T Cells Regulate Peripheral Naive Mature B Cell Survival by Cell–Cell Contact Mediated Through SLAMF6 and SAP

T Cells Regulate Peripheral Naive Mature B Cell Survival by Cell −Cell Contact Mediated through SLAMF6 and SAP

This information is current as Lihi Radomir, Sivan Cohen, Matthias P. Kramer, Eszter of September 23, 2021. Bakos, Hadas Lewinsky, Avital Barak, Ziv Porat, Richard Bucala, Polina Stepensky, Shirly Becker-Herman and Idit Shachar J Immunol 2017; 199:2745-2757; Prepublished online 13

September 2017; Downloaded from doi: 10.4049/jimmunol.1700557 http://www.jimmunol.org/content/199/8/2745

Supplementary http://www.jimmunol.org/content/suppl/2017/09/13/jimmunol.170055 http://www.jimmunol.org/ Material 7.DCSupplemental References This article cites 46 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/199/8/2745.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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

T Cells Regulate Peripheral Naive Mature B Cell Survival by Cell–Cell Contact Mediated through SLAMF6 and SAP

Lihi Radomir,*,1 Sivan Cohen,*,1 Matthias P. Kramer,* Eszter Bakos,* Hadas Lewinsky,* Avital Barak,* Ziv Porat,† Richard Bucala,‡ Polina Stepensky,x Shirly Becker-Herman,* and Idit Shachar*

The control of lymphoid homeostasis is the result of a very fine balance between lymphocyte production, proliferation, and apoptosis. In this study, we focused on the role of T cells in the maintenance/survival of the mature naive peripheral B cell population. We show that naive B and T cells interact via the signaling lymphocyte activation molecule (SLAM) family receptor, SLAMF6. This interaction induces cell type–specific signals in both cell types, mediated by the SLAM-associated protein (SAP) family of adaptors. This signaling results in an upregulation of the expression of the cytokine migration inhibitory factor in the T cells and augmented expression of its receptor CD74 on the B cell counterparts, consequently enhancing B cell survival. Downloaded from Furthermore, in X-linked lymphoproliferative disease patients, SAP deficiency reduces CD74 expression, resulting in the pertur- bation of B cell maintenance from the naive stage. Thus, naive T cells regulate B cell survival in a SLAMF6- and SAP-dependent manner. The Journal of Immunology, 2017, 199: 2745–2757.

he survival of peripheral naive mature B cells is dependent is modiﬁed by the addition of chondroitin sulfate, and this form of on three key cascades: 1) BCR tonic signals (e.g., Iga and CD74 is expressed on the surface of APCs (including monocytes and http://www.jimmunol.org/ T Syk) (1, 2); 2) the B cell activating factor receptor, which B cells) and epithelial cells (7). It was previously shown that mac- binds the B cell activating factor, belonging to the TNF family (the rophage MIF binds to the CD74 extracellular domain, a process that B cell activating factor is also known as BLyS/TALL-1/THANK/ results in the initiation of a signaling pathway in these cells (8). zTNF4) (3); and 3) CD74 (invariant chain) expressed on B cells, CD74 stimulation by MIF induces a signaling cascade leading to and its cognate ligand, macrophage migration inhibitory factor NF-kB activation, as well as transcription of genes that regulate (MIF), which is secreted by almost all cell types. These pathways the entry of the stimulated B cells into the S phase, an increase in have complementary roles in B cell survival (4, 5). DNA synthesis, cell division, and augmented expression of anti- CD74 is a type II integral membrane protein that acts as a chaperone

apoptotic proteins (5, 9, 10). The CD74 receptor induces a similar by guest on September 23, 2021 for MHC class II protein expression (6). A small proportion of CD74 survival cascade in oncogenically transformed cells derived from chronic lymphocytic leukemia (CLL) patients (11). To deﬁne the molecules whose expression is modulated by CD74 to regulate *Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel; †Department of Biological Services, Weizmann Institute of Science, Rehovot 76100, CLL cell survival, we previously screened for CD74 target genes. Israel; ‡Department of Internal Medicine, Yale University School of Medicine, New One molecule, whose expression was strongly upregulated by x Haven, CT 06520; and Pediatric Hematology-Oncology and Bone Marrow Transplan- CD74 activation, is signaling lymphocyte activation molecule tation Unit, Hadassah Hebrew University Medical Center, Jerusalem 91120, Israel 1 (SLAM)F5 (CD84), a member of the SLAM Ig superfamily (12). L.R. and S.C. contributed equally to this work. The SLAM family of receptors includes homophilic and het- ORCIDs: 0000-0002-0289-5261 (M.P.K.); 0000-0003-3059-181X (Z.P.); 0000-0003- 0004-6159 (I.S.). erophilic receptors that modulate the behavior of immune cells (13– 15). These receptors share a common ectodomain organization: a Received for publication April 19, 2017. Accepted for publication August 10, 2017. membrane-proximal Ig-like constant domain, and a membrane- This work was supported in part by the Binational Science Foundation, the Cooper- ation Program in Cancer Research of the Deutsches Krebsforschungszentrum distal Ig variable domain that is responsible for ligand recognition. and Israel’s Ministry of Science, Technology and Space, the Quinquin Foundation, and SLAM receptors interact with SLAM-associated protein (SAP)–related the Rubenstein Charitable Foundation. I.S. is the incumbent of the Dr. Morton and A. Kleiman Professorial Chair. molecules, a group of SRC homology 2 (SH2) domain adaptors. The SAP family is comprised of three members: SAP, Ewing’s L.R., S.C., and M.P.K. designed research, performed research, analyzed data, and wrote the paper; E.B., L.R., H.L., A.B., and Z.P. designed research, performed sarcoma–associated transcript 2 (EAT-2), and, in rodents, EAT-2– research, and analyzed data; R.B. and P.S. provided essential reagents and wrote related transducer (16, 17). SAP controls signal transduction path- the paper; S.B.-H. designed research and analyzed data; and I.S. designed research, analyzed data, and wrote the paper. ways downstream of the SLAM family receptors, and it is a key Address correspondence and reprint requests to Dr. Idit Shachar, Department of regulator of normal immune function in T, NK, and NKT cells (15, Immunology, Weizmann Institute of Science, Herzl Street, Rehovot 76100, Israel. 18). However, B cells do not express SAP (19), and EAT-2 was E-mail address: [email protected] suggested to serve as its functional homolog in these cells (20, 21). The online version of this article contains supplemental material. The SLAM receptors and their adaptor molecules were shown to Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; CLL, chronic be required for germinal center development and humoral memory lymphocytic leukemia; EAT-2, Ewing’s sarcoma–associated transcript 2; MIF, macrophage migration inhibitory factor; PB, peripheral blood; SAP, SLAM-associated (22–24). However, their role in naive B cell maintenance has not protein; SH2, SRC homology 2; siRNA, small interfering RNA; SLAM, signaling been assessed in detail. Lymphocyte populations derived from lymphocyte activation molecule; WT, wild-type; XLP, X-linked lymphoproliferative SAP-deﬁcient mice are grossly normal, although occasional mu- disease. tant animals exhibit a higher percentage of T and NK cells, as well Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 as a lower percentage of B cells in the spleen (25). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700557 2746 CD4+ T CELLS REGULATE NAIVE B CELL SURVIVAL

