NK-B Cell Interactions Receptors and Counterreceptors Involved In

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Receptors and Counterreceptors Involved in NK-B Cell Interactions Ning Gao, Tam Dang, Wesley A. Dunnick, John T. Collins, Bruce R. Blazar and Dorothy Yuan This information is current as of October 8, 2021. J Immunol 2005; 174:4113-4119; ; doi: 10.4049/jimmunol.174.7.4113 http://www.jimmunol.org/content/174/7/4113 Downloaded from References This article cites 50 articles, 29 of which you can access for free at: http://www.jimmunol.org/content/174/7/4113.full#ref-list-1

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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 © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Receptors and Counterreceptors Involved in NK-B Cell Interactions1

Ning Gao,* Tam Dang,* Wesley A. Dunnick,† John T. Collins,† Bruce R. Blazar,‡ and Dorothy Yuan2*

In addition to the well-documented effect of NK cells on B cell differentiation via their ability to secrete IFN-␥, NK cells can also induce, via direct cell-cell interactions, germline transcripts (I␥2a) necessary for switch recombination to IgG2a. Analysis of the ligand-receptor pairs that could be involved in this induction revealed that the expression of CD48 on B cells is crucial for the induction. NK cells from mice with targeted deletions of either the CD2 or the CD244 gene, both of which encode ligands for CD48, are compromised in their ability to induce B cell I␥2a expression. Interestingly, although CD244 can bind to CD48 with a higher .affinity, the ability of NK cells from CD244؊/؊ mice to stimulate I␥2a is not as compromised as NK cells from CD2؊/؊ mice Despite the difference between cell surface receptors that are stimulated by NK cells vs those stimulated by the combination of LPS and IFN-␥, we show in this study that the initiation of ␥2a germline transcription is regulated by similar cis-acting elements Downloaded from located at the 3؅ end of the IgH locus. However, NK cells cannot induce the final steps of switch recombination resulting in the production of mature mRNA from recombined DNA. Our findings suggest that these different signaling pathways converge on regulatory elements that are common to germline transcription; however, because NK induction does not result in the final steps of switch recombination, some signals initiated by LPS plus IFN-␥ are not induced by NK cells. The Journal of Immunology, 2005, 174: 4113–4119. http://www.jimmunol.org/ atural killer cells constitute one of the key components first step for switch recombination to IgG2a (6, 7). The Ig locus is of the innate immune system in that they can be rapidly the site of two types of rearrangements: V(D)J assembly that gen- activated without the need for expansion of Ag-specific erates the V region exons at the H and L chain loci during B cell N 3 clones. There is increasing awareness of the importance of their development and class switch recombination (CSR) at the H role in modulation of the immune system via their ability to pro- chain (IgH) locus after antigenic stimulation of specific B cell duce a number of cytokines as well as via nonlytic interactions clones. CSR specifically alters C region genes through a deletional with target cells (1–3). B lymphocytes, by contrast, are important process, whereby a downstream C region gene is brought to prox- constituents of the specific immune system due to the presence of imity of a rearranged VDJ gene, allowing expression of one of the clonally distributed Ag receptors and the persistence of the prog- downstream isotypes (IgG, IgE, or IgA). CSR is preceded by by guest on October 8, 2021 eny of specific clones that can continue to produce the relevant germline transcription of target switch sequences located upstream Abs. An understanding of the interactions between these two cell of all of the C region genes except the C␦. Activation and targeting types should provide important insights into the influence of the of CSR is correlated with the ability of certain mitogens and cy- rapid but transient response to pathogens on the specific immune tokines to induce or suppress germline transcription of each spe- response that takes longer to initiate but is longer lasting. cific C region genes (8, 9) Accurate splicing of germline transcripts Studies in both human and murine systems have shown that NK is also critical for the efficiency of CSR (10). In addition to the cells, on their own, exert minimal effects on IgM secretion of rest- cis-regulatory elements located upstream of the I promoters, a se- ing B cells. Only B cells that are preactivated in vivo can be en- ries