggggcggaatttacgtag-39) and subjected to basic local alignment search tool analysis.

Plasmid constructs, transductants, and transfectants Full-length FCRL5 was ligated into pMXs-IRES-EGFP. FCRL5-expressing Ba/F3 transductants were generated as described previously (6). To express truncated versions of FCRL5, specified BALB/c FCRL5 Ig domains were PCR-spliced to the mature N terminus of Thy1.1 as previously described (1). FCRL5 D1-5 was amplified from MSGSF…to…ISIFD; D1 from MSGSF… to…EFSED; D2 from FLVLQ…to…VQVQE; D3 from LFPRP…to… IPVQR; D1-3 from MSGSF…to…IPVQR; D1-2 from MSGSF…to… VQVQE; and D2-3 from FLVLQ…to…IPVQR. The FCRL5 leader peptide from MSGSF…to…VNGQH was spliced to the N terminus of D2 and D3. Amplicons were spliced to Thy1.1, cloned into pcDNA3 (Invitrogen), and transfected into 293T cells. Soluble FCRL-Fc fusion constructs were produced by amplifying FCRL1 from MLPWL…to...SLGLT and FCRL5 from MSGSF…to…ISIFD, then cloned into pFUSE-hIgG1-Fc1 (Invivogen, San Diego, CA). A 6-His tag was cloned onto the 39 end of the Fc to generate pFUSE-FCRL-Fc-His plasmids.

Recombinant protein production rOMCP, MULT1, RAE1d, and West Nile virus glycoprotein domain III (DIII) were produced and biotinylated when indicated as previously reported (1, 7, 8). Soluble FCRL1-Fc and FCRL5-Fc fusion proteins were produced by 293T cells transfected with pFUSE-FCRL-Fc-His plasmids and purified from spent supernatant with Ni-NTA beads (Qiagen, Valencia, CA).

Abs and flow cytometry Mouse IgG2k-PE, streptavidin-PE, streptavidin-APC, anti-mouse CD11b-PE, B220-PE, NKG2D (CX5), CD3-FITC, Thy1.1-PE, anti-human CD14- FITC, and CD19-PE were purchased from eBioscience (San Diego, CA). Rat IgG1k, rat IgG2a, anti-human NKG2D (1D11), anti-mouse CD21-FITC, CD23-PE, and NK1.1-PE were from BD Biosciences (San Jose, CA). Anti- mouse CD5-PE and CD19-FITC were from Biolegend (San Diego, CA). Anti- FIGURE 1. OMCP binds to B cells and monocytes/macrophages inde- FCRL5 clone 9D10 has been described (9). For blocking experiments, cells pendently of NKG2D. C57BL/6 splenocytes (A), peritoneal cells (B), or were incubated with Abs or recombinant soluble proteins for 30 min prior to human PBMCs (C) were stained with the indicated leukocyte subset markers. OMCP tetramer addition. Data were collected on an FACSCalibur (BD Bio- The gated cells were additionally stained with control DIII tetramers (line 1) sciences) and analyzed with FlowJo (Tree Star, Ashland, OR). or OMCP tetramers (line 2). OMCP tetramer staining following pre- incubation with 5 mg/ml isotype control mAb (line 3), 5 mg/ml anti-NKG2D ELISA (line 4), 2 mM DIII (control recombinant protein) (line 5), or 2 mM recombi- + OMCP or RAE1d was plated at 0.2 mg/ml in ELISA Coating Buffer and nant soluble MULT1 (line 6) is also shown. NK1.1 cells were excluded from blocked with Assay Diluent (eBioscience), followed by incubation with the the CD11b and B220 gates (A, top panels). Results are representative of at indicated FCRL-Fc concentration, then with 2 mg/ml anti-human IgG Fc- least three independent experiments. HRP (Southern Biotechnology Associates, Birmingham, AL). Tetramethyl- benzidine (eBioscience) was added to visualize bound proteins, and the reac- tion was stopped with sulfuric acid. OMCP tetramer staining. Binding was not due to NKG2D Results expression because neither anti-NKG2D mAb nor soluble OMCP binds to B cells and monocytes/macrophages MULT1 (lines 4 and 6, respectively, in Fig. 1A, top and middle panels, and 1B) inhibited OMCP tetramer binding in contrast To determine