Growth-Factor Receptor-Bound Protein-2 (Grb2) Signaling in B Cells Controls Lymphoid Follicle Organization and Germinal Center Reaction

Growth-factor receptor-bound protein-2 (Grb2) signaling in B cells controls lymphoid follicle organization and germinal center reaction

Ihn Kyung Janga,1,2, Darran G. Cronshawb,2, Luo-kun Xieb, Guoqiang Fangb, Jinping Zhanga,3, Hyunju Oha, Yang-Xin Fuc, Hua Gua,4,5,6, and Yongrui Zoub,4,5

aDepartment of Microbiology and Immunology, Columbia University, New York, NY 10032; bThe Feinstein Institute for Medical Research, North Shore-Long Island Jewish Health System, Manhasset, NY 11030; and cDepartment of Pathology, University of Chicago, Chicago, IL 60637

Edited* by Klaus Rajewsky, Immune Disease Institute, Boston, MA, and approved March 24, 2011 (received for review November 4, 2010)

Grb2 (growth-factor receptor-bound protein-2) is a signaling adap- network and GC formation (15). Currently, how the CXCR5 signal tor that interacts with numerous receptors and intracellular sig- is connected to LT expression still remains elusive. naling molecules. However, its role in B-cell development and The mammalian growth-factor receptor-bound protein-2 (Grb2) fl function remains unknown. Here we show that ablation of Grb2 is a simple adaptor that consists of one central SH2 domain anked in B cells results in enhanced B-cell receptor signaling; however, by two SH3 domains (16). It is broadly expressed in many tissues and is essential for embryo development and multiple cellular mutant B cells do not form germinal centers in the spleen after – fi antigen stimulation. Furthermore, mutant mice exhibit defects in functions (17 19). Grb2 has been identi ed as a major mediator in splenic architecture resembling that observed in B-cell–specific Ras-MAPK activation induced by numerous receptors because of lymphotoxin-β–deficient mice, including disruption of marginal its association with son of sevenless, a GDP-GTP exchange factor − − for Ras (18–20). Studies using T-cell–specificGrb2-deficient mice zone and follicular dendritic cell networks. We find that grb2 / B show that Grb2 plays a broad role in early T-cell development as cells are defective in lymphotoxin-β expression. Although lympho- both thymic-positive and -negative selection are impaired in the toxin can be up-regulated by chemokine CXCL13 and CD40 ligand absence of Grb2. Grb2 is a positive regulator for TCR signaling, and stimulation in wild-type B cells, elevation of lymphotoxin expression −/− it does not directly affect the activities of Ras and Erk1/2, but rather in grb2 B cells is only induced by anti-CD40 but not by CXCL13. Our amplifies the activity of Lck, a member of the Src family of tyrosine fi results thus de ne Grb2 as a nonredundant regulator that controls kinases that functions at the top of the TCR signaling cascade (21). lymphoid follicle organization and germinal center reaction. Loss of In chicken B cells, it has been reported that Grb2 regulates Ca2+ Grb2 has no effect on B-cell chemotaxis to CXCL13, indicating that influx upon BCR activation (22). Mice deficient in the hemato- Grb2 executes this function by connecting the CXCR5 signaling poietic adaptor protein downstream of kinase-3 (Dok-3), an pathway to lymphotoxin expression but not to chemotaxis. adaptor that recruits Grb2 to the membrane, exhibit enhanced BCR-induced Ca2+ mobilization (23, 24), suggesting that Grb2 may B-cell signaling and activation | follicular dendritic cell development | participate in BCR signaling also in mammalian B cells (for review, germinal center development | regulation of lymphotoxin expression | see refs. 18 and 19). However, the physiological role of Grb2 in B- chemokine signaling cell development and function remains unclear. Here we show that − − Grb2 negatively regulates BCR signaling. grb2 / B cells are hy- erminal center (GC) reaction is critical for effective humoral perreactive; however, mutant splenic B cells did not form GCs. We find that Grb2 is necessary for CXCL13-induced LT expression in B Gimmune responses and for the generation of memory B lym- fi phocytes. This process requires close interactions between antigen- cells. Our ndings establish the CXCL13/CXCR5-Grb2-LT sig- specific B and T cells, and follicular dendritic cells (FDCs) (1–3). naling axis in B cells as a nonredundant pathway that controls Multiple B-cell intrinsic signals are involved in orchestrating this lymphoid follicle organization and GC reaction. process. The signal that initiates GC formation is delivered through Results the B-cell receptor (BCR), with a group of signaling proteins BKO modulating the strength of BCR signaling and immunological Grb2 Mutation Impairs B-Cell Maturation and Enhances B-Cell synapse formation (4–6). These proteins include Lyn, SHP1, and Responses to Activating Stimuli. To determine the function of – fi DOCK8, as well as the coreceptor CD19, which collectively drive Grb2 in B cells, we generated B-cell speci c grb2 knockout B-cell activation, proliferation, and differentiation to either memory B cells or antibody-producing plasma cells in GCs (7–9). In addition to BCR signaling, signals derived from the surrounding Author contributions: H.G. and Y.Z. designed research; I.K.J., D.G.C., L.-k.X., G.F., J.Z., and niche induce activated B cells to undertake the GC differentiation H.O. performed research; Y.-X.F. contributed new reagents/analytic tools; I.K.J., D.G.C., pathway and support them to complete the GC maturation pro- H.G., and Y.Z. analyzed data; and H.G. and Y.Z. wrote the paper. gram. B-cell–expressing molecules that are involved in this process The authors declare no conflict of interest. include ICOSL, CD40, PD-1 ligands, and cytokine receptors (10– *This Direct Submission article had a prearranged editor. 13). However, how these signaling pathways converge in B cells to Freely available online through the PNAS open access option. elicit an optimal GC response is not known. 1Present address: Department of Cancer Biology, The Scripps Research Institute, Jupiter, Only a small fraction of antigen-activated B cells will differen- FL 33458. tiate into GC cells. It is not clear what selects a B-cell to commit to 2I.K.J. and D.G.C. contributed equally to this work. the GC fate, although it is suggested that B cells compete for 3Present address: Institute of Biology and Medical Sciences, Soochow University, Soochow, antigens, cognate T-cell help, and for other growth and survival Jiangsu 215123, China. signals from the specialized follicular microenvironment to estab- 4H.G. and Y.Z. contributed equally to this work. lish GCs. Thus, the precise localization of B cells during immune 5To whom correspondence may be addressed. E-mail: [email protected], yzou@nshs. responses is decisive for GC maturation. Chemokine receptors edu, or [email protected]. CXCR5 and CXCR4, lymphotoxin (LT), and TNF play a crucial 6Present address: The Clinic Research Institute of Montreal, Montreal, QC, Canada role in organizing B-cell follicles and GCs (14). Of a particular W2H 1R7. interest, it has been shown that activation of CXCR5 induces naive This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. B cells to produce LT that is required for establishing the FDC 1073/pnas.1016451108/-/DCSupplemental.