In the present study, we hypothesized that the SLAM family EAT-2 (ON-TARGET plus SMART pool) and negative control siRNA (ON- might be involved in the regulation of naive B cell survival in the TARGETplus nontargeting pool) (Dharmacon). cross-talk between naive B and naive T cells in an Ag-independent Real-time RT-PCR analysis environment. Our findings demonstrate that interaction of B cells with T cells in a SLAMF6/SAP-mediated manner upregulates RNA extraction and quantitative RT-PCR were performed as previously described (31). Primers are summarized in Table I. CD74 cell surface expression on B cells, inducing their survival in vitro and in vivo. This study highlights a crucial role of SAP Stimulation of murine B cells with CD74 expression on T cells in regulating B cell survival and provides Activation of CD74 was performed as previously described (9). new insights into the requirements for cell collaboration during homeostasis. Blocking SLAMF6 on human B cells Human B cells (1 3 107) were incubated in the presence of 20 mg/ml monoclonal anti–NTB-A Ab (clone NT-7) or mouse IgG1 k isotype control Materials and Methods Abs (BioLegend), as previously described (32). Mice Coculture C57BL/6, CD74-deficient (26), MIF-deficient (27), SLAMF5-deficient (24), SH2D1A (SAP)-deficient (25) SLAMF6-deficient (28), and SLAMF1- Coculture of murine primary B and T cells was performed in 24-well plates with 10% FCS (w/v) Iscove’s medium. Transwell experiments were performed deficient mice (29) were used in this study. All animal procedures were m approved by the Animal Research Committee at the Weizmann Institute of in 24-well plates containing an insert with a 5- m pore polycarbonate Science. The animals in each experiment were aged and sex matched. membrane. B cells were seeded in the well, and T cells were seeded in the

insert. Downloaded from Cells Immunoprecipitation and Western blot analysis Human peripheral blood lymphocytes. X-linked lymphoproliferative dis- Immunoprecipitation of endogenously expressed proteins and their de- ease (XLP) patient samples were provided in compliance with the Insti- tection were performed on fresh mouse lymphocytes as previously de- tutional Review Board of the Hadassah Hospital. Healthy control samples scribed (12). For immunoprecipitation and blotting of EAT-2, anti–EAT-2 were provided by the Magen David Adom in Israel blood bank. Ab (N-14; Santa Cruz Biotechnology) was used. pTYR was detected with Cells were separated as previously described (11). T lymphocytes were the anti–p-Tyr (FL-293; Santa Cruz Biotechnology) Ab. Immunoprecipita- enriched by positive selection using anti-CD3 MACS beads (Miltenyi Biotec) http://www.jimmunol.org/ according to the manufacturer’s protocol. B lymphocytes were enriched in the tion of Ly108 was performed with anti-Ly108 Ab (13G3-19D; eBioscience). T cell depleted flow through. RAG adoptive transfer Mouse splenic lymphocytes. B lymphocytes were enriched by positive A mixture of B and T cells at a 1:1 ratio (total of 1 3 107 cells) in 300 mlof selection using anti-B220 beads (BD Biosciences), as previously described 2/2 (9). The CD4 T cell population was enriched from the B220-negative PBS per mouse was injected i.v. into the tail vein of RAG1 mice. fraction by positive selection using CD4 (L3T4) beads (Miltenyi Biotec) CD4 depletion according to the manufacturer’s protocol. The CD8-enriched population was obtained as the negative fraction of the CD4 enrichment. WT mice were injected with 150 mg/ml anti-CD4 mAb GK1.5 (BE0003-1; Bio X Cell) diluted in sterile PBS to a final volume of 200 ml, i.v. into the Flow cytometry tail vein. by guest on September 23, 2021 FACS analysis was performed using a FACSCanto flow cytometer (BD Statistical analysis Biosciences). Abs from the following sources were used: anti-CD74, Ly108, CD40L (eBioscience), CD19, B220, CD4, CD3, CD21, CD24, CD23, SLAMF5, Data analysis was performed as previously described (33), using GraphPad SLAMF1 (BioLegend), anti-MIF (Abcam), and anti–EAT-2 (Proteintech). Prism version 6.0f (GraphPad Software, La Jolla, CA). Data are reported For assessment of apoptosis, annexin V and 7-aminoactinomycin D (7-AAD) as mean 6 SEM. To determine significance of differences, we used either staining was performed (BD Pharmingen). Flow cytometry data analysis was the unpaired Student t test or one-way ANOVA test for mouse group ex- performed using FlowJo software (Tree Star). periments, and a ratio paired Student t test for treatments of human samples.