whether OMCP displays NKG2D-independent to effective blocking on NK, NKT, and CD8+ T cells (Fig. 1A, binding activity, primary mouse and human cell preparations bottom panels) (1). Similar results were obtained with splenic were costained with rOMCP tetramers and leukocyte sub- and peritoneal cells from BALB/c, 129, and SJL mouse strains set markers. OMCP tetramer staining was observed on (data not shown). Examination of human PBMCs showed that C57BL/6 splenic CD11b+ macrophages and B220+ B cells OMCP tetramers bind to CD14+ monocytes and CD19+ (Fig. 1A, top panels). To examine murine splenic B cell subsets B cells (Fig. 1C). As was observed with murine B cells and more closely, follicular (FO) and MZ B cells were identified macrophages, preincubation with anti-NKG2D mAb did not based on CD21 and CD23 expression. This revealed a much prevent OMCP tetramers from binding to human B cells and higher level of OMCP tetramer binding to CD21hiCD23lo monocytes (Fig. 1C, line 4). Together, these data demonstrate MZ B cells than to CD21intCD23hi FO B cells (Fig. 1A, that B cells and monocytes/macrophages express one or more middle panels). Analysis of murine peritoneal B cells demon- 2 distinct non-NKG2D receptors for OMCP. strated that CD5+ B1a and CD5 B1b B cells also express relatively high levels of a receptor for OMCP (Fig. 1B). Although B cells do not express NKG2D, macrophages have FCRL5 is a receptor for OMCP on innate B cells been reported to express this receptor in certain circumstances To identify the receptor on B cells recognized by OMCP, we (10, 11). To exclude NKG2D as the receptor responsible for transduced the OMCP tetramer-negative cell line C1498 with OMCP binding on B cells and macrophages, cells were incu- a retroviral cDNA library derived from WEHI231 cells, a bated with either soluble MULT1, a high-affinity host- mouse B cell line with high cell-surface OMCP-binding activ- encoded NKG2D ligand (7), or anti-NKG2D mAb prior to ity. After four successive rounds of enrichment for OMCP 30 CUTTING EDGE: OMCP binds FCRL5 on Innate B cells tetramer-positive cells by flow cytometric sorting (Fig. 2A), Having established a specific interaction between OMCP retroviral inserts were obtained from individual clones by and FCRL5, we proceeded to test whether the binding of PCR. A predominant amplicon (15 of 17 amplifiable clones) OMCP tetramers to primary B cells and macrophages could be of ∼1.8 kbp was identified by basic local alignment search blocked by anti-FCRL5 mAb (Fig. 2E,2F). OMCP tetramers tool analysis as FCRL5, a protein expressed by WEHI231 were completely inhibited from recognizing MZ, B1a, and cells, MZ, B1a, and B1b B cells (Supplemental Fig. 1) (9, 12). B1b B cells after preincubation with anti-FCRL5 mAb. As To confirm the interaction between OMCP and FCRL5, expected, anti-FCRL5 mAb did not inhibit OMCP tetramer Ba/F3 cells were transduced with FCRL5 amplified from binding to NK cells, which express the OMCP receptor murine spleens. FCRL5 is polymorphic, with C57BL/6 and NKG2D. Consistent with the lack of FCRL5 expression by BALB/c strains representing the two distinct alleles (9). Ex- FO B cells and macrophages (Supplemental Fig. 1) (9, 12), pression of either FCRL5 allele endowed Ba/F3 transductants, pretreatment with anti-FCRL5 mAb also did not inhibit but not untransduced cells, with the ability to bind OMCP OMCP tetramer binding to these subsets. Thus, OMCP tetramers, but not control tetramers (Fig. 2B). The specificity binds specifically to FCRL5 expressed by murine innate of the