of DNase I hypersensitive sites located 2–32 kb 3Ј of the C␣ hanced by NK cells to increase both IgM and IgG synthesis. Fur- gene and downstream of the IgH locus, also affects both germline thermore, activated B cells can induce NK cells to up-regulate transcription as well as the process of switch recombination itself IFN-␥ secretion (reviewed in Ref. 4). We have previously shown (8, 9, 11, 12). Following the initiation of germline transcription, that, although NK cells cannot induce IgM secretion, they can switch recombination involving breakage and joining of DNA seg- initiate limited differentiation of resting cells as detected by the ments requires additional factors, the most important of which is induction of germline transcripts for IgG2a (I␥2a) (5), a necessary activation-induced cytidine deaminase (AID) (13), a member of the RNA-editing deaminase family. However, neither the target(s) of AID nor its potential cofactor(s) are precisely known and the *Department of Molecular Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75390; †Department of Microbiology and Immunology, Univer- exact role of AID is still under investigation. Interestingly, despite sity of Michigan Medical School, Ann Arbor, MI 48109; and ‡Department of Pedi- the ability of NK cells to stimulate production of appropriately atrics, Division of Blood and Marrow Transplantation, and Cancer Center, University ␥ of Minnesota Hospital and Cancer Center, Minneapolis, MN 55455 spliced I 2a transcripts as well as AID mRNA, they do not stimulate production of ␥2a transcripts from recombined DNA (5). Received for publication August 26, 2004. Accepted for publication January 24, 2005. Therefore, NK cells induce the germline transcripts that precede The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance switch recombination, but not the recombination event itself. Other with 18 U.S.C. Section 1734 solely to indicate this fact. inducers, such as LPS plus IFN-␥ or activated T cells, induce both 1 This work was supported by National Institutes of Health Grants AI38938 (to D.Y.), CA39068 (to W.A.D.), and R01 CA72669 (to B.R.B.). 2 Address correspondence and reprint requests to Dr. Dorothy Yuan, Department of Mo- 3 Abbreviations used in this paper: CSR, class switch recombination; AID, activation- lecular Pathology, University of Texas Southwestern Medical Center, 5323 Harry Hines induced cytidine deaminase; BAC, bacterial artificial chromosome; FL, fluorescein; Boulevard, Dallas, TX 75390. E-mail address: [email protected] PI-PLC, phosphatidylinositol-specific phospholipase C.

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 4114 NK-B CELL INTERACTIONS germline transcription and switch recombination. To test a poten- scribed (26). Rat anti-B220, hamster anti-CD48 (BCM1), goat anti-IgM Ј tial mechanistic basis for this difference, we examined the depen- F(ab )2, and mouse anti-DX5 were purchased from BD Biosciences. Rat ␥ anti-CD28 (29), anti-Fc␥R (2.4G2; Ref. 30), hamster anti-TCR␣␤ (31), rat dence of 2a germline transcription induced by NK cells and by ␥ ␥ Ј ⑀ anti-IFN- (R4-6A2; Ref. 32), and rat anti-CD48 (1G10) Abs (originally LPS plus IFN- on a set of regulatory elements 3 of the C gene. obtained from Dr. J. Bromberg, Mount Sinai School of Medicine, New In human systems, both CD11a (14, 15) as well as CD40- York, NY) were purified from hybridoma culture supernatants using Gam- CD40L (16) interactions have been shown to be important for NK maBind (Pharmacia Fine Chemicals) according to the manufacturer’s sug- Ј induction of B cell responses; however, the interaction molecules gestions. The F(ab )2 of rat anti-CD28 and the rat anti-CD48 Ab, 1G10, were prepared as previously described (33). Recombinant IFN-␥ was pur- for mouse cells have remained largely unknown. Some indicators chased from BioSource International. Phosphatidylinositol-specific phos- of possible NK-B cell interaction molecules are suggested by stud- pholipase C (PI-PLC) was purchased from Sigma-Aldrich. ies in which cells transfected with B cell ligands can be shown to elicit cytotoxic activity of NK cells (17, 18). Because the induction Semiquantitative RT-PCR analysis of I␥2a transcription by NK cells can occur without the addition of RNA was prepared using the TRIzol reagent (Invitrogen Life Technolo- other factors, it is a sensitive measure that can be used for the iden- gies), and RT-PCR was performed as previously described (34). Primers tification of the ligand-receptor pairs that may be involved in the for assessment of ␥2a germline transcription (I␥2a) and ␮M mRNA abun- NK-B cell interaction. We will show in this report that the CD48-CD2 dance have been