Cell conjugation assay Results 2 2 Naive wild-type (WT) B cells (5 3 106) were cultured with WT or SAP / Direct contact between B and T cells is essential for B cell 6 T cells (5 3 10 ) and incubated for 24 h in 24-well plates. Conjugates were survival enumerated by flow cytometry after the cell mixture was stained for CD3/ CD4/CD8 and B220 at 4˚C. To determine the role of SLAMs and their adaptor molecule, SAP, which is expressed in T cells, in the regulation of B cell mainte- Quantification of B and T cell conjugates by imaging flow nance, a coculture experiment was first performed. In this experi- cytometry ment, WT B220+ cells, which are mostly naive mature B cells, were Cells were examined by imaging flow cytometry using the ImageStreamX cultured alone or in the presence of WT or SAP-deficient naive 5 Mark II (Amnis [part of EMD Millipore], Seattle, WA). At least 10 cells T cells. As shown in Fig. 1A, the presence of WT T cells signifi- were collected from each sample. Images were analyzed using IDEAS 6.0 software (Amnis). Cells were gated for the cells in focus using the gradient cantly increased the survival of naive B cells compared with B cells root mean square feature (30). cultured alone by ∼3-fold, whereas incubation with SAP-deficient B220+ B/CD3+ T cell conjugates were gated according to their area and T cells supported less B cell survival. No significant change was aspect ratio to include doublets of relevant size. To eliminate doublets that observed in WT and SAP2/2 T cell survival rates in this coculture did not consist of viable cells, or cells that were too far apart, only cells setting (Supplemental Fig. 1A). These results suggest an SAP-dependent with the correct distance (3–7 mm) were gated. All the gates were visually inspected to verify the classifications. beneficial effect of naive T cells on naive B cell survival. To examine whether cell–cell contact or soluble factors are SAP and SLAMF6 downregulation by small interfering RNA responsible for the T cell–dependent regulation of B cell survival, Human cells. Purified T cells and B cells (5 3 106) were subjected to similar cultures were established, but direct B cell–T cell inter- electroporation as previously described (12). The cells were treated with actions were prevented by a Transwell membrane. As shown in the following small interfering RNAs (siRNAs): SAP, NTB-A, CD74 (ON- TARGETplus SMARTpool) and negative control siRNA (ON-TARGET Fig. 1A (open bars), B and T cell contacts were required for the plus nontargeting pool) (Dharmacon). T cell control of the B cell live population. Abrogation of cell Mouse B cells. B cells (5 3 106) were subjected to electroporation as contacts by the Transwell membrane reduced cell survival to previously described (12). The cells were treated with the following siRNAs: levels observed in B cells cocultured with SAP-deficient T cells. The Journal of Immunology 2747 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 1. Direct B cell–T cell contact regulates naive B cell survival. (A) Purified naive WT B cells were cultured alone or at 1:1 ratio with 5 3 106 WT or SAP2/2 T cells in regular or Transwell apparatus wells (open bars). After 24 h, B cells were analyzed by flow cytometry for B cell survival by annexin V/7-AAD staining. Results are shown as the percentage of live B cells (double-negative annexin V/7-AAD); right panels exhibit representative dot plots of annexin V/7-AAD staining. n =7.(B) Purified naive WT or SAP2/2-derived B cells were cultured alone or at a 1:1 ratio with 5 3 106 WT or SAP2/2 T cells. After 24 h, the cells were analyzed for T cell/B cell conjugates by flow cytometry of double-positive staining for B220+ and CD3+. Results are shown as the percentage of conjugates. Right panels exhibit representative dot plots of B220/CD3 staining. n =4.(C and D) WT B cells were cultured together with WT or SAP2/2 T cells. B cell/T cell conjugates were analyzed by imaging flow cytometry (ImageStream, n = 6). (C) Representative images of WT B cells cocultured with WT T cells. (D) Percentage of B cell/T cell doublets of total B and T cell populations. Results are shown as the percentage of T cell/B cell conjugates. n = 4. Each dot represents a biological repeat, and bars show SEM. ***p , 0.001.

It was previously shown that SAP2/2 T cells show impaired were cocultured with WT T cells for 24 h, and the cocultures were ability to generate stable interactions with B cells following im- stained for CD3 and B220. As shown in Fig. 1C and 1D, T cell munization (22). To determine whether SAP deficiency affects (CD3+)/B cell (B220+) doublets were detected in this setting, naive B cell/T cell conjugates, WT or SAP2/2-derived B cells suggesting direct contact between the cells. To further resolve the were cultured with WT or SAP2/2 T cells (25). After 24 h, the role of SAP in this interaction, and to determine whether forma- cells were analyzed for double-positive staining of CD3+ and tion of doublets is inhibited in the absence of SAP in T cells, B cells B220+, as was previously described (22). Surprisingly, as seen in were cocultured with SAP-deficient T cells, and the number of Fig. 1B, SAP2/2 T cells formed similar numbers of T cell/B cell doublets was analyzed. As shown in Fig. 1D, SAP deficiency did conjugates as WT T cells. We further validated our results and the not affect the percentage of T cell/B cell doublets, suggesting that existence of naive T cell/B cell conjugates by looking for naive in the absence of SAP, T cells can still interact with the B cells T cell/B cell doublets using image-based quantification. B cells but their downstream cascade is altered. Because SAP deficiency in 2748 CD4+ T CELLS REGULATE NAIVE B CELL SURVIVAL

Table I. Primer sets for quantitative RT-PCR

Gene Direction Primer Sequence Bcl-2 Forward 59-GCCAACTGAGCAGAGTCTC-39 Reverse 59-GGACAACATCGCCCTGTG-39 CD74 Forward 59-GGAGTACCCGCAGCTGAAGGGG-39 Reverse 59-GAAGATAGGTCTTCCATGTCCAGTG-39 SAP Forward 59-TCATGGGGCTTTCATTTCAGGCAGACATCAGG-39 Reverse 59-GACGCAGTGGCTGTGTAT-39 NTB-A Forward 59-TCCTTCTCTGTCTCTGCCCA-39 Reverse 59-GCCCTGTGTTCGCTGAGTAG-39

B cells did not cause any intrinsic defect on their cell survival from SAP-deﬁcient mice were slightly reduced (Supplemental Fig. (Supplemental Fig. 1B), these results suggest that T cells support B 1C) whereas almost no differences were detected in SLAMF5 maintenance in an SAP-dependent manner. (Supplemental Fig. 1D) and SLAMF1 (Supplemental Fig. 1E) cell surface levels. These results suggest that SLAMF6 may have a SAP-dependent B and T cell interaction regulates CD74 more important role in support of B cells by naive T cells. surface expression on B cells We then analyzed the role of these receptors expressed on T cells