OMCP–FCRL5 interaction was verified by prein- B cells. cubating the cells with anti-FCRL5 mAb prior to OMCP tetramer staining. Although preincubation with isotype con- OMCP binds to the three N-terminal Ig domains of FCRL5 trol mAb did not affect OMCP tetramer binding to either FCRL5 is composed of five Ig domains (9, 12), termed D1– Ba/F3-FCRL5 transductants or WEHI231 cells, OMCP tet- D5, with D1 being the most N-terminal domain. To identify ramers were unable to bind either cell type after anti-FCRL5 the domains that bind OMCP, truncated versions of FCRL5 mAb blockade, establishing the specificity of the interaction were cloned and expressed in 293T cells. To ensure surface between OMCP and FCRL5 (Fig. 2C). The OMCP–FCRL5 expression of the shortened constructs, different combinations interaction was further validated with an ELISA, which of FCRL5 Ig domains were fused to the mature N terminus of showed that rFCRL5-Fc, but not FCRL1-Fc, bound to the GPI-linked protein Thy1.1 to generate a series of FCRL5 plate-immobilized OMCP (Fig. 2D). FCRL5-Fc did not ectodomain-Thy1.1 transfectants. Neither FCRL5 D1, D2, bind to plate-immobilized RAE1d, a host-encoded NKG2D or D3 nor Thy1.1 alone bound OMCP tetramers (Fig. 3). ligand. Together, these results demonstrate that FCRL5 is 293T cells expressing FCRL5 D1–3 retained OMCP tetramer- specifically targeted by OMCP. binding ability, indicating that D4 and D5 are dispensable

FIGURE 2. OMCP binds specifically to mouse FCRL5. A, Untransduced or library-transduced C1498 cells were stained with control (left panels) or OMCP (right panels) tetramers after the indicated round of sorting. Note that quadrants are not drawn equivalently on each population because cells were stained 2 to 3 d postsorting and therefore were stained on different days. However, control tetramer staining is shown for comparison on each day. B, Ba/F3 cells transduced with C57BL/6 or BALB/c FCRL5 were stained with nothing (top panels), control tetramers (middle panels), or OMCP tetramers (bottom panels). Transductants are 2 EGFP+; untransduced EGFP Ba/F3 cells were included in each stain as an internal negative control. C, Ba/F3-FCRL5 (B6 allele) transductants or WEHI231 cells were stained with control tetramers (line 1) or OMCP tetramers (line 2). OMCP tetramer binding following preincubation of cells with 50 mg/ml isotype control mAb (line 3) or anti-FCRL5 mAb (line 4) is also shown. D, Plate-bound OMCP or RAE1d was incubated with the indicated concentrations of FCRL1-Fc or FCRL5-Fc. Bound Fc fusion proteins were detected with anti-human IgG Fc-HRP. Splenic (E) or peritoneal (F) cells were stained with control tetramers (line 1) or OMCP tetramers (line 2). OMCP tetramer staining following cellular preincubation with 50 mg/ml isotype control mAb (line 3) or anti-FCRL5 mAb (line 4) are also shown. Results are representative of three independent experiments. The Journal of Immunology 31

intracellular domain of human FCRL5 (another FCRL with dual ITIM and ITAM-like sequences) inhibits BCR-induced calcium mobilization in a B cell line (15). A similar phenom- enon has been observed with murine FCRL5 (9). However, we have found no functional impact of soluble, monomeric OMCP on FCRL5+ B cell proliferation, BCR-induced calcium flux, or FCRL5 cell-surface levels (data not shown). Interest- ingly, despite 64% identity and 75% similarity over the major- ity of the mouse and human FCRL5 intracellular domains, an abrupt sequence divergence and truncation occurs just prior to the mouse C terminus, causing loss of a tyrosine moiety critical to inhibitory signaling by human FCRL5 (15). Thus, FIGURE 3. The three N-terminal Ig domains of FCRL5 are necessary and we hypothesize that FCRL5 may use its ITAM-like sequence to sufficient for OMCP binding. 