7926–7931 | PNAS | May 10, 2011 | vol. 108 | no. 19 www.pnas.org/cgi/doi/10.1073/pnas.1016451108 Downloaded by guest on October 1, 2021 (Grb2BKO) mice by crossing grb2 floxed mice to CD19-Cre hanced proliferation in response to anti-IgM or anti-IgM plus transgenic mice (Fig. S1) (21). Grb2 deficiency did not perturb B- anti-CD40 (twofold), or IL-4 plus anti-CD40 (threefold) (Fig. cell development at an early stage (Table S1 and Fig. S2), but 1B). When B cells were stimulated with LPS, proliferation impaired further maturation of B cells after immature B cells responses of both mutant and WT B cells were drastically in- egressed from the bone marrow (BM) into the spleen. The pe- creased, and even then, mutant B cells showed stronger proliferation (Fig. 1B). These same stimuli also induced increased ripheral transitional T1 and T2 B cells in mutant mice were sig- − − nificantly reduced compared with that of WT mice (Fig. 1A and proliferation in grb2 / immature B cells, even though responses Table S1). As a result, follicular B cells declined to ∼70% of the of both WT and mutant immature B cells to these stimuli were number in WT mice (Fig. 1A and Table 1), and the subset of overall much lower than that of mature B cells (Fig. S3). − mature recirculating B cells (B220hiIgM+/ ) in the mutant BM Next, we sought to determine which BCR signaling event was was only 40% of the WT counterpart (Table S1 and Fig. S2). We interfered with by Grb2 deficiency. BCR stimulation activates the noted that the populations of marginal-zone B cells and B1 B cells tyrosine kinase Lyn, which then phosphorylates immunoreceptor α β were only mildly altered in the spleen of mutant mice. These tyrosine-based activation motifs (ITAM) in Ig and Ig . The results indicate that Grb2 exerts a differential regulatory role in B- phosphorylated ITAMs recruit and activate Syk kinase, leading to cell lineage development, with follicular B cells being most pro- subsequent tyrosine phosphorylation of signaling effector mole- BKO cules, including BLNK/p65 and PLCγ2 (25). We first examined foundly affected by the Grb2 mutation. fi To evaluate the role of Grb2 in B-cell activation, we compared whether Grb2 de ciency affected proximal BCR signaling. We −/− found that while the kinetics and the level of tyrosine phosphor- the proliferative responses of grb2 and WT B cells to various −/− activating stimuli. We found that mature B cells exhibited en- ylation of total proteins in grb2 and WT B cells were comparable, the active form of Lyn phosphorylation in mutant B cells was slightly reduced than that of WT B cells after anti-IgM stimulation (Fig. S4). When distal signaling transduction molecules were examined, we found that phosphorylation of PLCγ2, activation of MAP kinases Erk1/2, p38, and JNK, and mobilization of − − Ca2+ were all significantly elevated in grb2 / B cells (Fig. 1 C and D). These data indicate that Grb2 is a negative regulator and regulates BCR signaling at the top of the BCR signaling cascade. How does Grb2 negatively regulate BCR signaling? It has been previously shown that Grb2 is associated with CD22, a membrane receptor that negatively regulates BCR signaling (26). Upon BCR stimulation, CD22 becomes tyrosine phosphorylated at its cytoplasmic tail by Lyn, which then recruits tyrosine phosphatase SHP-1 to the membrane to suppress BCR downstream signaling molecules, including BLNK and Igα/Igβ (27). Therefore, we examined this negative regulatory circuit of BCR signaling. We found that tyrosine phosphorylation of CD22 in the mutant B cells was reduced compared with that occurring in WT cells (Fig. 1E). These results thus indicate that Grb2 is involved in the regulation of CD22 tyrosine phosphorylation, and suggest that Grb2 may control BCR signaling by integrating the CD22-SHP-1 negative- feedback loop in B cells.