We next followed the SAP-dependent molecular mechanism reg- in the regulation of naive B cell survival. Naive B cells were Downloaded from ulating splenic B cell survival. Our hypothesis was that because cocultured with naive WT or SLAMF6/SLAMF5/SLAMF1-deficient CD74 is a survival receptor expressed on B cells, its expression or T cells (24, 28, 29) for 24 h, and B cell survival was then analyzed. function might be modulated by an SAP-dependent T cell–B cell Although deficiency of each of these receptors interfered with the interaction. Therefore, CD74 cell surface expression was analyzed support induced by T cells, SLAMF6 deficiency caused the most on B cell splenic populations derived from WT and SAP-deficient significant reduction in B cell survival (Fig. 3B). Additionally, the

mice. A significant reduction in surface CD74 expression was abrogation of T cell support to B cells following disruption of the http://www.jimmunol.org/ + int low detected on total B cells, mature (B220 , CD21 CD24 ) and interaction was independent of whether the B cells or the T cells + low high transitional B cell (transitional 1, B220 ,CD21 CD24 ; were SLAMF6 deficient (Supplemental Fig. 1F), SLAMF5 defi- high high high transitional 2, CD21 CD24 CD23 ) populations derived cient (Supplemental Fig. 1G), or SLAMF1 deficient (Supplemental from SAP-deficient mice (Fig. 2A). Fig. 1H). Based on these results, we decided to further focus on the 2/2 Next, WT or SAP -derived B cells were cultured alone or role of SLAMF6 in the T cell–induced survival support. with naive WT or SAP-deficient T cells for 24 h, and expression of Next, to determine the role of SLAMF6 expression in T cell– 2/2 surface CD74 was analyzed. B cells (WT or SAP ) cultured dependent B cell survival, murine splenic WT or SAP2/2-derived alone or with SAP-deficient T cells exhibited significantly lower B cells were cultured alone or with naive WT or SAP-deficient T cells cell surface expression levels of surface CD74 compared with for 24 h, and expression of SLAMF6 was analyzed. SLAMF6 ex- by guest on September 23, 2021 B cells cocultured with WT T cells (Fig. 2B). Furthermore, lower pression was significantly upregulated on B cells cultured with T levels of CD74 were observed in the Transwell setting, in which cells, compared with its levels on freshly isolated B cells (Fig. direct interaction between WT B and T cells was inhibited 3C). This elevation did not occur in the presence of SAP2/2 Tcells (Fig. 2B, open bars). (Fig. 3C, 3D). Moreover, no change in SLAMF6 expression levels The lower cell surface expression of CD74 seen in the absence of on T cells was detected under these conditions (Supplemental Fig. SAP-expressing T cells might result from an upregulation of its 1I). These results suggest a T cell–dependent elevation of SLAMF6 levels on B cells in the presence of T cells or by downregulation of expression on B cells mediated through SAP. When B cell–T cell 2/2 its levels due to the lack of a proper signal from the SAP T cells. interactions were prevented by a Transwell membrane, CD74 cell To address this point, the levels of cell surface CD74 on freshly surface levels on B cells remained at the low levels observed in isolated splenic B cells were compared with its levels detected on B cells incubated with SAP-deficient T cells (Fig. 3D, open bars). B cells cocultured for 24 h with T cells. As seen in Fig. 2C, We further investigated the role of SLAMF6 in T cell–dependent following coculture with WT T cells, elevated levels of cell sur- regulation of CD74 expression and B cell survival. First, CD74 face CD74 were detected on the B cells, whereas incubation with expression levels were analyzed on the SLAMF62/2 cells. Similar 2/2 SAP T cells resulted in a milder elevation. These results surface levels of CD74 were detected on freshly isolated WT and suggest a mechanism whereby T cells upregulate CD74 cell sur- SLAMF62/2 B cell populations (Supplemental Fig. 1J). Next, face levels on B cells in an SAP-dependent manner. splenic WT or SLAMF62/2 B cells were cocultured with splenic WT or SLAMF62/2 T cells and levels of CD74 and B cell survival CD74 regulation by SAP signaling is mediated by SLAMF6 were analyzed (Fig. 3E, Supplemental Fig. 1K). Abolishing SLAMF6- SAP transmits signals induced by the SLAM family of receptors mediated interaction between T and B cells led to a significant (16). To determine which SLAM member regulates the T and decrease in CD74 surface expression on B cells, accompanied by a B cell contact that results in survival of B cells, the effects of three downregulation in B cell survival (Supplemental Fig. 1F). SLAMF5 SLAM members, SLAMF6 (Ly108 or NTB-A in humans), deficiency also resulted in a reduction in CD74 upregulation SLAMF5 (CD84), and SLAMF1 (CD150), were analyzed. These (Supplemental Fig. 1L), whereas SLAMF1 deficiency showed no receptors were shown to work in synergy in the regulation of Ab effect at all (Supplemental Fig. 1M). responses and were identified as mediators of the B cell–T cell CD74 activation induces B cell survival (9, 10) (Supplemental contact in germinal centers (24, 34). We first analyzed the ex- Fig. 2A) and upregulates SLAMF6 expression on B cells (Fig. pression of these receptors on splenic B cells. As shown in 3F). SAP2/2 mice exhibit lower levels of CD74 on B cells (Fig. Fig. 3A, high levels of SLAMF6 were detected on the surface of 2A) and a lower response of these B cells to CD74 Ab stimulation B cells, whereas SLAMF1 and SLAMF5 levels were significantly (data not shown). These results suggest that the SLAMF6 B cell– lower. Additionally, SLAMF6 expression levels on B cells derived T cell interaction elevates CD74 expression on B cells, which The Journal of Immunology 2749 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 2. SAP-dependent B and T cell interactions regulate CD74 expression on the B cell surface. (A) WT and SAP2/2 splenocytes were analyzed by flow cytometry for surface CD74 expression on B cell populations. Total B cells (B220+), mature population (B220+, CD21intCD24low), transitional 1 (B220+,CD21lowCD24high), and transitional 2 (B220+,CD21highCD24+CD23+) are shown; graph shows CD74 MFI levels. n =4.(B) Naive WT and SAP2/2 derived B cells and WT or SAP2/2 T splenocytes were isolated. B cells were cultured alone or at a 1:1 ratio with 5 3 106 WT or SAP2/2 Tcellsin regular or Transwell apparatus wells. After 24 h, cells were analyzed by flow cytometry for CD74 surface expression on B cells. Graph shows CD74 mean fluorescence intensity (MFI) levels. Right panels exhibit representative histograms of CD74 staining on B220+ cells. n =4.(C) Purified WT or SAP2/2 Bcells were cultured for 24 h alone or at a 1:1 ratio with 5 3 106 WT or SAP2/2. T cells were analyzed for CD74 surface expression before (B cells before co-culture) or after (B cells after co-culture) coculture. CD74 MFI levels are shown in the graph. In all of the graphs, each dot represents a biological repeat. n =3.Bars show SEM. **p , 0.01, ***p , 0.001, ****p , 0.0001. ns, not significant (p $ 0.05). results in B cell survival. Additionally, CD74 induces SLAMF6 2 plays a role in the B cell side of this cascade. To this end, we first levels, which can further induce/enhance the process. established the expression of EAT-2 protein in splenic B cells. Using a flow cytometry analysis, EAT-2 levels in B cells were EAT-2 is recruited to SLAMF6 compared with its levels in NK cells, which are known to express B cells do not express SAP; instead, they express the mRNA of the EAT-2, and in T cells, which express low levels of the adaptor (15, adaptor molecule, EAT-2 (16, 35). To characterize the SLAMF6- 18). As shown in Fig. 3G, B cells express the EAT-2 protein. Next, induced cascade in B cells, we wanted to determine whether EAT- to determine whether EAT-2 is involved in the interaction via 2750 CD4+ T CELLS REGULATE NAIVE B CELL SURVIVAL Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 3. SAP-dependent B and T cell interaction regulates SLAMF6 expression and function in B cells. (A) Fresh WT mature naive B cells were analyzed by flow cytometry for SLAMF6, SLAMF5, and SLAMF1 surface expression. Results are shown as the levels of SLAM receptor mean fluorescence intensity (MFI). n =3.(B) WT B cells were cultured at a 1:1 ratio with 5 3 106 WT, SLAMF62/2, SLAMF52/2, or SLAMF12/2 T cells. After 24 h, B cells were analyzed by flow cytometry for B cell survival by annexin V/7-AAD staining. Results are shown as the percentage of live B cells (double negative for annexin V/7-AAD). n =6.(C) Fresh WT or SAP2/2 (B cells before co-culture) and purified naive WT B cells were cultured for 24 h alone at a 1:1 ratio with 5 3 106 WT or SAP2/2 T cells (B cells after co-culture) and were analyzed for SLAMF6 expression. SLAMF6 MFI levels are shown in the graph. n =3.(D) Naive WT or SAP2/2-derived B cells and WT or SAP2/2 T splenocytes were cultured alone or at a 1:1 ratio with 5 3 106 WT or SAP2/2 T cells in regular or Transwell apparatus (open bars) wells. After 24 h, cells were analyzed by flow cytometry for SLAMF6 expression on B cells (B220+ gate). Results are shown as SLAMF6 MFI; right panels display representative histograms of SLAMF6 staining on B220+ cells. n =4.(E) Naive WT B splenocytes were cultured alone or at a 1:1 ratio with 5 3 106 WT or SLAMF62/2 T cells. After 24 h, cells were analyzed by flow cytometry for CD74 expression, shown by CD74 MFI. n =3.(F) Naive mature WT B cells were activated with anti-CD74 or IgG control Abs for 18 h. Cells were then analyzed for SLAMF6 expression on the B220+ gate. n =3.(G) Fresh B cells, NK cells, and T cells were analyzed for EAT-2 expression by (Figure legend continues) The Journal of Immunology 2751