293T cells transfected with Thy1.1 or the indi- function as an activating or costimulatory receptor. In this sce- cated FCRL5-Thy1.1 fusion construct were stained with negative control DIII nario, OMCP would serve as a competitive antagonist of tetramers (line 1), OMCP tetramers (line 2), isotype control mAb (line 3), or FCRL5 in a manner analogous to that for NKG2D (1). anti-Thy1.1 mAb (line 4). Results are representative of three independent experiments. Mouse FCRL5 is highly expressed by splenic MZ and peritoneal B1 B cells (Supplemental Fig. 1) (9, 12). MZ B cells are located in the splenic MZ where blood exits the circu- for this activity. Cells expressing the D1-2 and D2-3 constructs lation via the marginal sinus, allowing MZ B cells immediate were incapable of recognizing OMCP. Together, these experi- access to blood-borne Ags (16). B1 B cells are also differen- ments demonstrate that the three N-terminal Ig domains of tially distributed but are located in the peritoneal and pleural FCRL5 are necessary and sufficient for binding to OMCP. cavities. In contrast to follicular B cells, MZ and B1 B cells exhibit an activated phenotype (17, 18) and participate in the Discussion early immune defense against bacteria and viruses (19–23) This study reveals a novel interaction between an FCRL family through production of low-affinity/high-avidity IgM, termed member and a virally encoded, MHC-like immunomodulator. natural Ab (16, 24). Interestingly, mice deficient in natural Ab The EBV nuclear Ag 2 transcription factor has been shown to exhibit increased viral titers in the blood, liver, and kidneys induce expression of human FCRL5 (13), but OMCP is the when infected with vaccinia virus (23), implying that MZ and first known virally encoded protein to recognize an FCRL B1 B cells may contribute to control of orthopoxvirus infec- member directly. Furthermore, it is the first identified ligand tion. If OMCP indeed prevents FCRL5 from interacting with of either microbial or host origin for FCRL5. The function its activating natural ligand(s), this may inhibit or delay MZ of OMCP with regard to FCRL5, however, remains to be and B1 B cells from mounting antiviral responses. of identified. mice with an rOMCP-null CPXV may be used to test the One possibility involves OMCP acting as a cross-linker cumulative impact of OMCP on its target cells. However, between cells expressing different OMCP receptors. For exam- because OMCP targets multiple receptors, the in vivo effect + ple, OMCP could link an NKG2D+ NK cell to an FCRL5+ of OMCP on FCRL5 cells will best be directly evaluated by B cell to induce cytolysis against the B cell, formally pos- infection with rCPXV expressing mutant OMCP unable to sible because FCRL5 and NKG2D recognize independent bind FCRL5, but still capable of interacting with its other OMCP binding sites (not shown). However, given the rapid targets. Generation and use of these recombinant viruses is in dissociation kinetics of OMCP from mouse NKG2D (1), progress. such cross-linking would require dissociation kinetics of the Murine macrophages and FO B cells bind OMCP tetramers OMCP–FCRL5 interaction that are uncommonly slow for in an FCRL5- and NKG2D-independent manner (Figs. 1, 2). receptors of this type. Experimentally, OMCP neither enhan- A likely explanation