Grb2BKO Mice Have Disrupted Lymphoid Follicles and Cannot Form GCs in the Spleen. To directly assess the impact of Grb2 deﬁciency on T-cell–dependent (TD) antibody responses, we immunized mice with a TD antigen NP-KLH (4-hydroxy-3- nitrophenylacetyl–keyhole limpet hemocyanin). At day 10 after immunization, WT B cells had undergone extensive clonal ex- pansion and some of the antigen-activated B cells differentiated into GC cells (28). Grb2BKO mice generated an equivalent number of BrdU+ B cells in the spleen during 4-h BrdU pulse, even though they have ∼40% fewer mature B cells compared with controls. Thus, proliferating B cells were proportionally increased ∼twofold in Grb2BKO mice (Fig. 2A). Although mutant B cells

− − responded strongly to antigen stimulation, these cells did not give Fig. 1. Altered development and antigen-receptor signaling of grb2 / B rise to GC cells (Fig. 2B). We further analyzed the antigen-specific + BKO cells. (A) Dot plots show B220 -gated splenic cells of WT and Grb2 mice. B-cell compartment for the generation of IgG1lowCD38low GC (Top) Mature (AA4.1loCD24lo) and immature (AA4.1hiCD24hi) B cells. (Middle) high + hi hi lo lo and the IgG1 CD38 memory B cells (29, 30) and found that transitional T1 (CD21 CD23 ) and T2 (CD21 CD23 ) B cells within the both subsets were reduced in the spleen of the mutant mice (Fig. AA4.1hiCD24hi-gated immature B-cell population. (Bottom) marginal-zone B hi lo hi hi lo lo 2C). As a consequence of the enhanced antigen-activated B-cell (CD21 CD23 ), follicular B (CD21 CD23 ), and B1 B (CD21 CD23 ) cells proliferation but an impaired GC reaction, Grb2BKO mice gen- within the AA4.1loCD24lo-gated mature B cells. Percentages of each subset + lo lo hi erated more NP-specific IgM-producing and fewer IgG-producing are indicated in the plots. (B) Purified B cells (B220 AA4.1 CD24 CD21 IMMUNOLOGY hi plasma cells in the spleen (Fig. 2D). CD23 ) were stimulated with anti-IgM F(ab)2, anti-CD40, IL-4, and LPS either alone or in combination as indicated. Cell proliferation was measured by 3H- To determine whether the lack of GC responses impaired long- 2+ term humoral immunity, we tested primary and recall responses thymidine incorporation; error bars with SD (n = 3). (C) Intracellular Ca in B BKO fl to TD antigens in Grb2 and WT mice. When the serum level cells was measured by uorescence intensity of Fluo-4 vs. Fura-red (n = 8). (D fi and E) Purified B cells from WT or Grb2BKO mice were stimulated with anti- of NP-speci c antibodies was measured at different time points IgM for various periods. Tyrosine phosphorylation of PLCγ2 and CD22 was during the primary immune response, we found that the NP- determined by immunoprecipitation followed by Western blotting against specific IgM titer was slightly higher in the mutant mice compared phosphotyrosine (4G10). Active forms of Erk1/2, JNK, and p38 were directly with that of controls (Fig. S5A). To our surprise, anti-NP re- determined using specific antibodies against individual phosphorylated sponses of other Ig isotypes including IgG1, IgG2b, IgG2c, and kinases. The results represent more than three independent experiments. IgG3 were unaffected by the loss of Grb2 (Fig. S5A). Generation