SLAMF6, a coimmunoprecipitation experiment was performed, in T cells is essential for B cell survival in an SLAMF6- and CD74- which SLAMF6 was pulled down by anti-SLAMF6 Ab from dependent manner. CD74 expression on B cells cultured with B cell lysates and EAT-2 levels were analyzed in the immuno- T cells deﬁcient in SAP, SLAMF6, or MIF did not induce an precipitated proteins. As seen in Fig. 3H (top panel), SLAMF6 upregulation of CD74 expression on B cells, suggesting that all of recruited EAT-2 in B cells, suggesting that EAT-2 transmits the these molecules contribute to the T cell support of B cell survival SLAMF6 signal in B cells. Following the recruitment of EAT-2 to (Supplemental Fig. 2G). the receptor, it is phosphorylated on tyrosine residues, which ac- Following immunization or infection, binding of CD40L on the tivates it to transmit downstream signaling (14). We next wanted T cells to CD40 on B cells promotes B cell proliferation, germinal to identify the presence of phosphorylated EAT-2 in naive B cells. center development, and the differentiation of B cells into Ab- EAT-2 was pulled down from naive B cell lysate, and the secreting plasma cells (36). It was previously shown that SAP immunoprecipitated proteins were analyzed for p-Tyr. As dem- downregulates the expression of CD40L on T cells, as SAP2/2 onstrated in Supplemental Fig. 2B, phosphorylated EAT-2 was T cells exhibit higher levels of CD40L (23). We therefore deter- detected in the lysates of naive B cells. To directly demonstrate mined CD40L expression on naive T cells in the unstimulated recruitment of phosphorylated EAT-2 to SLAMF6, SLAMF6 was coculture setting. WT/SLAMF62/2/SAP2/2 CD4+ T cells were pulled down and the immunoprecipitated proteins were analyzed. cultured alone or with B cells, and CD40L was analyzed on the Phosphorylated EAT-2 was pulled down with SLAMF6 (Fig. 3H, T cells. As described previously, SAP2/2 CD4+ T cells exhibited bottom panel), suggesting that in B cells, EAT-2 can be recruited elevated levels of CD40L compared with WT cells (Supplemental to SLAMF6 and is activated. To directly show the involvement of Fig. 2H). However, the presence of B cells in the coculture had

