Marginal zone (MZ) and B1 B cells have the capacity to respond to foreign Ags more rapidly than conventional B cells, providing early immune responses to blood-borne pathogens. Ly9 (CD229, SLAMF3), a member of the signaling lymphocytic activation molecule family receptors, has been implicated in the development and function of innate T lymphocytes. In this article, we provide evidence that in Ly9-deficient mice splenic transitional 1, MZ, and B1a B cells are markedly expanded, whereas development of B lymphocytes in bone marrow is unaltered. Consistent with an increased number of these B cell subsets, we detected elevated levels of IgG3 natural Abs and a striking increase of T-independent type II Abs after immunization with 2,4,6-trinitrophenyl-Ficoll in the serum of Ly9-deficient mice. The notion that Ly9 could be a negative regulator of innate-like B cell responses was supported by the observation that administering an mAb directed against Ly9 to wild-type mice selectively eliminated splenic MZ B cells and significantly reduced the numbers of B1 and transitional 1 B cells. In addition, Ly9 mAb dramatically diminished in vivo humoral responses and caused a selective downregulation of the CD19/CD21/CD81 complex on B cells and concomitantly an impaired B cell survival and activation in an Fc-independent manner. We conclude that altered signaling caused by the absence of Ly9 or induced by anti-Ly9 may negatively regulate development and function of innate-like B cells by modulating B cell activation thresholds. The results suggest that Ly9 could serve as a novel target for the treatment of B cell–related diseases.
Marginal zone (MZ) and B1 B cells are distinct B lymphocyte subsets that differ from conventional follicular (FO) B cells both developmentally and functionally. These two cell types have been termed innate B lymphocytes because they share many properties with innate immune cells and serve as a bridge between the rapidly occurring innate responses and the slower adaptive immunity (1). Because of their anatomical location, MZ and B1 B cells are the earliest lymphocytes to encounter invading viruses and bacteria acquired through the bloodstream and the gut/peritoneum. These B cell subsets have evolved to provide a first line of defense against pathogens by mounting quick and potent humoral responses, characterized by the production of Abs with a broad reactivity (2). They play an important role in T-independent (TI) Ab responses, particularly to TI type II (TI-2) Ags (3). Ab responses to these Ags are essential for generating protective immunity against the cell-wall polysaccharides expressed by a number of capsulated bacterial pathogens, such as Streptococcus pneumoniae (4). Despite numerous insights into the understanding of these humoral responses, the molecular mechanisms regulating TI-2 Ag responses and MZ and B1 B cell homeostasis remain only partially understood (5).
Leukocyte cell-surface molecules are required for the appropriate development, activation, and effector functions of lymphocytes. Most of these transmembrane molecules mediate adhesion and elicit intracellular signals that positively or negatively regulate immune responses. Among the different families of cell-surface molecules, the signaling lymphocytic activation molecule (SLAM) family receptors have been shown to exert crucial immunomodulatory functions in the regulation of several immunological processes such as lymphocyte development and survival, cytotoxicity, cell adhesion, and humoral immunity (6, 7). Recently, several reports have demonstrated that the SLAMF receptors are crucial to the development of innate-like T lymphocytes, such as invariant NKT (iNKT) cells (8, 9).
Ly9, also known as CD229 or SLAMF3, is one of the nine members of the SLAM family. It is expressed on all T and B lymphocytes (10). Interestingly, its highest expression levels are found on innate-like lymphocytes such as iNKT cells and MZ B cells (11). Ly9 is also expressed in virtually all chronic lymphocytic leukemias (12). Its expression is not lost during B cell differentiation into plasma cells, and it is highly expressed on multiple myeloma cells (13). Ly9 has four extracellular Ig-like domains, and as has been shown with other SLAMF members, the N-terminal Ig domain of Ly9 mediates homophilic adhesion (14). Its cytoplasmic tail contains two copies of the conserved immunoreceptor tyrosine-based switch motif (ITSM), which is a docking site for the adapter molecules SLAM-associated protein (SAP) and Ewing’s sarcoma–associated transcript 2 (EAT-2), and also for phosphatases such as SHIP and SH2 domain–containing phosphatase (SHP-2) (15, 16). Recently, we have shown that in contrast with SLAMF1 and SLAMF6, Ly9 has a negative rather than a positive role in the signaling pathways required for iNKT development and innate-like CD8 T cell expansion in the thymus (17). Moreover, aged Ly9-deficient mice spontaneously develop features of systemic autoimmunity, such as splenomegaly and production of autoantibodies, indicating that the Ly9 cell-surface receptor is involved in the maintenance of immune cell tolerance (18). All these data support the view that Ly9 differs from the other SLAMF members by acting as a nonredundant inhibitory molecule. To date, however, the regulatory effects of Ly9 on B cell biology remain elusive.