described (5). The primers for assessment of cDNA from switched mRNA (S␥2a), consists of the forward primer (5Ј-CCAACCAT interaction is important for the activation of B lymphocytes by NK GGGATGGAGCTTC) corresponding to the V region of the A/J ARS- cells. The significance of these findings will be discussed. specific gene used for the BAC transgenic mice, and the reverse primer (5Ј-GGCCAGGTGCTCGAGGTT) located in the first exon of the ␥2a Materials and Methods gene. The 635-bp RT-PCR product derived from appropriately spliced mRNA was confirmed by restriction enzyme mapping. Ly49 primers are Downloaded from Transgenic mice consensus sequences for most Ly49 family members including both We prepared mice with a 230-kb transgene (Fig. 1A) of the entire H chain BALB/c and C57BL/6 strains (S. Anderson, unpublished observations) Ј Ј C region locus from strain 129 mice (Igha) (19). The transgenic mice were (forward, 5 -ATCATCCCAAGATGAGTGAGC; reverse, 5 -TTAGAT ϫ GGGCCATTGTCAATC). All primers were ascertained to span intronic generated using (SJL C57BL/6)F2 fertilized eggs, and are maintained by backcrosses to C57BL/6. Thus, the endogenous locus is Ighb. Using ho- regions. To quantify RT-PCR products, at least one of each primer pair was Ј ␥ 32 mologous recombination in Escherichia coli (20), we inserted an assem- 3 end-labeled with [ - P]ATP and used to spike reaction mixtures. Am- plified products were quantified using the ImagQuant software package bled VDJ 2 segment that encodes anti-arsonate binding activity (21) into http://www.jimmunol.org/ H (Molecular Dynamics). For all primer pairs, titration curves were per- the germline JH region of the 230-kb construct. We named this parent bacterial artificial chromosome (BAC) “ARS/Igh.” The ARS/Igh BAC in- formed to ascertain that the cycle number used fell within the linear range ϳ ␮ ␦ as cDNA concentrations were increased. cludes 14 kb upstream of the assembled VDJH2 segment, C ,C , the four C␥ genes, C⑀,C␣, the four known H chain 3Ј enhancers, and ϳ15 kb downstream of the last known 3Ј enhancer, HS4. We also used homologous FACS analysis recombination in E. coli to insert 4 bp (AATT) in the EcoRI site in I␥2a. Cultured cells were stained and analyzed using the FACScan flow cytometer This insertion creates an AseI site in the transgene that allows us to dis- (BD Biosciences) as previously described (35). PE-conjugated (PE)-mouse ␥ tinguish germline transcripts of transgenic 2a genes from germline tran- anti-IgMa, FL-mouse anti-IgMb, PE-rat anti-CD2, PE-mouse anti-CD244, PE- scripts of the endogenous genes. Among the founders with the transgenic rat anti-CD48, FL-rat anti-CD19, PE-mouse anti-IgD, FL-anti-hamster Ig, and VDJ exon, we identified two founders that included truncations of the 3Ј PE-rat isotype control Abs were purchased from BD Biosciences. end of the transgene. One line (822) retained the C⑀ gene, but deleted S␣ by guest on October 8, 2021 and everything 3Ј of it. A second line (1001) retained the ␥2a gene, but deleted C⑀ and everything 3Ј of it. Thus, both of these truncated lines lack Results the four known 3Ј enhancer elements. Role of 3Ј enhancer region in the regulation of induction of ␥2a germline transcripts by NK cells Cell preparation and culture Although IFN-␥ is an important cytokine in driving preferential For B cell preparations, T lymphocytes were depleted from splenocytes of Ϫ Ϫ switch recombination to the ␥2a exons, we have shown that both CD2 / (Ref. 22; bred and kindly provided by Dr. M. Bennett, University ␥Ϫ/Ϫ ␥ of Texas Southwestern Medical Center), and BALB/c-IfgϽtm1 (IFN- T and NK cells from IFN- mice can induce I 2a germline ␥Ϫ/Ϫ; Ref. 23) mice (The Jackson Laboratory), or BAC transgenic lines, transcripts that necessarily precede switch recombination. In con- and fractionated by Percoll gradient centrifugation as previously described trast to stimulation by LPS plus IFN-␥ or by T cells, however, NK (24). The high density fraction (25) was further purified by binding to cells cannot induce the final step of gene rearrangement required Ј fluorescein-conjugated (FL)-CD43 (BD Biosciences) and F(ab )2 of FL- goat anti-mouse IgG (Southern Biotechnology Associates) before incuba- for expression of switched transcripts (2). The failure of NK cell tion with anti-fluorescein-conjugated magnetic beads (Miltenyi Biotec), for stimulation to induce switch recombination might be due to the depletion of remaining non-B cells and IgGϩ cells, respectively. B cells fact that NK cells induce factors in B cells that act through dif- were found routinely to be Ͼ90% positive for the CD19 marker. For in Ϫ Ϫ ferent cis-acting elements than do those