EAT-2 in B cell survival, EAT-2 levels were knocked down in no effect on CD40L cell surface expression. Furthermore, com- Downloaded from using siRNA against EAT-2. siEAT-2– or siControl-treated B cells parable levels of CD40L expression were observed on WT CD4+/ were cultured with naive T cells for 48 h. A reduction of ∼40% in CD8+ or SLAMF62/2 CD4+ T cells (Supplemental Fig. 2I). EAT-2 protein levels was detected in siEAT-2–treated B cells Thus, CD40L does not play a part in the naive T cell–B cell (Supplemental Fig. 2C) (Table I). In this setting, SLAMF6 interaction. (Fig. 3I) and live B cell (Fig. 3J) levels showed a significant re- SAP and SLAMF6 regulate B cell survival in vivo duction of ∼20%. This further suggests that EAT-2 regulates http://www.jimmunol.org/ SLAMF6 expression and B cell survival. To determine the in vivo role of SAP and SLAMF6 in naive T cell– + B cell interactions, as well as regulation of B cell survival, purified B cell survival is regulated by CD4 T cells WT splenic B cells were adoptively transferred together with Next, we wanted to identify which T cell subpopulation, CD4+ or purified WT or SAP2/2 splenic T cells into lymphocyte-deficient CD8+, is involved in the maintenance of naive B cells. First, as RAG12/2 recipients, which lack mature B and T cells. The mice seen in Supplemental Fig. 2D, CD4 T cells express higher levels of were sacrificed 24 h after the cell transfer. CD74 (Fig. 5A) and SLAMF6 than do CD8 T cells. Next, WT, SAP2/2,orSLAMF62/2 SLAMF6 (Fig. 5B) cell surface expression levels were signifi- CD4+ or CD8+ T cells were purified (Supplemental Fig. 2E) and cantly lower on B cells cotransferred with SAP-deficient naive cocultured with B cells. As shown in Fig. 4A, a greater number of T cells, compared with their levels in the presence of WT T cells. by guest on September 23, 2021 conjugates of B cells with CD4+ T cells compared with CD8+ Additionally, the percentage of the live B cell population was T cells were detected. Additionally, only WT CD4+ Tcellsman- downregulated when B cells were transferred together with SAP- aged to support B cell survival (Fig. 4B) and SLAMF6 expression deficient T cells (Fig. 5C). Moreover, to directly show the role of on B cells (Fig. 4C). Thus, CD4+ T cells regulate B cell survival in CD4+ T cells in vivo, WT naive B cells were adoptively trans- an SAP- and SLAMF6-dependent manner. ferred into RAG12/2 alone or with WT CD8+ T cells, and WT or Because CD74 expression is regulated by the SLAMF6- SAP2/2 CD4+ T cells. As seen in Fig. 5D, only WT CD4+ T cells mediated T cell–B cell interaction, we wanted to determine supported B cell survival. whether the SLAMF6/SAP-dependent B and T cell interaction To further investigate the role of CD4+ T cells in the mainte- regulates the expression of CD74 ligand, MIF, in T cells. As nance of naive B cells, CD4+ cells were depleted by injection of shown in Fig. 4D, CD4+ T cells expressed higher levels of intra- anti-CD4 Ab (Supplemental Fig. 2J) (37). After 4 d, spleens were cellular MIF compared with its levels in CD8+ T cells and B cells. harvested and analyzed for cell surface markers. SLAMF6 levels Next, regulation of MIF expression levels in T cells by splenic were significantly reduced on B cells derived from anti-CD4– B cells was determined. Whereas B cells elevated MIF levels in treated mice compared with control-injected mice (Fig. 5E), WT CD4+ T cells in the coculture setting, MIF levels in CD4+ suggesting a role for SLAMF6 expression on CD4+ cells in B cell T cells lacking SAP or SLAMF6 were not upregulated by B cells maintenance. CD74 expression levels were not altered at this time (Fig. 4E). This elevation was specific to the CD4+ T cells and was point (data not shown). These results show that naive CD4+ T cells not detected in the CD8+ population (Supplemental Fig. 2F). are required in vivo for the maintenance of B cells in an SAP- To directly determine whether MIF plays a role in this crosstalk, dependent manner. murine WT splenic B cells were cultured alone or with naive WTor MIF-deficient T cells (27) for 24 h. FACS analysis revealed a The role of SAP and SLAMF6 in human B cell survival reduced B cell survival (Fig. 4F), CD74 cell surface expression We next wanted to determine whether a similar SAP-dependent (Fig. 4G), and SLAMF6 (Fig. 4H) on B cells incubated with MIF- T cell–induced survival pathway exists in human B cells; to this deficient T cells. These results imply that MIF secreted from end, SAP expression in healthy human peripheral blood (PB)

intracellular staining and flow cytometry analysis. Results are shown as representative histograms of EAT-2 expression on the various populations. (H) Purified WT B splenocytes were lysed, and SLAMF6 was immunoprecipitated. Proteins were separated by 12% SDS-PAGE and analyzed for EAT-2 expression (top panel, n = 3), and for p-Tyr (bottom, n = 2). (I and J) siRNA control or EAT-2–treated naive B cells were were cocultured with untreated naive T cells. After 48 h, the cells were stained for SLAMF6 (I) and annexin V and 7-AAD (J). n = 2. In all graphs, each dot represents a biological repeat; n represents the number of experiments. Bars show SEM. *p , 0.05, ***p , 0.001, ****p , 0.0001. ns, not significant (p $ 0.05). 2752 CD4+ T CELLS REGULATE NAIVE B CELL SURVIVAL Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 4. Naive CD4+ T cells induce B cell survival regulation via MIF secretion. (A) Purified naive WT B cells were cultured at a 1:1 ratio with 5 3 106 WT CD4+ or CD8+ T cells. After 24 h, the cells were analyzed for T cell/B cell conjugates by flow cytometry by double-positive staining for B220+ and CD3+. Results are shown as the percentage of conjugates. (B and C) Naive WT B splenocytes were cultured alone or at a 1:1 ratio with 5 3 106 WT or SAP2/2 CD4+ T or CD8+ T cells. After 24 h, cells were analyzed by flow cytometry for annexin V/7-AAD (n =4)(B) or SLAMF6 surface expression (C)on B cells (n = 4). (D) MIF expression was analyzed 5 h following monensin treatment by FACS staining. Histograms show MIF expression by CD4+ T cells, CD8+ T cells, and B cells compared with MIF expression on MIF2/2 splenocytes (background). (E) Naive WT/SAP2/2/SLAMF62/2 T splenocytes were cultured alone or at a 1:1 ratio with 5 3 106 WT B cells. After 24 h, expression of MIF in CD3+CD4+ was analyzed by flow cytometry. n =4(F–H) Naive WT B splenocytes were cultured alone or at a 1:1 ratio with 5 3 106 WT or MIF2/2 T cells. After 24 h, cells were analyzed by flow cytometry for annexin V/7-AAD (F), CD74 (G), or SLAMF6 (H) surface expression on B cells. n = 3. Results are shown as percentage (F) or mean fluorescence intensity (MFI) (G and H). In all experiments, each dot represents a biological repeat; n represents the number of experiments. Bars show SEM. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. ns, not significant (p $ 0.05).