Our main goal was to elucidate the function of Ly9 in B cell development and homeostasis. This study demonstrated that Ly9 negatively regulates innate B lymphocyte homeostasis and function. Importantly, it also shows that Ab-mediated targeting of Ly9 hampers B cell responses.
Materials and Methods
Wild-type (WT) BALB/c and C57BL/6 mice were obtained from Charles River Laboratories (Saint-Aubin-lès-Elbeuf, France). Ly9−/− (129 × B6) mice, kindly provided by Dr. D.J. McKean (La Jolla Institute for Allergy and Immunology, San Diego, CA) (19), were backcrossed to BALB/c or C57BL/6 backgrounds for at least 12 generations. All animals were used at 8–12 wk of age and housed under specific pathogen-free conditions.
Mice were immunized i.p. with 100 μg 2,4,6-trinitrophenyl (TNP)-keyhole limpet hemocyanin (KLH) conjugate (TNP31-KLH; Biosearch Technologies) in CFA (Sigma) at day 0 and boosted at day 14 with 100 μg TNP31-KLH diluted in PBS. For assessment of TI responses, mice received i.p. 50 μg TNP0.3-LPS or 50 μg TNP65-Ficoll (Biosearch Technologies) in PBS. Serum samples were collected from the tail vein at the indicated time points after immunization.
For the detection of TNP-specific Abs, high binding plates (Costar) were coated with 4 μg/ml TNP18-BSA (Biosearch Technologies) diluted in PBS. For the detection of natural Abs, plates were coated with either 8 μg/ml Pneumo23 vaccine (Sanofi Pasteur) or 5 μg/ml PC16-BSA (Biosearch Technologies) in PBS. For the detection of total IgG or IgM levels in serum, plates were coated with 3 μg/ml anti-mouse IgG (Sigma) or 3 μg/ml F(ab′)2 fragments anti-IgM (Jackson Immunoresearch). Serum samples were added at an optimal dilution chosen from a previous titration curve. Bound Abs were detected using either anti-mouse IgG peroxidase (Sigma) or biotin-conjugated goat anti-mouse IgM, IgG1, IgG2a, IgG2b, and IgG3 (Jackson Immunoresearch) and streptavidin-peroxidase (Roche). Plates were developed with o-Phenylenediamine dihydrochloride (Sigma) and read on an Epoch plate reader at 405 nm.
Cell suspensions of lymphocytes from spleen, blood, peritoneum, and bone marrow were treated with RBC lysis buffer (0.15M NH4Cl, 0.01M Tris HCL), washed, and incubated in 20% heat-inactivated rabbit serum before being stained with fluorophore-labeled Abs. The following anti-mouse mAbs were purchased from BD Pharmingen: CD11b-PE and FITC (M1/70), CD21/CD35-FITC (7G6), CD23-PE (B3B4), CD43-FITC (S7), CD86-PE (GL1), CXCR5-biotinylated (2G8), IgM-biotinylated (R6-60.2), and TNP-PE (G235). The mAbs B220-PB and allophycocyanin (RA3-6B2), CD11c-PECy7 (N418), CD19-A647 (6D5), CD69-PerCP (H1.2F3), CD81-PE (Ewing’s sarcoma–associated transcript 2), CD138-PECy7 (281-2), and integrin β 7-allophycocyanin (FIB504) were obtained from Biolegend. CD5-PECy7 (53-7.3), CD62L-allophycocyanin (MEL-14), CD93-PerCP-Cy5.5 (AA4.1), and specific intracellular adhesion molecule-grabbing nonintegrin receptor-1 (SIGN-R1)-allophycocyanin (eBio22D1) were purchased from eBioscience. Anti-mouse CD49d (integrin α 4) VioBlue (R1-2) and MHC class II (MHC-II) FITC (REA528) were obtained from Miltenyi Biotec. Anti-mouse sphingosine 1-phosphate receptor 1 (S1P1)-allophycocyanin (713412) was purchased from R&D Systems; anti-mouse CD18-FITC (C71/16) was from Sigma Immunochemicals; and biotinylated anti–MOMA-1 was from Abcam. The Abs anti-mouse IgM-FITC (polyclonal) and IgD-PE (11-26c) were obtained from Southern Biotech. Streptavidin brilliant violet 421 and streptavidin PECy5 were obtained from Biolegend. Data were acquired with FACSCanto II cytometer and analyzed with FlowJo software (Tree Star).
In vitro stimulation assays
Spleen single-cell suspensions were depleted of RBCs and cultured in RPMI 1640 media supplemented with 10% FCS, 2 μM l-glutamine, 50 U/ml penicillin, and 50 μg/ml streptomycin. Cells were stimulated with goat F(ab′)2 anti-IgM (10 μg/ml; Jackson Immunoresearch) for 6 h, and upregulation of activation markers was assessed by flow cytometry.