is that these cell types express yet another ces NK cell-mediated killing of WEHI231 cells nor increases receptor(s) targeted by OMCP. We hypothesize that the conjugate formation between NKG2D+ and FCRL5+ cells human ortholog of this putative third receptor may mediate (data not shown). Therefore, it appears unlikely that OMCP binding of OMCP to human B cells and monocytes. Candi- provides a physical link between cytolytic effectors and innate date human receptors include the six transmembrane FCRL B cells. Rather, we favor the hypothesis that OMCP blunts family members (FCRL1–6) expressed on human leukocytes signaling through FCRL5 by serving as a competitive antag- (25). Indeed, three of these members (FCRL3–5) contain onist for its natural ligand(s). domains with homology to the three N-terminal Ig domains FCRL5 possesses canonical ITIM and ITAM-like sequences of mouse FCRL5 necessary for recognition of OMCP (Fig. (9, 12), but it remains unclear if it functions primarily as an 3). However, when we tested the ability OMCP tetramers to inhibitory or an activation receptor in vivo. Human FCRL3 recognize human FCRL molecules ectopically expressed on provides an example of a receptor containing both ITIM and 293T cells, none of these six individual human FCRL mole- ITAM-like sequences with the potential to transmit either acti- cules bound OMCP tetramers (data not shown). Additional vating or inhibitory signals. Phospho-FCRL3 associates in vitro studies to resolve this issue are currently underway. with both activating kinases (Syk and ZAP70) and inhibitory Complete understanding of the contributions of OMCP and phosphatases (Src homology region 2 domain-containing FCRL5 to poxvirus pathogenesis and host immunity awaits phosphatase-1 and Src homology region 2 domain-containing identification of OMCP’s remaining target receptor(s) and phosphatase-2) (14). Coligation with mAbs of BCR and the the natural ligand(s) of FCRL5. However, the targeting of 32 CUTTING EDGE: OMCP binds FCRL5 on Innate B cells

FCRL5 by a microbial immunoevasin implies that FCRL5 11. Buhtoiarov, I. N., A. L. Rakhmilevich, L. L. Lanier, E. A. Ranheim, and P. M. Sondel. 2009. Naive mouse macrophages become activated following recognition of plays an important role in antiviral immunity and establishes L5178Y lymphoma cells via concurrent ligation of CD40, NKG2D, and CD18 CPXV infection as in vivo model with which to probe FCRL5 molecules. J. Immunol. 182: 1940–1953. biology. 12. Davis, R. S., R. P. Stephan, C. C. Chen, G. Dennis, Jr., and M. D. Cooper. 2004. Differential B cell expression of mouse Fc receptor homologs. Int. Immunol. 16: 1343–1353. 13. Mohan, J., J. Dement-Brown, S. Maier, T. Ise, B. Kempkes, and M. Tolnay. 2006. Disclosures Epstein-Barr virus nuclear antigen 2 induces FcRH5 expression through CBF1. R.S.D. holds a patent on the mouse FCLR5 sequence. Blood 107: 4433–4439. 14. Xu, M. J., R. Zhao, H. Cao, and Z. J. Zhao. 2002. SPAP2, an Ig family receptor containing both ITIMs and ITAMs. Biochem. Biophys. Res. Commun. 293: 1037– 1046. References 15. Haga, C. L., G. R. Ehrhardt, R. J. Boohaker, R. S. Davis, and M. D. Cooper. 2007. 1. Campbell, J. A., D. S. Trossman, W. M. Yokoyama, and L. N. Carayannopoulos. Fc receptor-like 5 inhibits B cell activation via SHP-1 tyrosine phosphatase recruit- 2007. Zoonotic orthopoxviruses encode a high-affinity antagonist of NKG2D. ment. Proc. Natl. Acad. Sci. USA 104: 9770–9775. J. Exp. Med. 204: 1311–1317. 16. Pillai, S., A. Cariappa, and S. T. Moran. 