induced by LPS plus vivo modulation of CD48, IFN-␥ / mice were injected twice, on days Ϫ5 ␥ ␥ Ϫ ␮ IFN- . It is known that LPS plus IFN- -induced germline tran- and 3, with 300 g/animal of an ammonium sulfate cut of anti-CD48 ␥ (HM48-1) (26). On day 0, B cells were isolated from these mice. NK cells scription and switch recombination to 2a depends, to a large ex- were prepared by passage of spleen cells from BALB/c IFN-␥Ϫ/Ϫ, tent, on the set of enhancers 3Ј of C␥ (8, 9, 11, 12). We have C57BL/6 (The Jackson Laboratory), CD2Ϫ/Ϫ or CD244Ϫ/Ϫ (27) mice over studied transgenes of the entire H chain C region locus that un- nylon wool columns to remove adherent cells, and depleted of T cells by dergo germline transcription and switch recombination (19). We complement-mediated lysis. NK cells were then isolated by positive selection using anti-DX5 Abs and magnetic beads (Miltenyi Biotec). Purified recently identified transgenes that lack all four regulatory elements cells were cultured for 3–4 days in 1000 U/ml IL-2 as described previously 3Ј of C⑀ (HS3b, HS1/2, HS3b, and HS4). Mice expressing these (5), at which time the nonadherent cells were discarded, and the adherent transgenes express Ͻ5% the amount of LPS plus IFN-␥-induced cells were propagated for another 4–7 days. B lymphocytes were cultured ␥2a germline transcripts compared with transgenes with an intact ϫ 6 ϫ 6 either alone (1 10 /ml), or together with NK cells (0.5 10 /ml) in the 3Ј regulatory region.4 As illustrated in Fig. 1B, the various lines presence of 100 U/ml IL-2 with or without other additives in 24- or 48-well Falcon tissue culture plates (BD Biosciences). established with the ARS/Igh transgene display two patterns of allelic exclusion. In about one-half of the lines, 30–50% of B cells Abs and reagents

Hamster anti-CD40L (CD154; Ref. 28) Abs were kindly provided by Dr. 4 W. A. Dunnick, J. Shi, K. A. Graves, and J. T. Collins. Deletion of the 3Ј end of the R. Noelle (Dartmouth Medical School, Dartmouth, NH). Ammonium sul- murine heavy chain constant region locus results in dramatically reduced germline fate cut preparations of hamster anti-CD48 (BCM1) were prepared as de- transcription and switch recombination of the four ␥ genes. Submitted for publication. The Journal of Immunology 4115

express transgenic ␥2a germline transcripts; all of the I␥2a transcripts detected were resistant to AseI digestion. Identical results were obtained with a second transgenic line (line 1001), which also lacks the 3Ј end of the H chain locus (data not shown). Thus, like ␥2a germline transcripts induced by LPS plus IFN-␥, germline transcripts induced by NK cells depend on elements in the 3Ј end of the H chain locus. Fig. 1 also shows that mature, switched transcripts, which can be detected by primers specific for the transgenic V region, are also induced by LPS plus IFN-␥ activation of cells carrying the intact transgenes. Such postswitch transcripts are not induced in cells carrying the transgene with the deleted 3Ј enhancer (Fig. 1C).4 In contrast, activation by NK cells induced low to nondetectable levels of switched transcripts from cells carrying the intact transgenes despite the induction of I␥2a transcripts. These results are similar to our previous findings using nontransgenic B cells (5). Therefore, it is possible that other cis-acting elements, perhaps located in the promoter region of the ␥2a germline gene, bind transcription factors that are differentially stimulated by LPS plus IFN-␥ vs NK cells. Downloaded from Inhibition of induction of I␥2a transcription by Abs against determinants expressed by B and NK cells To identify the ligand/receptors that may be involved in the NK cell induction of B cell I␥2a transcription, we first used a number of Abs against possible candidates for the NK-B cell interaction in an attempt to block the induction. NK cells propagated from IFN- http://www.jimmunol.org/ ␥Ϫ/Ϫ mice were used to ensure that the induction does not involve IFN-␥. Fig. 2A shows representative semiquantitative RT-PCR analyses of RNA obtained from cocultures in the presence of various Abs. The relative level of I␥2a induction in each coculture