MZ B cell isolation and phosphoflow analysis
MZ B cells were purified with the Marginal Zone and Follicular B Cell isolation kit (Miltenyi Biotec) following manufacturer’s protocol. For the assessment of BCR signaling events, cells were simulated for 5 min in the presence of 10 μg/ml F(ab′)2 anti-IgM (Jackson Immunoresearch). After the incubation time, cells were fixed immediately by adding 3% formaldehyde directly into the culture medium to obtain a final concentration of 1.5% formaldehyde. Cells were incubated in this fixation buffer for 10 min at 37°C and then pelleted. Cells were permeabilized with ice-cold Perm Buffer III (BD) at 4°C for 30 min, then washed in FACS wash buffer (PBS with 2% FCS and 0.01% NaN3) and resuspended in staining buffer. The detection of phosphorylated epitopes was carried out by using the mAbs Btk(pY223)-A647 (N35-86), JNK(pT183/pY185)-PE (N9-66), and p38 (pT180/pY182)-PE (36/p38), all from BD.
Tissue staining and confocal microscopy
WT BALB/c mice received 250 μg i.p. of Ly9.7.144 or control Ab (IgG1). Twenty-four hours later, spleens were collected and frozen in Tissue-Tek OCT Compound (Sakura). Five-micrometer sections were cut in a cryostat microtome (Leica). Slides were fixed in acetone and stored at −80°C. Before staining, samples were extensively washed with PBS and blocked with 6% FBS in PBS during 30 min at room temperature. Endogenous biotin was blocked by using an Avidin/Biotin blocking kit (Vector Laboratories) following the manufacturer’s protocol. Samples were incubated overnight at 4°C with anti-mouse IgM-FITC (polyclonal; Southern Biotech), anti–SIGN-R1–allophycocyanin (clone eBio22D1; eBioscience), biotinylated anti–MOMA-1 (MOMA-1; Abcam), biotinylated anti–VCAM-1 (Caltag), or anti–ICAM-1 PE (3E2; BD) followed by incubation with Alexa Fluor 555–conjugated streptavidin (Life Technologies) or anti-hamster rhodamine (Jackson Immunoresearch). Laser scanning confocal images were acquired using a Leica TCS SL laser scanning confocal spectral microscope (Leica Microsystems) equipped with argon and HeNe lasers and a Leica DMIRE2 inverted microscope. FITC, Alexa555, and allophycocyanin emission were acquired sequentially with a triple dichroic beam-splitter (TD 488/543/633 nm); excitation at 488/543/633 nm and emission detection ranges 500–540 nm, 555–625 nm, and 643–700 nm, respectively. Images were obtained using HCPLFLUOTAR10× Dry (numerical aperture, NA 0.3), HCPLFUOTAR 20× Dry objective lens (NA 0.5) equipped with phase-contrast optics and the confocal pinhole set at 1 Airy unit. Image assembly, processing, and quantification were performed using ImageJ software.
TNP-Ficoll capture in the MZ
Ly9−/− and age-matched WT mice were injected i.v. with 50 μg TNP65-Ficoll (Biosearch Technologies). After 30 min, TNP binding to spleen B cells was assessed by flow cytometry. Distribution of TNP+ cells was assessed by immunofluorescent analysis; tissues were processed as described earlier and stained with Abs against TNP, IgM, and SIGN-R1.
In vivo treatment with Ly9.7.144
For analyzing spleen B cell subsets, BALB/c mice received i.p. 250 μg Ly9.7.144 (IgG1) mAb, generated in our laboratory (17), or an isotype control (IgG1) mAb diluted in PBS. F(ab′)2 were generated using the Pierce Mouse IgG1 Fab and F(ab′)2 Micro Preparation Kit (Thermo Scientific). Spleen, blood, and bone marrow populations were analyzed by FACS either 1 or 26 d later. To assess humoral responses after anti-Ly9 treatment, we i.p. injected BALB/c or CD1d−/− mice with 250 μg Ly9.7.144 or 250 μg mouse IgG1 control 24 h before being immunized with T-dependent (T-D), TI type I (TI-1), and TI-2 Ags as described earlier. BALB/c mice received i.p. 50 μg whole Ly9.7.144, F(ab′)2 Ly9.7.144, or isotype control mAb, to assess F(ab′)2 Ly9.7.144 in vivo effects on B cell populations. Twenty-four hours later, spleen B subsets were analyzed by FACS. For analysis of TI-2 responses after treatment with F(ab′)2 Ly9.7.144 fragments, BALB/c mice received 50 μg Ly9.7.144, F(ab′)2 Ly9.7.144, or isotype control mAb i.p. on days 0, 3, and 6. On day 1, mice were immunized i.p. with 50 μg TNP65-Ficoll in PBS. On day 8, mice were sacrificed and anti-TNP–specific Ab levels in the serum were determined by ELISA as described earlier.