T cells was knocked down by SAP siRNA (Supplemental Fig. 3A). human B cells cocultured with SAP-deficient T cells, compared SAP-deficient T cells were then cocultured with untreated human with their levels when incubated with T cells treated with a control PB B cells, and their SLAMF6 expression levels were analyzed. As siRNA. shown in Fig. 6A and Supplemental Fig. 3B, lower SLAMF6 cell Next, we determined whether reduced SAP, SLAMF6, and CD74 surface expression levels were detected on B cells cultured with levels lead to cell death in human B cells. Incubation of B cells with SAP-deficient T cells compared with B cells cultured with T cells SAP knockdown T cells significantly reduced their Bcl-2 mRNA treated with control siRNA. CD74 mRNA (24 h; Fig. 6B) and levels (Fig. 6D) and induced their death (Fig. 6E). Collectively, protein (48 h; Fig. 6C, Supplemental Fig. 3C) levels were then these results suggest that SAP plays a dominant role in T cell analyzed in B cells incubated with T cells deficient in SAP. CD74 induction of peripheral human B cell survival by regulating sur- message and protein levels were significantly reduced in healthy face CD74 and SLAMF6 expression. The Journal of Immunology 2753 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 5. SAP-mediated signaling in T cells regulates CD74 expression and B cell survival in vivo, and anti-CD4 downregulates SLAMF6 expression in vivo. (A–D) Naive WT B cells were injected into the tail vein of RAG12/2 mice together with WT or SAP2/2 T cells. After 24 h, splenocytes of RAG1 recipients were harvested and analyzed by ﬂow cytometry for (A) CD74, (B) SLAMF6, and (C) live cells. (A–C) Left panels, Graphs show fold of changes in the expression; right panels show representative histograms and plot. All results are shown as x-fold of WT B cells and WT T cells. (D) Naive WT B cells were injected into the tail vein of RAG12/2 mice together with 5 3 106 WT or SAP2/2 CD4+ or CD8+ T cells. After 24 h, splenocytes of RAG12/2 recipients were harvested and analyzed by ﬂow cytometry for B cell survival. Results are shown as x-fold of WT B cells and WT CD4+ T cells. n =3.(E) WT mice were injected i.v. into the tail vein with 150 mg of anti-CD4 neutralizing Ab or with PBS alone as control. After 4 d following the injection, PB was collected from the tail vein and analyzed for SLAMF6 expression on B cells. n = 3. Results are shown as fold change relative to the control group; bars indicate SEM. In all results, each dot represents a biological repeat; n represents the number of experiments. *p , 0.05, **p , 0.01,***p , 0.001, ****p , 0.0001.