FIGURE 1. Induction of I␥2a transcription by NK cells is regulated by elements in the 3Ј end of the H chain C region locus. A, Schematic of the transgenes in the 847, 858, and 822 lines. B, The three lines of transgenic by guest on October 8, 2021 mice were stained, along with the parental strain, C57BL/6, with allotypes- specific anti-IgM, as indicated, to assess the extent of allelic exclusion. C, Resting B cells from each of the indicated strains were cultured for 48 h either with LPS plus IFN-␥ or with NK cells from IFN-␥Ϫ/Ϫ mice. RNA extracted from each culture was analyzed by RT-PCR assay using specific primers for transcripts derived from germline (I␥2a) or the productively rearranged gene (S␥2a; 635 bp). The I␥2a PCR product (399 bp) from the transgene was distinguished from that from the endogenous locus by digestion with AseI, which yielded a smaller species of 319 bp. express transgenic IgMa and not IgMb of the endogenous (C57BL/6) genes, and the remainder of the B cells express the endogenous IgMb and not the transgenic IgMa (lines 847 and 822). Other lines exhibit virtually complete allelic exclusion by the transgenes and all of the B cells express IgMa (line 858). The lines with truncated transgenes allow us to test the depen- dence of the NK cell-induced ␥2a germline transcription on the 3Ј enhancer region (Fig. 1). To distinguish ␥2a germline transcripts FIGURE 2. Requirement for CD48 expression for induction of I␥2a of the endogenous locus from ␥2a germline transcripts of the trans- transcription by NK cells. Column-purified, resting B cells from IFN-␥Ϫ/Ϫ genic locus, we digested the amplified product with AseI, which BALB/c mice were cultured for 2 days with or without day 7–10 IL-2- cuts only in the products derived from the transgene. As demon- propagated NK purified from the same mice (NK:B ratio ϭ 0.5:1) in the strated by the intact, 399-bp band, all mice express ␥2a germline presence of various additives as indicated. A, Left and right panels show transcripts from their endogenous genes. The presence of the the semiquantitative RT-PCR analysis of two representative experiments ␥ 32 319-bp fragment derived by AseI digestion of products from lines using - P-labeled primers. B, Products fractionated on agarose gels were ␥ visualized, and band intensities were quantified on the ImagQuant. The 847 and 858 indicates that I 2a transcripts are expressed from the ␥ Ј relative level of amplified I 2a cDNA was determined by normalization to transgenic locus with an intact 3 regulatory region (compare with that of amplified ␮M cDNA for each sample. Percent change was calcu- products from cells from the nontransgenic strains, C57BL/6 and lated by dividing the relative I␥2a level in B cells cultured with NK cells Ϫ/Ϫ IFN-␥ ). Transgenic ␥2a germline transcripts were made in re- in the presence of additives by the relative level obtained from cultures sponse both to LPS plus IFN-␥ and to NK cells. In contrast, B cells without Abs. The average values obtained from three to four experiments from line 822, lacking the transgenic 3Ј regulatory region, did not are shown together with the SEM. 4116 NK-B CELL INTERACTIONS

FIGURE 3. PI-PLC treatment of B cells reduces the level of induction of I␥2a transcription in response to stimulation. Column-purified, resting B cells from IFN-␥Ϫ/Ϫ BALB/c mice were treated with various concentrations of PI-PLC for different time periods as indicated. A, After removal of Downloaded from the enzyme, cells were cultured overnight and analyzed on the FACS after staining with the reagents as indicated. Cells treated with 0.5 U of PI-PLC for 2 h and rested overnight were incubated with either LPS and IFN-␥,or with syngeneic IL-2 propagated NK cells for another 48 h, before RT-PCR analysis and quantification by ImagQuant analysis. B, The relative mean I␥2a transcript levels determined by normalization of the amplified product to that of ␮M cDNA for 3 experiments are shown. Numbers in brackets http://www.jimmunol.org/ indicate p values (Յ) from two-sample t tests assuming unequal variances testing for the probability that the two samples are the same. was determined as a function of the level of mRNA for membrane IgM (␮M mRNA). The effect of the inclusion of Abs are compared in Fig. 2B in which the relative levels of induced I␥2a transcripts is shown as a percentage of the levels induced in the absence of FIGURE 4. In vivo modulation of CD48 reduced the response of B cells by guest on October 8, 2021 inhibitors. Thus, neither anti-CD28 nor anti-CD40L, both hamster to NK induction. IFN-␥Ϫ/Ϫ mice were treated with two consecutive injec- Abs directed against ligands previously reported to be on NK cells tions (300 ␮g/animal) of anti-CD48 on days Ϫ4 and Ϫ2 before the PBL (16, 29, 36), altered the level of induction of I␥2a transcription nor was isolated and examined for the extent of depletion as well as Ab re- did a hamster Ab against TCR␣␤ chains, added as a specificity tention by FACS analysis (A). One day later, resting splenic B cells were control, because they are not expressed on the purified, IL-2-prop- prepared from the same mice, also analyzed by flow cytometry (B), and