Ex vivo analysis of proliferation, viability, and IgG production of anti-Ly9–treated cells
BALB/c mice received i.p. 250 μg Ly9.7.144 or IgG1 control mAb. Twenty-four hours later, spleen lymphocytes were isolated and stained with CellTrace CFSE (Life Technologies) following manufacturer’s instructions. A total of 106 lymphocytes were cultured in 0.5 ml complete RPMI 1640 in 24-well plates. Cells were cultured for 72 h in medium alone or in the presence of 10 μg/ml LPS (Sigma) or 10 μg/ml F(ab′)2 anti-mouse IgM (Jackson Immunoresearch). Viability was assessed by staining with Live/Dead Fixable far red dye (Invitrogen) following the manufacturer’s protocol.
IgG levels in the supernatants were determined by ELISA. Ninety-six–well plates were coated with 3 μg/ml anti-mouse IgG (Sigma). A purified in-house mouse IgG Ab was used as a standard. Bound IgG was detected with peroxidase-conjugated anti-mouse IgG (Sigma). The reaction was developed with o-phenylenediamine dihydrochloride substrate (Sigma) and read at 405 nm.
Differences between group means were assessed with GraphPad Prism 5, using unpaired two-tailed Student t tests unless otherwise indicated. The paired t test was used to compare Ig levels on each mouse before and after anti-Ly9 treatment.
Significantly higher numbers of splenic transitional 1, MZ, B1 B cells, and consequently increased production of natural Abs in Ly9-deficient mice
Analysis of B cell development in Ly9-deficient (Ly9−/−) mice showed no major alterations in bone marrow B cell subsets (data not shown). In contrast, the absence of Ly9 led to an expansion of splenic B220+ B cells compared with WT mice, both in percentages and absolute numbers (62.18 ± 1.07 versus 52.87 ± 0.62%; p > 0.0001; 71.92 ± 4.18 versus 53.18 ± 2.68 cell numbers ×106). This increase was also observed in the proportion of mature B cells in blood, lymph node, and bone marrow (data not shown). The most striking finding, however, was a marked expansion, ∼2-fold, of transitional and MZ B cells in the spleen of Ly9−/− mice (Fig. 1). The increase in transitional B cells (B220+ CD93+) was due to an expansion of transitional 1 (T1), but not T2 or T3, cells (Fig. 1A–C). Moreover, analysis of the mature B lymphocytes showed an increase in B1 cells (Fig. 1G–I). B1 cells were further subdivided into B1a and B1b cells, based on CD5 expression, showing that the increase in splenic B1 cells resulted from an expansion of B1a, but not B1b, cells (Fig. 1H, 1I). Surprisingly, no significant alterations in the peritoneal B1 cell populations were detected (data not shown). In conclusion, our results show that the absence of Ly9 promotes an expansion of innate-like B cells subsets in the spleen.
To gain insight into the nature of innate-like B cell expansion in the absence of Ly9, we decided to analyze the expression of various activation markers on splenic B cell subsets of Ly9−/− and WT mice, before and after BCR cross-linking. Ly9-deficient MZ and B1 B cells displayed higher expression of MHC-II in basal conditions, whereas Ly9−/− FO B cells presented normal levels (Supplemental Fig. 1A). These higher levels of MHC-II suggested that Ly9-deficient innate-like B cells had the potential to respond more potently to activating signals. However, no significant differences were seen in the viability and proliferation rate of Ly9-deficient B cells after 24 h of stimulation with anti-IgM or LPS (data not shown). Because BCR signaling strength influences the differentiation of transitional B cells into MZ or FO B cells, we examined signaling events downstream of the BCR in isolated MZ B cells from WT and Ly9−/− mice. Interestingly, Ly9-deficient MZ B cells expressed higher phosphorylation of Bruton’s tyrosine kinase (Btk) compared with WT cells, although this difference did not reach statistical significance (Supplemental Fig. 1B). After anti-IgM stimulation, upregulation of pBtk, pJNK, and pp38 in Ly9-deficient MZ B cells was similar to that observed in WT mice cells (Supplemental Fig. 1B), indicating that Ly9 absence does not significantly impair early BCR signaling events.
Because MZ B cells and B1a cells are known to be the major source of natural Abs in the absence of previous Ag contact, we assessed whether the serum levels of this Ab type were altered in Ly9-deficient mice. We tested the levels of IgM and IgG3, which are the predominant subclass of natural Abs, reactive with the Pneumo23 vaccine and phosphorylcholine. Pneumo23 vaccine is composed of a mixture of 23 purified capsular polysaccharides from Streptococcus pneumoniae. Our results show a significant increase of IgG3 natural Abs in Ly9-deficient mice compared with WT mice (Fig. 1J, 1K). No differences in IgM levels could be observed in either group of mice. Taken together, the data indicate that the presence of Ly9 prevents expansion of MZ and B1 cells.