Jang et al. PNAS | May 10, 2011 | vol. 108 | no. 19 | 7927 Downloaded by guest on October 1, 2021 Fig. 3. Defective spleen architecture and GC reaction in Grb2BKO mice. Ten days after SRBC immunization, sections of WT and Grb2BKO spleens and mesenteric lymph nodes were prepared for immunofluorescence histological analysis. (A and B) Spleen sections at 4× magnification. (C and D) Lymph- node sections at 10× magnification. (E) Marginal zones in the spleen of WT and Grb2BKO mice are highlighted by MOMA-1 staining (red). B-cell follicles (B220+) and T-cell (CD4+, CD8+) zones are shown in blue and green, respectively. Fig. 2. Impairment of GC reaction in Grb2BKO spleen. Data were obtained from WT and Grb2BKO mice (8- to 10-wk-old) at day 10 after NP-KLH im- − munization. (A)(Left) Contour plots show BrdU+IgD+ and BrdU+IgD pop- works in the spleen, further inspection of the spleen follicles ulations of the B220+-gated cells. (Right) Frequencies and absolute numbers revealed that the marginal zone was also severely disrupted in of BrdU+ B cells in WT (white bars) and Grb2BKO (gray bars) mice are shown Grb2BKO mice (Fig. 3E). This result thus indicates that Grb2 is as mean with SD (n = 7). (B) Plots are B220+-gated splenocytes. GC B cells are responsible for general organization of the spleen follicles. shown as either CD95+PNA+ population in contour plots or CD95+GL7+ cells in dot plots. (C) Splenocytes were stained with lineage markers (CD4, CD8, Naive grb2−/− B Cells Are Deficient in LTβ Expression. The pheno- Gr1, F4/80), anti-B220, NP, and anti-CD38. (Upper) B220 vs. NP staining BKO − types of Grb2 mice, such as defective marginal zone, dimin- of gated-lineage marker negative (Lin ) splenocytes. The percentages of − + + + ished FDC networks, and failure of GC formation in the spleen Lin NP-binding B220 cells are indicated. The frequencies of IgG1 CD38 but unaffected lymph-node architecture, bear a striking re- memory (Mem) and IgG1+CD38lo GC subsets within the antigen specific – fi β fi – + + semblance to that of B-cell speci cLT -de cient mice (31 33). (B220 NP ) B-cell compartment are shown in the dot plots (n = 7). (D) We hence decided to examine whether Grb2 was required for the Numbers of NP-specific IgM and IgG secreting plasma cells in the spleen of β WT (white bars) and Grb2BKO (gray bars) mice. Each symbol represents a expression of LT and other immediate members of the TNF family. Quantitative RT-PCR analysis shows that although mean value of triplicate samples of an individual mouse. − − grb2 / and WT naive B cells (IgM+ IgD+) had comparable levels of LTα and TNF-α transcripts, LTβ expression was clearly de- of high-affinity anti-NP antibody occurred normally in mutant fective in the mutant B cells compared with the WT counterparts mice (Fig. S5B), despite the apparent lack of splenic GCs. The (Fig. 4A). When cell-surface LT was examined by a soluble form β mutant mice also elicited normal recall responses (Fig. S5C), of the LT receptor, LT R-Ig, which binds to the membrane-anchored LTα1β2, we could not detect LT on the cell surface of suggesting that the development of memory B cells and long-lived −/− plasma cells is intact in Grb2BKO mice. grb2 B cells (Fig. 4B). These results demonstrate that Grb2- Our serological findings that Ig class switch and antibody-affinity mediated signaling is critical for LT expression in naive B cells. maturation occurred normally in Grb2BKO mice in the absence of splenic GCs suggested that these processes might take place in Grb2 Connects CXCR5 Signaling to LT Expression. Previous studies − − other tissues than in the spleen, or grb2 / GC B cells could not have shown that CXCR5 signaling is essential for the expression express the characteristic cell-surface markers. We, therefore, of LT on naive B cells to control the formation of lymphoid examined the GC structure in both spleen and lymph nodes by follicles (15), whereas CD40L and other stimulatory molecules immunohistology with anti-CD35 that highlights FDC clusters in further up-regulate LT expression on activated lymphocytes the light zone of the GC. At day 10 after immunization with sheep during immune responses (34, 35). To determine whether Grb2 red blood cells (SRBC), WT mice formed large GCs in both the connected signaling from CXCR5 or CD40 to LT up-regulation spleen and lymph nodes (Fig. 3 A–D), visualized as PNA+ B-cells in B cells, we examined LT expression after stimulating splenic B and CD35+ FDC clusters. In contrast, neither PNA+ B cells nor cells with the CXCR5 ligand CXCL13 or antibody to CD40. We found that anti-CD40 treatment drastically up-regulated LT ex- FDC networks were detected in the spleen of mutant mice (Fig. 3 − − pression on both WT and grb2 / B cells (Fig. 4C). In contrast, A and B). Interestingly, the GC structure was intact in lymph nodes − − of Grb2BKO mice (Fig. 3 C and D). These data demonstrate that B- CXCL13 induced LT expression only in WT but not in grb2 / cell–specific deletion of grb2 only affects GC formation locally in B cells (Fig. 4C and Fig. S6), demonstrating that Grb2 is an the spleen. The observed Ig switch recombination and antibody- essential adaptor downstream of the CXCR5 signal-transduction affinity maturation in mutant mice can be attributed to normal GC pathway leading to LT expression. It is important to note that − − reaction in lymph nodes. In addition to the absence of FDC net- chemotaxis of grb2 / B cells to either CXCL13 or CXCL12