2005. Marginal zone B cells. Annu. Rev. 2. Fang, M., L. L. Lanier, and L. J. Sigal. 2008. A role for NKG2D in NK cell- Immunol. 23: 161–196. mediated resistance to poxvirus disease. PLoS Pathog. 4: e30. 17. Oliver, A. M., F. Martin, G. L. Gartland, R. H. Carter, and J. F. Kearney. 1997. 3. Houchins, J. P., T. Yabe, C. McSherry, and F. H. Bach. 1991. DNA sequence Marginal zone B cells exhibit unique activation, proliferative and immunoglobulin analysis of NKG2, a family of related cDNA clones encoding type II integral secretory responses. Eur. J. Immunol. 27: 2366–2374. membrane proteins on human natural killer cells. J. Exp. Med. 173: 1017–1020. 18. Oliver, A. M., F. Martin, and J. F. Kearney. 1999. IgMhighCD21high lymphocytes 4. Bauer, S., V. Groh, J. Wu, A. Steinle, J. H. Phillips, L. L. Lanier, and T. Spies. enriched in the splenic marginal zone generate effector cells more rapidly than the 1999. Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible bulk of follicular B cells. J. Immunol. 162: 7198–7207. MICA. Science 285: 727–729. 19. Boes, M., A. P. Prodeus, T. Schmidt, M. C. Carroll, and J. Chen. 1998. A critical 5. Diefenbach, A., A. M. Jamieson, S. D. Liu, N. Shastri, and D. H. Raulet. 2000. role of natural immunoglobulin M in immediate defense against systemic bacterial Ligands for the murine NKG2D receptor: expression by tumor cells and activation of NK cells and macrophages. Nat. Immunol. 1: 119–126. infection. J. Exp. Med. 188: 2381–2386. 6. Morita, S., T. Kojima, and T. Kitamura. 2000. Plat-E: an efficient and stable system 20. Baumgarth, N., J. Chen, O. C. Herman, G. C. Jager, and L. A. Herzenberg. 2000. for transient packaging of retroviruses. Ther. 7: 1063–1066. The role of B-1 and B-2 cells in immune protection from influenza virus infection. 7. Carayannopoulos, L. N., O. V. Naidenko, D. H. Fremont, and W. M. Yokoyama. Curr. Top. Microbiol. Immunol. 252: 163–169. 2002. Cutting edge: murine UL16-binding protein-like transcript 1: a newly 21. Baumgarth, N., O. C. Herman, G. C. Jager, L. Brown, L. A. Herzenberg, and L. A. described transcript encoding a high-affinity ligand for murine NKG2D. J. Immu- Herzenberg. 1999. Innate and acquired humoral immunities to influenza virus are nol. 169: 4079–4083. mediated by distinct arms of the . Proc. Natl. Acad. Sci. USA 96: 8. Nybakken, G. E., T. Oliphant, S. Johnson, S. Burke, M. S. Diamond, and D. H. 2250–2255. Fremont. 2005. Structural basis of West Nile virus neutralization by a therapeutic 22. Choi, Y. S., and N. Baumgarth. 2008. Dual role for B-1a cells in immunity to antibody. Nature 437: 764–769. influenza virus infection. J. Exp. Med. 205: 3053–3064. 9. Won, W. J., J. B. Foote, M. R. Odom, J. Pan, J. F. Kearney, and R. S. Davis. 2006. 23. Ochsenbein, A. F., T. Fehr, C. Lutz, M. Suter, F. Brombacher, H. Hengartner, and Fc receptor homolog 3 is a novel immunoregulatory marker of marginal zone and R. M. Zinkernagel. 1999. Control of early viral and bacterial distribution and B1 B cells. J. Immunol. 177: 6815–6823. disease by natural antibodies. Science 286: 2156–2159. 10. Baba, T., A. Ishizu, S. Iwasaki, A. Suzuki, U. Tomaru, H. Ikeda, T. Yoshiki, and 24. Martin, F., and J. F. Kearney. 2000. B-cell subsets and the mature preimmune M. Kasahara. 2006. CD4+/CD8+ macrophages infiltrating at inflammatory sites: repertoire. Marginal zone and B1 B cells as part of a “natural immune memory”. a population of monocytes/macrophages with a cytotoxic phenotype. Blood 107: Immunol. Rev. 175: 70–79. 2004–2012. 25. Davis, R. S. 2007. Fc receptor-like molecules. Annu. Rev. Immunol. 25: 525–560.