Enhanced Ab responses against TI-2 Ags in Ly9-deficient mice
The observed alterations in B cell subsets in the absence of Ly9 prompted us to assess the role of Ly9 in the regulation of B cell function. To this end, we compared the response of WT and Ly9−/− mice with T-D (TNP31-KLH), TI-1 (TNP0.3-LPS), and TI-2 (TNP65-Ficoll) Ags. The absence of Ly9 did not impair Ab production against T-D or TI-1 Ags (Supplemental Fig. 2A, 2B). In contrast, TI-2 responses were enhanced in Ly9−/− mice. Upon TNP-Ficoll immunization, the serum of Ly9−/− BALB/c mice contained significantly higher levels of TNP-specific IgG2a, IgG2b, and IgG3 compared with those of their WT counterparts (Fig. 2). Similarly, serum anti-TNP IgG levels were significantly increased after TNP-Ficoll immunization of Ly9−/− C57BL/6 mice as compared with WT animals (data not shown). These data support the concept that the Ly9 receptor negatively regulates TI-2 B cell responses independently of the genetic background. Taken together, these data demonstrate that Ly9 functions as a negative regulator of TI humoral responses.
MZ architecture and Ag capture is not altered in Ly9 absence
MZ B cell retention and survival in the MZ is influenced by the structural framework and the survival signals delivered by MZ-related stromal and myeloid cells. We sought to determine whether the increased numbers of MZ B cells in Ly9−/− mice were due to alterations of the splenic MZ architecture caused by Ly9 absence. Thus, we analyzed the frequency and location of myeloid subsets and the expression of adhesion molecules in the spleens of Ly9−/− mice. Cell numbers of plasmacytoid dendritic cells (B220+ CD11c+), conventional dendritic cells (cDCs) (B220− MHC-II+ CD11c+), CD8+ cDCs (B220− MHC-II+ CD11c+ CD8+ CD11b−), and CD11b+ cDCs (B220− MHC-II+ CD11c+ CD8− CD11b+) were not altered in Ly9−/− spleens (Fig. 3A). Similarly, numbers of MOMA-1+ metallophilic macrophages and SIGN-R1+ MZ macrophages were normal in Ly9 absence (Fig. 3B). Tissue immunofluorescence analysis confirmed that Ly9−/− mice display a normal distribution of MOMA-1+ and SIGN-R1+ MZ macrophages (Supplemental Fig. 3A). Moreover, Ly9 deficiency did not lead to alterations in VCAM-1+ endothelial cells or ICAM-1+ marginal sinus cells in the MZ (Supplemental Fig. 3B).
Because Ly9-deficient mice display enhanced TI-2 Ab responses, we evaluated the ability of Ly9−/− MZ B cells and SIGN-R1+ macrophages to capture TI-2 blood–borne Ags. WT and Ly9−/− mice received an i.v. injection of TNP-Ficoll, and 30 min later, we examined TNP+ cell distribution. Immunofluorescence analysis of spleen sections stained with IgM, SIGN-R1, and TNP revealed that MZ B cells bound more TNP-Ficoll than did FO B cells, and TNP+ cells were located mostly in the MZ. The distribution of TNP+ cells in Ly9−/− mice was similar to that observed in their WT counterparts (Fig. 3C). To corroborate these observations, we performed FACS analysis to quantify Ag uptake by MZ B cells. Ly9-deficient cells were able to trap TNP-Ficoll as efficiently as WT MZ B cells did, as judged by TNP mean fluorescence intensity (MFI) (Fig. 3D, 3E).
Taken together, these data suggest that both the increased Ab response against TI-2 Ags and the expansion of MZ and B1 B cells observed in Ly9−/− mice are driven by a B cell–intrinsic mechanism triggered in Ly9 absence, rather than being a consequence of an altered splenic architecture.
Administering an mAb directed against Ly9 selectively depletes splenic MZ and B1 B cells
To further explore the role of Ly9 in the humoral immune response, we produced a mouse mAb directed against mouse Ly9 (Ly9.7.144) (Supplemental Fig. 4A, 4B). Upon administering this noncytotoxic IgG1 mAb, MZ B cells could not be detected in WT BALB/c mice (Fig. 4A–C). Second, 24 h after injecting anti-Ly9, the number of splenic B1 B cells was reduced by half (Fig. 4D–F). This depletion was selective, because anti-Ly9 did not affect other B lymphocyte subsets, for example, FO B cells, even though they express high levels of Ly9 (Supplemental Fig. 4C). The slight increase in the percentage of FO B cells was most likely due to the reduction in the proportion of MZ B cells (Fig. 4A–C). Further evidence for the specific effect of the Ly9.7.144 mAb came from the observation that its administration to Ly9−/− mice did neither alter MZ nor B1 B cell numbers (Fig. 4A, 4D). Consistent with the flow cytometry analyses, a significant reduction in IgM+ B cells located in the MZ was observed in spleen tissue sections of anti-Ly9–treated mice (Fig. 4G). Elimination of MZ B cells caused by Ly9.7.144 was further confirmed by the absence of CD1dhi CD23lo lymphocytes in the spleen (data not shown). Interestingly, no effect on B1 cells in the peritoneal cavity was observed (data not shown). Because CD21hi CD23lo cells were not detected in the peripheral blood at 4 and 24 h after administering anti-Ly9 (Fig. 4H, 4I), we reasoned that MZ B cells had been depleted rather than migrating into the bloodstream.