7928 | www.pnas.org/cgi/doi/10.1073/pnas.1016451108 Jang et al. Downloaded by guest on October 1, 2021 Fig. 5. Restoration of GC development of Grb2BKO B cells by WT B cells in BM chimeras. At day 10 after SRBC immunization, splenocytes from WT and Grb2BKO BM chimeras were examined for the generation of GC cells. Grb2BKO (CD45.2+) and WT (CD45.1+) B cells were gated, respectively, and displayed as contour plots for GC B cells (CD95+PNA+). (A) BM chimeras with mixed WT and Grb2BKO donor cells. (B) BM chimeras with only Grb2BKO donor cells. n =4.

mice is responsible for the defects of splenic architecture and GC reaction caused by Grb2 deficiency. Fig. 4. Regulation of CXCR5 signaling and LT expression by Grb2. (A) IgM+IgD+ naive splenic B cells were purified from WT and Grb2BKO mice by Discussion FACS. Transcripts of LTα,LTβ, and TNF-α were quantified by qRT-PCR and are Grb2 has been asserted to be a key adaptor for multiple cellular β shown as relative units after being normalized to the expression of -actin in functions by virtue of its physical association with a variety of the corresponding samples; P < 0.005, n =5.(B) Splenocytes were stained with anti-B220 and LTβR-Ig (n = 7). The percentages of cells within each quadrant are indicated in the plots. (C) Splenocytes were rested on ice for 8 h to disengage the chemokine signaling. Cells were then stimulated with either 3 μg/mL of CXCL13 or 10 μg/mL of anti-CD40 at 37 °C for 16 h and then subjected to LTβR-Ig and anti-B220 staining. Shown are percentages of LT- positive (B220+LTβR-Ig+) B cells in a B220+-gated population. n =4.

stimulation was normal (Fig. S7). These results indicate that Grb2 speciﬁcally controls a branch of CXCR5 signaling in B cells that induces LTβ transcription but not chemotaxis.

− − grb2 / B Cells Are Not Intrinsically Deficient in Differentiation into − − GC Cells. The above results let us posit that grb2 / B cells may undergo GC differentiation if the positive-feedback loop of LT and chemokines were restored in the spleen of Grb2BKO mice by WT B cells. To test this hypothesis, we established BM chimeras by cotransplanting an equal number of WT (CD45.1+) and Grb2BKO (CD45.2+) BM cells into lethally irradiated B6.SJL mice (CD45.1+) and examined GC formation after immunization. In this experimental setting, B cells derived from WT BM stem cells should express LT and, therefore, were expected to restore the splenic FDC networks. We found that indeed, in chimeras that received both WT and Grb2BKO BM cells, splenic − − GC cells contained not only WT but also grb2 / B cells (Fig. 5A). In contrast, no GC population could be found in the spleen of mice receiving only Grb2BKO BM cells (Fig. 5B). This finding − − thus indicates that grb2 / B cells are not intrinsically defective in GC differentiation. The GC defect caused by the Grb2BKO mutation can be rectified by the presence of WT B cells that presumably provide the necessary LT signals for the genesis of FDC networks and pattern formation of lymphoid follicles.