cultured with LPS plus IFN-␥ or with NK cells propagated from IFN-␥Ϫ/Ϫ agated NK cells. Therefore, it is unlikely that cross-linking of NK mice for 48 h. RNA extracted from the cultures along with cultures of NK cells to B cells by virtue of the binding of the Ig Fc portion to the and B cells from noninjected mice were analyzed by RT-PCR and quan- B cell FcRII affected the induction. The addition of anti-B220 Abs, tified by ImagQuant (C). The mean levels from three independent deter- which can recognize determinants on both B and NK cells, also minations are shown and are representative of two independent had no effect, further indicating that binding to FcRs alone does experiments. not affect I␥2a induction. In contrast, two different mAbs against CD48 significantly inhibited the interaction. Although the extent of inhibition by BCM1 (hamster Ab) was greater than that by 1G10 0.5 U of PI-PLC for 2 h effectively removed all of the CD48 but (rat Ab), the inhibition was partially reversed by the inclusion of did not affect the expression of IgD or CD19. Furthermore, CD48 anti-FcR Abs in the cocultures. Therefore, ligation of FcR on NK was not regenerated even after an incubation period of 24 h. The cells may have augmented the inhibition by these Abs. However induction of B cell I␥2a transcripts by IL-2-propagated NK cells Ј the F(ab )2 of the anti-CD48 Ab also significantly inhibited the was significantly reduced when the B cells were first treated with Ј induction, whereas neither the F(ab )2 of anti-CD28 nor that of PI-PLC (Fig. 3B). Induction of I␥2a transcripts by LPS and IFN-␥ anti-IgM caused significant decreases. was also reduced, probably due to the removal of the CD14 portion of the functional LPS receptor by PI-PLC (40); however, the re- ␥ Induction of I 2a requires the presence of CD48 on B cells tention of substantial induction by this pathway suggests that the B A problem with inhibition studies with anti-CD48 Abs is that this cells were not rendered totally inert by the treatment with PI-PLC. receptor is expressed by both B and NK cells; thus it is not clear It is also possible to modulate CD48 in vivo by the injection of which cell type is affected. We therefore sought an alternate anti-CD48 Abs (26). Examination of the PBL from animals in- method to remove CD48. Because CD48 is known to be a GPI- jected with anti-CD48 showed that virtually all of the detectable linked protein (37), we attempted to remove it from B cells by CD48 Ag on all cells was eliminated by this treatment (Fig. 4A). enzymatic treatment with PI-PLC. The number of known GPI- Furthermore, as indicated by staining with an anti-hamster Ab, linked B cell surface Ags is limited (38). Other than CD48, those very little of the Ab remained on the cell surface (3.6%) of the expressed on B cells include CD24 and CD14, the LPS receptor, modulated cells. We therefore prepared resting B cells from ani- and a minor subset of IgD (39). Fig. 3A shows that treatment with mals treated in this manner. FACS analysis after purification The Journal of Immunology 4117 showed that, although 5% of the CD19-positive cells remained resistant to the modulation (Fig. 4B), the B cells were dramatically compromised (78% inhibition) in their ability to express I␥2a transcripts in response to stimulation by NK cells (C). In comparison, the response to LPS plus IFN-␥ induction was affected to a much lesser degree (36% inhibition). Thus, removal of CD48 from only B cells by two alternative methods resulted in a compromised response to NK cells. Fig. 4C also shows that the low level of I␥2a expressed by B cells cultured on their own was not increased when the B cells were obtained from anti-CD48-treated mice, indicating that the in vivo encounter with the Ab did not enhance I␥2a FIGURE 6. Expression of ligands for CD48 on propagated NK cells transcription. from mutant strains. Day 7 IL-2-propagated NK cells from C57BL/6, CD244Ϫ/Ϫ, or CD2Ϫ/Ϫ mice were stained with either PE-rat isotype con- Ligands on NK cells involved in the induction of I␥2a trol, PE-rat anti-CD2, or PE-mouse anti-CD244 Abs and analyzed by flow transcripts cytometry. There are at least two receptors expressed on NK cells for CD48, CD2 and CD244. To determine whether either could be involved in the NK-B cell interaction, we prepared NK cells from animals merous experiments performed with NK cells obtained from var- with deletion mutations of the genes encoding CD2 and CD244. ious strains. Although accompanied by some variability due prob- Downloaded from Because the induction of I␥2a by NK cells is amplified by the ably to the use of different preparations of propagated NK cells, the Ϫ/Ϫ addition of IFN-␥, we also included anti-IFN-␥ in the cocultures to mean level of I␥2a transcription derived from CD2 NK cell restrict the effect to that mediated by cell-cell interactions. Fig. 5A stimulation was consistently lower than that from B6 NK cells shows that, in comparison to induction by NK cells propagated both in the presence ( p Յ 0.0003) and absence of anti-IFN-␥ ( p Յ from B6 cells, the level of I␥2a transcripts induced by NK cells 0.002). In the presence of anti-IFN-␥, the stimulatory activity of Ϫ/Ϫ from CD2Ϫ/Ϫ animals was significantly compromised. Induction CD2 NK cells was also significantly lower than that by Ϫ Ϫ / http://www.jimmunol.org/ by NK cells propagated from CD244Ϫ/Ϫ animals was also re- CD244 NK cells ( p Յ 0.003). Although the stimulatory activ- Ϫ/Ϫ duced, although not to an equivalent extent. The titration of Ly49 ity of CD244 NK cells was in general lower than that by B6 mRNA levels expressed in NK cells in the cocultures did not re- NK cells, the difference did not achieve statistical significance veal significant differences between the strains, indicating that the ( p Յ 0.065). As shown previously, NK cells propagated from Ϫ/Ϫ lower level of induction by CD2Ϫ/Ϫ NK cells was not due to IFN-␥ mice in general induced somewhat lower levels of I␥2a reduced viability of these cells. Fig. 5B summarizes results of nu- transcripts and approximated that induced by B6 NK cells in the presence of anti-IFN-␥. Induction by either CD2Ϫ/Ϫ or CD244Ϫ/Ϫ NK cells in the presence of anti-IFN-␥ was significantly decreased ( p Յ 0.0003 and p Յ 0.007) compared with induction of I␥2a by by guest on October 8, 2021 IFN-␥Ϫ/Ϫ cells. Because absence of neither CD2 nor CD244 reduced the level of induction to below background levels, it is possible that both ligands play a role. However, CD2 appears to be the major inducer. There is a possibility that deletion of either CD2 or CD244 altered the level of expression of the alternate receptor for CD48 in each strain. Therefore, we evaluated the propagated NK cells from each of the mutant strains for the expression of CD244 and CD2. Fig. 6 shows that, when compared with C57BL/6 NK cells, no significant difference in the expression of CD2 on CD244Ϫ/Ϫ cells or CD244 on CD2Ϫ/Ϫ cells was found. The lack of expression of the appropriate Ag on each mutant strain was also confirmed. Because only CD2, but not CD244, is expressed on B cells there is a possibility that the differential response to the CD2 vs CD244 ligand is modulated by the expression of CD2 on B cells. How- ever, we found that the level of induction of I␥2a was not affected when B cells from CD2-deficient mice were cocultured with NK cells (data not shown). Therefore, CD2 on B cells does not play a FIGURE 5. The absence of CD2 expression on NK cells reduces their role in the interaction. ability to stimulate B cell-I␥2a transcription. IL-2-propagated NK cells from CD2Ϫ/Ϫ, CD244Ϫ/Ϫ, or C57BL/6 mice were cultured with column- Discussion Ϫ Ϫ purified, resting B cells from BALB/c IFN-␥ / mice in the presence of We have previously shown that both NK and activated T cells can anti-IFN-␥ for 48 h. A, A representative ethidium bromide-stained gel of induce B cell expression of ␥2a germline transcripts in the absence 2-fold stepwise dilutions of cDNA obtained from each culture amplified of IFN-␥ (5). Thus, despite the requirement for IFN-␥ in the in- with ␥-32P-labeled primers specific for I␥2a, ␮M, or Ly49 sequences. B, Ϯ duction of IgG2a by LPS, stimulation by other Ags may not re- The relative mean level ( SD of the mean) of at least three experiments ␥ from cells cultured either with or without anti-IFN-␥, as indicated, were quire IFN- (41). LPS stimulates B cells via TLR, but the nature quantified by ImagQuant. Experiments from IFN-␥Ϫ/Ϫ NK cells were also of the surface ligand(s) involved in NK stimulation of B cells was, added as additional controls. Two-sample t tests assuming unequal vari- until now, not clear. By the use of a number of Abs, including ances yielded two-tailed p values testing for the probability that the two many that are directed toward determinants expressed on NK and samples are the same. B cells, we determined that the only reagents that consistently 4118 NK-B CELL INTERACTIONS demonstrated inhibition of the induction of I␥2a by NK cells are possibly different intracellular signaling pathways, must ultimately two anti-CD48 Abs (Fig. 2). The use of Abs to inhibit cell-cell result in induction of common transcription factors necessary for interactions can be problematic due to interactions with the FcR of binding to the cis-regulatory sequences located in the 3Ј end of the