We further assessed the long-term effects of a single injection of 250 μg anti-Ly9 Ab. Twenty-six days after a single anti-Ly9 dose, the numbers of T1 transitional B cells were reduced. Higher numbers of T2 and T3 transitional B cells were found, concomitant with the decrease in T1 transitional B cells (Fig. 5A, 5B). MZ B cells were still absent after 26 d (Fig. 5C, 5D), and a significant reduction in the percentage and absolute number of B1 cells was also maintained (Fig. 5E, 5F).
Moreover, levels of total serum IgM and IgG decreased approximately 40% (Fig. 5G). Although B220int CD138hi plasma cells in the spleen were not affected, the number of plasma cells in the bone marrow was significantly reduced after this time (Fig. 5H). Thus, anti-Ly9 negatively regulates the number of innate-like B lymphocytes.
Pretreatment with anti-Ly9 reduces humoral responses
Because anti-Ly9 treatment caused a severe decrease in MZ and B1 cells, which are involved in TI Ab responses, we next analyzed the response to TNP65-Ficoll Ag in mice pretreated with Ly9.7.144 mAb. Injection of Ly9.7.144 caused a significant reduction in hapten-specific Ab production against TNP65-Ficoll. This treatment affected all tested isotypes except IgM (Fig. 6A).
Remarkably, anti-Ly9 administration also reduced the levels of anti-TNP IgG1, IgG2a, IgG2b, and IgG3 after immunization with T-D and TI-1 Ags (Fig. 6B, 6C). Because NKT cells have been shown to play a role in T-D responses, and we have previously reported that Ly9 influences NKT development in the thymus (17), we sought to assess whether the impaired Ab production against T-D Ags was due to the effect of Ly9.7.144 over NKT cells. To this end, we determined humoral response against TNP31-KLH in NKT-deficient CD1d−/− mice pretreated with either anti-Ly9 or a control Ab. Ly9.7.144 administration dampened the production of TNP-specific IgG1, IgG2b, IgG3, and IgM in CD1d−/− mice (Fig. 6D), demonstrating that Ly9 targeting is able to impair T-D responses independently of the presence of NKT cells. We conclude that anti-Ly9 mAbs are able to downmodulate Ag-induced Ab responses independently of Ag type.
In vivo anti-Ly9 treatment induces a downregulation in the expression of the CD19 coreceptor complex and several adhesion molecules
The observation that anti-Ly9 treatment had an effect on the T-D responses, which were not altered in Ly9-deficient mice, prompted us to analyze the consequence of Ab treatment on the remaining FO B cells. First, we assessed the expression of different cell-surface molecules on spleen B cells (B220+) after in vivo treatment with Ly9.7.144. Anti-Ly9 administration decreased surface protein levels of the B cell coreceptor complex formed by CD19, CD21, and CD81 (Fig. 7A, 7B). This significant reduction was selective, as the levels of other B cell surface proteins such as B220, IgM, and IgD were not significantly affected after treatment (Fig. 7A, 7B). Other cell-surface molecules such as CD1d, CD2, CD20, CD22, CD23, CD24, CD38, CD40 CD44, CD45, CD79b, and CD84 were not affected or only minimally altered (data not shown). These effects were detected 24 h after administering anti-Ly9 (Fig. 7A, 7B) and were maintained over 26 d after a single injection of Ab (data not shown). Ly9.7.144 also downmodulated the expression of several adhesion molecules such as CD62L, integrin β7, integrin α4, and CD18 on B220+ lymphocytes (Fig. 7C). In contrast, anti-Ly9 treatment did not alter the surface levels of other molecules involved in B cell adhesion and migration such as S1P1 or CXCR5 (Fig. 7C).
Impaired B cell survival, proliferation, and differentiation in anti-Ly9–treated mice
Because anti-Ly9 administration reduced the surface expression of the CD19 coreceptor complex, which is critical for B cell activation, we next sought to determine whether B cell viability, proliferation, and differentiation were impaired after treatment. To this end, 24 h after in vivo administration of Ly9.7.144 or an isotype control Ab, spleen cells were CFSE-labeled and cultured ex vivo over 72 h in the presence of LPS or anti-IgM. A significant reduction in cell viability was observed in all culture conditions, as judged by percentage of Live/Dead+ cells (Fig. 7D). The proliferation of anti-Ly9–treated B cells was significantly impaired, especially after LPS stimulation (Fig. 7E). Moreover, LPS-induced IgG production was reduced (Fig. 7F). Taken together, these data suggest that anti-Ly9 treatment has an important impact on B cell activation.