Enforced LTβ Expression Restores Splenic Defects in Grb2BKO Mice. To further test whether enforced expression of LTβ in Grb2BKO IMMUNOLOGY mice is sufficient to restore the FDC networks and GC reaction, BKO Fig. 6. Enforced LTβ expression restores GC reaction and FDC networks in we reconstituted Grb2 BM cells with either murine stem cell BKO BKO β the spleen of Grb2 mice. WT and Grb2 BM cells were transduced with virus (MSCV) or LT expressing MSCV retroviral vector and MSCV empty (MSCV) or LTβ-expressing (MSCV-LTβ) vector and transplanted generated BM chimeras. At day 11 after immunization, we ex- −/− fl into irradiated RAG-1 recipients. Two months after transplantation, BM amined GC B cells by ow cytometry and spleen architecture by chimeras were immunized with SRBC. (A)(Upper) Enforced LT expression in immunofluorescent staining. As shown in Fig. 6, both GCs and WT and Grb2BKO splenocytes. (Lower) B220+-gated plots. The percentages FDC networks were fully restored in the BM chimeras trans- of GC B cells are indicated (n = 4). (B) Spleen sections from WT and Grb2BKO duced with LTβ-MSCV but not with empty MSCV vector. This BM chimeras were stained with anti-B220, -CD35 together with anti-CD4 BKO result clearly shows that impaired LTβ expression in Grb2 and -CD8 antibodies.

Jang et al. PNAS | May 10, 2011 | vol. 108 | no. 19 | 7929 Downloaded by guest on October 1, 2021 receptors and their downstream signaling molecules. However, organization and a diminishment—but not absence—of GC re- its function in the immune system has not been fully character- action in the spleen (15, 39, 40). In vitro data have shown that ized. While Grb2 positively regulates TCR signaling, it acts as chemokines other than CXCL13 can also up-regulate LT ex- a negative regulator in B-cell activation. Although the detailed pression in naive B cells (15). It is therefore likely that ablation mechanism by which Grb2 negatively regulates BCR signaling is of Grb2 in B cells disconnects signaling from all chemokine still unclear, our observation of a reduction of CD22 phos- receptors to LT expression (41), hence completely eliminating −/− phorylation in grb2 B cells after BCR stimulation may provide splenic FDC networks and GCs. an explanation. CD22 is an inhibitory receptor for BCR signal- Grb2BKO mutation selectively affects humoral immunity in the ing. It has been shown that Lyn phosphorylates CD22 after BCR spleen but not lymph nodes. It remains unclear whether such stimulation. Phosphorylated CD22 then recruits the tyrosine a differential regulation has any biological impact on immunity phosphatase SHP-1 to the CD22/BCR complex to exert sup- against pathogens or autoantigens. It is generally believed that 2+ fl pressive effects on Ca ux and MAPK activation. Thus, it is lymph nodes normally mediate immunity against pathogens from likely that Grb2 is required for strengthening the Lyn-CD22- their draining peripheral tissues. In contrast, the spleen encounters SHP-1 negative-feedback loop. Our data showing that the active mostly blood-borne antigens. Interestingly, B-cell–specificLTβ- form of Lyn was slightly reduced in the mutant B cells is con- deficient mice, which have normal lymph nodes but a similar defect sistent with this notion. in the splenic architecture as Grb2BKO mice, fail to develop ef- It has been shown that a strong BCR signal in immature B fective humoral responses against a low dose of vesicular stomatitis cells may lead to apoptosis, a mechanism being used to eliminate virus (33). In this regard, splenic GC reaction may play a more autoreactive B cells. Notably, Grb2BKO mice show about 60% profound role in immune responses against those blood-borne loss of immature B cells at T1 and T2 stages, despite normal B- pathogens. In addition to immunity against pathogens, we have cell genesis. Because Grb2 deficiency enhances BCR signaling BKO without affecting BAFF dependent survival of B cells, it is pos- noticed that Grb2 mice produce high titers of anti-dsDNA IgM − − sible that a strong BCR signal in grb2 / immature B cells and have a high incidence of antibody deposits in kidneys, but assumes negative selection and purge ∼60% of immature B cells do not develop lupus-like nor other autoimmune diseases. It is in the periphery of Grb2BKO mice. Alternatively, it is also likely therefore worthwhile to examine whether lack of splenic GC that the splenic microenvironment of Grb2BKO mice is less function in these mice may attenuate autoimmune responses. − − supportive for grb2 / B cells. Consistent with this idea, we ob- In summary, our data demonstrate that Grb2 plays an important −/− role in B cells via integrating various signaling transduction pathways served that grb2 B cells were preferentially increased com- BKO pared with the WT counterpart in Grb2BKO and WT mixed BM of the BCR and CXCR5. Our Grb2 mice thus provide a useful chimeras (Fig. S8), suggesting that the presence of WT B cells model to further dissect the signalosomes downstream of these may correct the defective spleen environment (such as the lack of receptors. Importantly, we additionally reveal a Grb2-containing FDCs and perhaps also other accessory cells). More experiments CXCR5 signalosome, distinct from those required for chemotaxis, are needed to clarify this issue. which regulates LT expression. In this regard, targeting Grb2 may While progressing through the T2 stage, maturing B cells migrate differentially modify chemokine receptor signaling and immune to and reach the B-cell follicles. After encountering antigens and responses to achieve more selective therapeutic goals. with T-cell help, mature follicular B cells initiate GC reaction and develop into either memory B cells or long-lived plasma cells. While Materials and Methods much progress has been made in the past decades, the mechanism Mice, Adoptive Transfer, and Immunization. grb2-floxed (grb2fl) mice, gener- that governs B cells into these two developmental pathways has ated in our laboratory, have been back crossed to C57BL/6 for more than 12 not been fully understood. It is surprising that Grb2 deficiency in generations, and further crossed to CD19-Cre transgenic mice to generate B- B cells abrogates GC formation in the spleen, even though mutant cell–specific grb2 knockout (grb2fl/fl CD19-Cre heterozygous) mice, termed B cells respond to antigens vigorously. Our further studies dem- here as Grb2BKO mice. Grb2BKO mice were healthy with no obvious abnor- onstrate that Grb2-mediated signaling is not involved in dictating mality. To generate BM chimeras, 106 BM cells from WT B6.SJL and Grb2BKO the GC-cell fate of antigen-activated B cells; rather, it delivers mice were injected intravenously, either alone or at 1:1 ratio, into 750 Rad- signals from CXCR5 to induce expression of LT, a well-known irradiated B6.SJL recipients (The Jackson Laboratory). To immunize mice

lymphokine that is essential for the development of proper lym- with TD antigens, 50 μgofNP30-KLH (Biosearch Technologies) precipitated in phoid follicle structures, including the marginal zone, FDC net- Imject Alum (Pierce) were injected into mice intraperitoneally. To immunize − − works, and GCs. Indeed, grb2 / B cells are able to form GCs when mice with SRBC, 200 μL of PBS-washed 10% SRBC were injected into each coexisting with WT B cells, suggesting that the CXCR5-Grb2-LT mouse intraperitoneally. All animals were housed in the speciﬁc pathogen- signaling axis in WT naive B cells is required for laying out func- free barrier facility at Columbia University in accordance with institution tional spleen architecture that accommodates mutant B cells to approved protocols. − − undergo the GC maturation program. Because grb2 / B cells can form GCs, our results thus indicate that reverse signaling from the Biochemistry and Antibodies. For Western blot analysis, 50 to 100 μg of total membrane-anchored LT, if any, is not required for the GC reaction. proteins were loaded and size-fractionated on a 12% SDS-PAGE gel by Why is the defective development of FDC and GC conﬁned to electrophoresis and transferred to a PVDF membrane. For immunoprecipi- BKO the spleen but not to lymph nodes of Grb2 mice? One ex- tation, 1 to 2 μg of antibody and 50 μL of protein G agarose beads was planation for such a phenotype is that deletion of Grb2 in B cells added to total cell lysate from 2 to 5 × 106 cells and rotated at 4 °C over- impairs LT expression only in B cells but not in other lineages of night. After washing, proteins were denatured by boiling and fractionized cells. It is known that different secondary lymphoid organs re- on 12% SDS-PAGE gel. Immunobloting was performed according to our quire different LT- and LTβR-expressing hematopoietic and previous protocol (42). The following antibodies were used for biochemical

stromal cells for proper organogenesis. For example, the archi- study: anti-IgM F(ab)2 (Biosource); anti-phsphotyrosine (4G10) (Upstate Bio- tecture of the spleen but not of lymph nodes relies on B-cell– tech), anti–p-Erk, anti-Erk1/Erk2, anti-pJNK, anti-JNK, anti-p38, anti-Grb2, derived LT (31–33). On the other hand, LT produced by lym- and anti-PLCγ2 (Santa Cruz); and anti–p-p38 (Biosource). phoid tissue-inducer cells during days 11 to 16 gestation is pivotal for lymph node development (36, 37); however, lack of lymphoid In Vitro 3H-Thymidine Incorporation Assay. Mature (B220+AA4.1loCD24lo) tissue-inducer cells did not affect the formation of splenic B-cell splenic B cells were puriﬁed by FACS sorting, and seeded in triplicates into follicles (38). We will be interested to determine whether Grb2 a 96-well plate at 5 × 104 cells per well, in the presence of goat anti-IgM F

also functions in other cell lineages to control LT expression for (ab)2 (Organon Teknica Corp.; 10 μg/mL), anti-IgM F(ab)2 plus anti-CD40 (BD the formation of lymphoid follicles and GCs in lymph nodes. It Pharmingen; 10 μg/mL), or IL-4 (Biosources; 20 U/mL), anti-CD40 plus IL-4, or should also be noted that although our results establish Grb2 as LPS (Sigma-Aldrich; 30 μg/mL). Forty-eight hours later, 3H-thymidine (1 μCi) an essential integrator of CXCR5 signaling pathways, there are was added to each well and incubated for an additional 6 h. Cells were BKO phenotypic differences between Grb2 mice and CXCL13- or harvested on a cell harvester and 3H-thymidine incorporation was measured CXCR5-mutant mice, as the latter show more distorted follicular on a β-counter.