either B or NK cells that can result in either inhibition or stimu- H chain locus (Fig. 1). In contrast to induction by LPS plus IFN-␥ lation of the response. Therefore, it is significant that the inhibition or activated T cells, however, NK cell induction does not result in Ј of the response was also apparent when F(ab )2 of anti-CD48 Abs productive switch recombination to IgG2a. The absence of were used although the extent of decrease was somewhat less. The switched ␥2a sequences in the presence of increased abundance of reduced level of inhibition may be attributed to anti-CD48 stimu- germline transcripts further confirms that the induction by NK lation of NK cells that also express the ligand. cells is not attributed to expansion of small numbers of preacti- The removal of CD48 from B cells by either PI-PLC treatment vated B cells in the cocultures. The finding that NK cells, by them- (Fig. 3) or by in vivo modulation with anti-CD48 Abs (Fig. 4) selves, can only stimulate the first few steps of switch recombina- significantly reduces the level of induction of I␥2a transcription by tion without inducing the terminal steps is not unreasonable, NK cells. This is the best indication that the CD48 on B cells (not because one would not expect any encounter of NK cells with B that on NK cells) is critical for the induction. Whereas the reduc- cells in vivo to result in IgG2a secretion. It is likely that this in- tion of LPS responsiveness in B cells treated with PI-PLC can teraction only skews the B cells to this response upon interaction most likely be attributed to the removal of the LPS receptor, CD14, with cognate Ag. Using monoclonal B cells from the quasi-mon- the reason for the lower LPS response of the anti-CD48-modulated oclonal mouse (48), we have recently obtained evidence for this B cells is not clear. Although the B cell response to LPS was only hypothesis (unpublished observations). This priming step may ex- minimally compromised in the CD48Ϫ/Ϫ mouse, other aspects of plain in vivo findings that many viral infections tend to result in Ig B cell function have not been examined (42). responses that are skewed toward IgG2a (49). Thus, in vivo stim- Downloaded from Further evidence for the importance of CD48 in the NK stimulation of NK cells by poly(I:C), which results from stimulation by ulation of B cells is derived from our finding that the induction of similar cytokines as those resulting from viral infections, also I␥2a was significantly compromised when NK cells from mice causes preferential increases in Ag-specific IgG2a production (50). defective in expression of CD2, the murine ligand for CD48. CD48 Now that we have some information regarding the receptor-ligand is anchored to the membrane via a GPI linkage. In the absence of pairs involved in the interaction between NK and B cells, it may be the cytoplasmic domain, how the signal arising from interaction possible to determine their relative importance in the in vivo re- http://www.jimmunol.org/ with the CD2 ligand is transmitted to B cells is not clear. However, sponse to antigenic stimulation. GPI-anchored receptors have been shown to coimmunoprecipitate with various members of the SRC family of tyrosine kinases in Acknowledgments glycolipid-enriched microdomains (43) as well as G protein sub- We thank Drs. John Schatzle and Michael Bennett (University of Texas South- units (44). Inasmuch as the removal of CD48 from B cells by either western Medical Center) for stimulating discussions and for supply of various Ab modulation or PI-PLC treatment did not reduce the induction to mouse strains as well as the anti-244 Ab. The hybridoma HM48-1 was kindly background levels, other compensatory interactions may be in- provided by Dr. Hideo Yagita. volved. Considering the weak charge-charge interactions of CD2 by guest on October 8, 2021 with its ligands, which nonetheless exhibit strong specificity (45), Disclosures one possibility is that any encounter between CD2 and CD48 only The authors have no financial conflict of interest. serves to initiate the interaction between the two cell types such that another receptor-ligand pair can provide the final stimulation. References 1. Hoshino, T., R. T. Winkler-Pickett, A. T. Mason, J. R. Ortaldo, and H. A. Young. In the absence of CD48 or its ligand, other interaction molecules 1999. IL-13 production by NK cells: IL-13-producing NK and T cells are present may continue to function, albeit much less efficiently. For exam- in vivo in the absence of IFN-␥. J. Immunol. 162:51. ple, CD48 has been shown to deliver an accessory signal for 2. Biron, C. A., K. B. 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