Ly9 Ab–mediated functions are Fc independent
In most cases the effects of mAbs are mediated by their Fc region through the interaction with complement or Fc receptors. To evaluate this possibility, we compared the effect on B cell subsets of anti-Ly9 mAb F(ab′)2 fragments with that of the intact Ab. As shown in Fig. 8, F(ab′)2 fragments were equally efficient in deleting MZ and B1 B cells (Fig. 8A–C), reducing TI-2 Ab responses (Fig. 8D), and downmodulating the CD19 coreceptor complex (Fig. 8E). These results exclude a role of complement activation or Ab-dependent cell-mediated cytotoxic reaction as a mechanism explaining these anti-Ly9 effects.
In this study, we provide evidence that the absence of the cell-surface receptor Ly9 induces a selective expansion of splenic MZ and B1 cells without affecting the development of conventional B cells or myeloid cells. These two subsets of innate-like B cells are primarily involved in rapid Ab production to TI-2 Ags. These Ags are largely associated with extrafollicular B cell responses and represent a powerful first line of defense against pathogens entering the spleen (3, 20). Consistent with this, Ly9-deficient mice showed an increased production of IgG2a, IgG2b, and IgG3 against TI-2 Ags (TNP65-Ficoll).
Mice deficient in several cell-surface molecules known to positively regulate BCR signaling, such as CD19 or the BAFF-R, present reduced MZ and B1 cells numbers (21–23). In contrast, the opposite phenotype, an increase in the MZ and B1 cell compartment and an enhanced response to TI Ags, is observed upon transgenic overexpression of these two stimulatory molecules (24, 25). However, little is known about the cell-surface molecules that negatively regulate the homeostasis of innate-like B cells. Mice deficient in cell-surface molecules that negatively regulate BCR signaling such as CD22, Siglec G, or CD72 do not show an expansion of MZ B cells (26, 27). These mice present alterations in the activation and development of conventional B cells or increased numbers of B1 cells in both spleen and peritoneum. This is clearly different from what we observed in mice lacking Ly9.
The regulation on innate-like B cell homeostasis is especially relevant as the activation of these cells requires a tight regulation because they have very low activation thresholds and are known to produce polyreactive Abs that are potentially self-reactive (28). Moreover, because these cells are potent responders to microbial Ag, they may also cause collateral tissue injury (29). Importantly, the expansion of MZ and B1 cells has been associated with autoimmune disorders and malignancy in both mice and humans (21, 30, 31). In the NOD mouse strain, splenic MZ B cell expansion correlates with the development of type 1 diabetes (32, 33). An enlarged MZ B cell pool is also found in different mouse models of Sjögren syndrome (29, 34–36). In BAFF transgenic mice, MZ-like B cells infiltrate the salivary glands and are thought to contribute to tissue damage (34). Consistent with these observations, we have recently reported that aged Ly9-deficient mice spontaneously develop features of systemic autoimmunity. In this study, we show that young Ly9-deficient mice presented a striking increase in natural Abs of the IgG3 isotype. Natural Abs, which recognize multiple conserved microbial epitopes, are mainly produced by MZ and B1a cells and present an important autoimmune potential (37).
MZ cell survival and Ab responses are influenced by their interaction with myeloid and stromal populations in the spleen. The frequency of main myeloid subsets and the distribution of MZ macrophages and adhesion molecules were not altered in Ly9 absence. Furthermore, Ly9−/− mice showed selectively increased Ab responses against TNP-Ficoll, which is able to trigger Ab production by B lymphocytes without T cell help. Collectively, these data support the idea that Ly9 restrains the size of MZ and B1 cell subsets and regulates the extent of TI-2 responses in a B cell–intrinsic manner. Nevertheless, further experiments will be required to confirm the B cell–autonomous nature of the innate-like B cell expansion and enhanced Ab production observed in Ly9−/− mice.
SLAMF receptors have been shown to modulate lymphocyte function by recruiting activating and inhibitory SH2 domain-containing proteins to ITSMs present in their cytoplasmic tails (6, 7, 38). In cells lacking SLAM-associated protein (SAP), such as B cells, tyrosine phosphatases can bind to the ITSMs and transmit negative activation signals (39). In this study, we hypothesize that the homophilic interactions of the Ly9 receptor established between adjacent B cells prevent the spontaneous activation of these innate-like B cells by recruiting SHP-2 and SHIP tyrosine-phosphatases (15, 16). These phosphatases are responsible for the negative signals transmitted by most inhibitory coreceptor molecules (40). Together, the phenotype of the Ly9-deficient mice suggests that Ly9 negatively regulates innate-like B lymphocyte activation and homeostasis.