7930 | www.pnas.org/cgi/doi/10.1073/pnas.1016451108 Jang et al. Downloaded by guest on October 1, 2021 Flow Cytometry and BrdU Labeling. Single-cell suspensions were prepared Transwell Assay. Total splenic cells were suspended in DMEM con- from total BM, spleen, or mesenteric lymph nodes. After staining, cells were taining 10% FCS (107 cells/mL). Cells (100 μL) were loaded into the upper analyzed on a BD LSR II. The following antibodies are used for the staining: chamberofa3-μM pore-size transwell (Corning Costar). The lower anti-B220, anti-CD21, anti-CD23, anti-CD24, anti-CD38, anti-GL7, anti-AA4.1, chamber was filled with 600 μL of medium or medium containing either anti-IgG1 (BD Pharmingen); anti-IgM, anti-IgD (Southern Biotech); PNA CXCL13 (1 μg/mL) or CXCL12 (500 ng/mL) (PeproTech). After incubation at (Vector); NP-PE (gift from M. Shlomchik, Yale University, New Haven, CT). For 37 °C for 3 h, cells in the lower chamber were counted and examined by cell-surface LT staining, cells were incubated for 1 h on ice with staining flow cytometry. solution containing 10% goat and rat serum to block Fc receptors. After washing, cells were incubated with 50 μLofLTβR-Ig (10 μg/mL) on ice for 45 ELISA and ELISPOT. For ELISA, serum was collected from mice at different time min. Stained cells were further revealed by anti-human IgG (eBioscience). points after immunization. Samples were diluted serially and added to 96-well For BrdU-labeling, mice were injected with BrdU (1 mg per mouse) in- μ traperitoneally. Four hours later, mice were killed for analysis. BrdU staining plates (50 L per well) precoated with NP30-BSA (Biosearch Technologies; 10 μ was performed using a kit (BD Pharmingen) according to the manufac- g/mL). After incubation at 37 °C for 1 h, bound antibodies were revealed by turer’s instructions. AP-conjugated anti-mouse Ig of different isotypes (Southern Biotech). Results were presented as relative units taking the average of day-7 immunized-WT Ca2+ Mobilization. Splenic B cells were labeled with Fluo-4 and Fura-red samples as a control. For an affinity maturation assay, the plates were coated μ (Molecular Probes) according to the manufacturer’s instructions. Cells were with either NP30-BSA or NP2–3-BSA (Biosearch Technologies; 10 g/mL). The

then stimulated with anti-IgM F(ab)2 at 37 °C and subjected to FACS analysis. serum titers of IgG1 against each conjugates were determined as above. 2+ fl Intracellular Ca concentration was presented as the ratio of uorescence Results were presented as ratios of IgG1 titers against NP2–3-BSA vs. NP30-BSA. intensities of Fluo-4 vs. Fura-red. For ELISPOT, 96-well MultiScreen filtration plates (Millipore) were coated 4 with NP30-BSA. Total splenic cells were seeded into the plate (10 cells per Immunofluorescence Histology. Cryosections of spleen and mesenteric lymph well) and cultured overnight at 37 °C. After washing, AP-conjugated anti- nodes were fixed in 4% paraformaldehyde at room temperature for 10 min, mouse IgM or IgG (Southern Biotech) was added to the plates and incubated blocked with 10% goat serum in PBS containing 0.2% Triton X-100 for 30 min at 37 °C for 1 h. Antibody bound plaques were revealed using a BCIP/NBT kit at room temperature, and then stained with corresponding antibodies in (Vector) and quantified under a microscope. PBS containing 0.1% Tween-20 in a humidity chamber at RT for 1 h. Antibodies used are: anti-CD4, anti-CD8, anti-CD35, and B220 (BD Pharmingen); PNA- – – ACKNOWLEDGMENTS. We thank M. Shlomchik for kindly providing the biotin (Vector); anti Moma-1 (Serotec Inc.), anti-IgD (clone 1.3 5; gift from NP-PE, C. Swanson and R. Pelanda for anti-mouse IgD, M. McHeyzer-Williams R. Pelanda, University of Colorado, Denver, CO), streptavidin-Alexa 568 or for discussion, and B. Diamond and A. Davidson for reading and comments on streptavidin-Cy5 (Molecular Probes). Data were obtained with a Nikon TE- the manuscript. This work was supported by an Irene Diamond Fund (to H.G.) 2000E microscope using NIS-Elements 2.3 software. and by an Institution Fund (to Y.Z.).

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