We next decided to study the in vivo capacity of an mAb against Ly9 to affect innate-like B cell homeostasis and activation, because this could be a potential approach for treating B cell–related diseases. We found that injecting mice with Ly9 mAb resulted in the complete deletion of MZ B cells. It is not clear whether this Ab treatment leads to MZ B cell death or to their migration to other sites. We could not observe the migration of MZ B cells to the blood, as has been observed after treatment with anti-VLA4 mAbs (41). Moreover, the expression of S1P1 receptor and CXCR5, which are known to regulate MZ B cell localization in MZ and migration into follicles, was not altered in the anti-Ly9–treated mice (42, 43). We cannot completely exclude the possibility that these cells have changed their phenotype. However, this is highly unlikely, because the deletion of MZ B cells was confirmed using an alternative combination of markers.
Splenic B1 cell numbers were also reduced after Ab treatment, albeit to a lesser extent. However, the numbers of B1 cells in the peritoneum were not affected. Interestingly, the resistance of B cells in the mouse peritoneal cavity has also been reported during CD20 immunotherapy in mice (44). Reduced numbers of T1 cells could be observed only after prolonged anti-Ly9 treatment, but not 24 h after administration of the mAb. Notably, the B cell subsets that were eliminated or reduced by anti-Ly9 treatment (MZ, B1, and T1 cells) are the ones we found expanded in the Ly9-deficient mice. This supports the notion that Ly9.7.144 mAb has an agonistic effect. Furthermore, anti-Ly9 treatment diminished circulating levels of IgG and IgM, and this decrease was concomitant with a reduction in the numbers of bone marrow plasma cells.
A key finding of our study was that treating animals with Ly9 mAb reduced the in vivo production of Abs not only against TI-2 Ags, but also against TI-1 and T-D Ags. The reduction of T-D humoral responses was independent of the effect of Ly9.7.144 over NKT cells. This therefore demonstrates that Ly9 mAb treatment can reduce Ab production by both innate-like and conventional B cells. This is consistent with our observation that Ly9 treatment caused a reduction in B cell survival, proliferation, and differentiation. The ability of anti-Ly9 mAb to hinder humoral immune responses could be a promising therapeutic option in diseases involving the pathogenic production of Abs (45).
Analysis of a large panel of cell-surface molecules showed that anti-Ly9 treatment induced a fast and selective reduction of the CD19 complex expression, without significantly affecting the levels of molecules that constitute the BCR (IgM, IgD, and CD79b) or other B cell–associated molecules. This is a surprising result because other B cell–directed treatments, such as CD22, induce a reduction not only of CD19 and CD21, but also of IgM, CD79b, or CD20 (46). Moreover, the effect of CD22 Abs was mediated by an Fc/FcR-dependent membrane transfer from B cells to effector cells via trogocytosis, whereas the reduction of the CD19 complex induced by anti-Ly9 injection is not only more selective, but also Fc independent (46). The observation that the anti-Ly9–mediated effects are Fc independent also excludes the possible participation of complement system or Ab-dependent, cell-mediated cytotoxic reaction in the observed phenotype. Although more experiments are required to explain the mechanism of action of anti-Ly9 Abs, a downmodulation in the expression of the CD19 complex may represent the main molecular mechanism responsible for the alterations in B cell subsets and humoral response deficiency observed in the treated mice. No alteration in the expression of the CD19 complex could be observed in the Ly9-deficient mice. Thus, we hypothesize that the wide-ranging effect of the anti-Ly9 as compared with the phenotype of the Ly9-defcient mice is potentially due to an impairment in the CD19-dependent signaling that affects the function of the conventional B cells. The CD19 complex is critical for B cell activation and the homeostasis of MZ and B1 cells by modulating signal transduction through the BCR or by inducing survival tonic signals (47). Moreover, elevated CD19 has been correlated with susceptibility to autoimmunity in both mice and humans (48, 49).
In contrast with B cell Ab depletion therapies, inhibition of signaling pathways and selective elimination of MZ and B1 cells with minimal reduction of the conventional B cell compartment should be regarded as a promising new therapeutic option.
We thank A. Lázaro and I. Azagra for technical assistance.
This work was supported by Ministerio de Educación y Ciencia Grant SAF2012-39536 (to P.E.), National Institutes of Health Grant PO1 AI 065687 (to C.T. and P.E.), and Ministerio de Educación, Cultura y Deporte Grant AP2010-1754 (to M.C.).
The online version of this article contains supplemental material.
Abbreviations used in this article:
Bruton’s tyrosine kinase
conventional dendritic cell
immunoreceptor tyrosine-based switch motif
keyhole limpet hemocyanin
mean fluorescence intensity
MHC class II
specific intracellular adhesion molecule-grabbing nonintegrin receptor-1
signaling lymphocytic activation molecule
sphingosine 1-phosphate receptor 1
TI type I
TI type II
The authors have no financial conflicts of interest.