To further examine the role of SLAMF6 in B cells, SLAMF6 PB B cells by CD74 siRNA (Supplemental Fig. 3F). Reduced expression in healthy human PB T cells was knocked down by expression levels of SLAMF6 were detected on B cells with SLAMF6 siRNA (Supplemental Fig. 3D). SLAMF6-deficient knockdown of CD74 (Fig. 7D). T cells were then cocultured with human PB B cells. CD74 surface levels were analyzed after 48 h. As shown in Fig. 7A and SAP and SLAMF6 regulate survival of XLP B cells Supplemental Fig 3E, lower levels of surface CD74 were detected XLP is a severe immunodeficiency associated with mutations of the on B cells incubated with SLAMF6 knockdown T cells. Addi- SH2D1A gene that encodes SAP. Although, the absolute number of tionally, healthy total PB cells (T and B cells) were incubated in total B cells in XLP patients is normal (38), these patients exhibit a the presence of either SLAMF6 neutralizing (32) or control IgG marked reduction in their PB memory B cell population (39–41) Abs. Reduced T cell–B cell interactions through SLAMF6 resul- (Fig. 7E, 7F). ted in lower CD74 cell surface levels on B cells when compared To determine whether the lack of SAP in these patients regulates with IgG-treated cells (Fig. 7B). Furthermore, blocking SLAMF6 B cell maintenance through the control of CD74 expression, CD74 reduced Bcl-2 protein levels (Fig. 7C). These results suggest that expression was analyzed in XLP patient–derived PB B cells from SLAMF6 regulates CD74 cell surface expression and human three patients. CD74 cell surface expression on total PB B cells B cell survival. (CD19+ cells; Fig. 7G) and on naive (CD19+CD24lowCD38int; Finally, to determine whether CD74 autoregulates SLAMF6 Fig. 7H) and memory (CD19+CD24hiCD38low; Fig. 7I) PB B cells expression, CD74 expression was downregulated in healthy human derived from the XLP patients was significantly reduced compared 2754 CD4+ T CELLS REGULATE NAIVE B CELL SURVIVAL Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 6. SAP-dependent B cell–T cell crosstalk regulates the expression of CD74, SLAMF6, and B cell survival in human cells. (A–D) Healthy human PB B cells were cocultured with healthy human PB T cells that were treated with control scrambled or SAP siRNA. Following 24 h of incubation, RNA was puriﬁed, and mRNA was analyzed by quantitative RT-PCR for CD74 mRNA (B) and Bcl2 (D). After 48 h, cell surface expression of SLAMF6 (A, right panel shows representative SLAMF6 histograms, n = 3), CD74 (C, right panel shows representative CD74 histograms), and annexin V (E) on CD19+ positive population was determined by FACS. Results are shown as fold change relative to the siControl group; bars indicate SEM. Each dot represents a biological repeat; n represents the number of experiments. *p , 0.05, **p , 0.01. with its levels on WT human B cells. Almost no change in CD74 Discussion + expression was observed on the transitional subpopulation (CD19 Peripheral B cell numbers are tightly regulated by various ho- CD24hiCD38hi; Fig. 7J). The reduced expression levels of CD74 meostatic mechanisms to maintain a functional and selective humoral in the naive state of the B cells suggest that SAP expression in immune response. Our present study provides evidence that naive T cells regulates B cell survival from the naive stage. mature B cell survival is not only an intrinsic process, but its The Journal of Immunology 2755 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 7. The role of SLAMF6 in CD74 regulation on human B cells in vitro and in vivo. (A) Healthy human B cells were cocultured with healthy human PB T cells that were treated with SLAMF6 or control siRNA. After 48 h, CD74 surface expression was analyzed by FACS. Right panel shows representative CD74 histograms. n =3.(B and C) Purified PB B and T cells were incubated in the presence of anti-SLAMF6 or control IgG Abs. After 24 h, (B) CD74 surface expression was analyzed by flow cytometry. (C) Purified B cells were lysed and analyzed for Bcl2 and tubulin by Western blot analysis (n = 3). (D) PB B cells were treated with control scrambled or CD74 siRNA and cocultured with nontreated healthy PB T cells. After 48 h, the cells were analyzed by flow cytometry for SLAMF6. n = 3. Results are shown as fold change relative to the siControl group; bars indicate SD. Each dot represents one sample; n represents the number of experiments. (E and F) Lymphocytes were isolated from PB derived from XLP and healthy patients and stained for B cell populations. Results are shown as fold change relative to the control group; bars indicate SD. Each repeat represents a different blood sample from the same patient. (E) Memory B cells (patient 1, n = 8; patient 2, n = 6; patient 3, n = 5). (F) Representative contour plots of B cell population in healthy control and in XLP patients. (G–J) CD74 expression was analyzed on B cell populations derived from subjects with XLP and compared with healthy controls. CD74 surface expression on the total CD19+ population (patient 1, n = 8; patient 2, n = 7; patient 3, n =4)(G), mature B cell population (patient 1, n =7; patient 2, n = 6; patient 3, n =3)(H), memory B cell population (patient 1, n = 7; patient 2, n = 6; patient 3, n =3)(I), and transitional B cell population (patient 1, n = 5; patient 2, n = 6; patient 3, n =3)(J). *p , 0.05, ***p , 0.001, ****p , 0.0001. ns, not significant (p $ 0.05). 2756 CD4+ T CELLS REGULATE NAIVE B CELL SURVIVAL regulation requires CD4+ T cells in an SLAM/SAP-dependent B cells in XLP patients is normal (38), these patients exhibit a manner. marked reduction in their PB memory B cell population (39–41). SLAMs and SAP mediate many immunological processes, in- However, previous analysis of the B cells in XLP patients revealed cluding the interaction of CD4+ T cells with B cells in the germinal an increase in the circulating immature/transitional B cells (46). centers, whereas SAP2/2 CD4+ T cells are unable to form long- Our results suggest that the elevation of the immature population term conjugates with cognate B cells, leading to perturbed germinal may result from a constant death of the mature B population that center formation (22–24). Our study followed an Ag-independent needs to be constantly replenished. interaction between naive B and T cells. Our results reveal a novel Taken together, our results suggest that T cells have the ability to role for SAP and SLAMF6 in regulation of mature naive B cell regulate peripheral B cell survival through SLAMF6/SAP. This survival. T cells lacking SAP cannot support the SLAMF6-induced pathway, in turn, increases MIF secretion, which activates CD74. B cell survival pathway. The two cell types can interact through This consequently enhances cell survival and SLAMF6 expression, SLAMF6, but the absence of SAP leads to lower CD74 expression thereby forming a positive feedback loop supporting B cell on B cells and their reduced survival. maintenance (Supplemental Fig. 4). The adaptor molecule SAP controls signal transduction pathways downstream of the SLAM family receptors and is a key Acknowledgments regulator of normal immune function in T, NK, and NKT cells (15, We thank members of the Shachar laboratory for fruitful discussions 18). However, B cells do not express SAP (19), and EAT-2 was and support. We thank Dr. Z. Shulman for suggestions and help with described as a functional homolog in these cells (20, 21). Our reagents. results show that EAT-2 protein is expressed in B cells. Activation Downloaded from of SLAMF6 induces EAT-2 binding to SLAMF6, resulting in Disclosures EAT-2 phosphorylation. The authors have no financial conflicts of interest. It was previously shown that the lymphocyte populations are grossly normal in SAP-deficient mice, although occasional mutant animals exhibit a lower percentage of B cells in the spleen (25). References 1. Kraus, M., M. B. Alimzhanov, N. Rajewsky, and K. Rajewsky. 2004. Survival of That the steady-state levels of B cells are not dramatically altered a b http://www.jimmunol.org/ 2/2 resting mature B lymphocytes depends on BCR signaling via the Ig / heter- in the SAP mice might be due to redundancy and constant odimer. Cell 117: 787–800. replenishment of the B population, and from the support provided 2. Srinivasan, L., Y. Sasaki, D. P. Calado, B. Zhang, J. H. Paik, R. A. DePinho, J. L. Kutok, J. F. Kearney, K. L. Otipoby, and K. Rajewsky. 2009. PI3 kinase by other cells in the environment. signals BCR-dependent mature B cell survival. Cell 139: 573–586. The SLAMF6-induced cascade autoregulates its own expression. 3. Mackay, F., W. A. Figgett, D. Saulep, M. Lepage, and M. L. Hibbs. 2010. B-cell Abrogation of stimulation via SLAMF6 and signaling through SAP stage and context-dependent requirements for survival signals from BAFF and the B-cell receptor. Immunol. Rev. 237: 205–225. leads to downregulation of SLAMF6 expression on B cells but not 4. Shachar, I., and M. Haran. 2011. The secret second life of an innocent chaper- on T cells. The lack of SAP in T cells results in reduced SLAMF6 one: the story of CD74 and B cell/chronic lymphocytic leukemia cell survival. expression levels on B cells due to the perturbed signaling cascade Leuk. Lymphoma 52: 1446–1454. 5. Bucala, R., and I. Shachar. 2014. The integral role of CD74 in antigen presen- induced by the T cell–B cell interaction in B cells. SLAMF6 el- tation, MIF signal transduction, and B cell survival and homeostasis. Mini Rev. by guest on September 23, 2021 evates CD74 expression levels, which increases the survival po- Med. Chem. 14: 1132–1138. 6. Stumptner-Cuvelette, P., and P. Benaroch. 2002. Multiple roles of the invariant tential of naive B cells. Beyond its role in B cell survival, CD74 chain in MHC class II function. Biochim. Biophys. Acta 1542: 1–13. regulates SLAMF6 expression levels. 7. Ong, G. L., D. M. Goldenberg, H. J. Hansen, and M. J. Mattes. 1999. Cell CD4+ T cells are the main players in supporting B cell survival. surface expression and metabolism of major histocompatibility complex class II + invariant chain (CD74) by diverse cell lines. Immunology 98: 296–302. In vivo depletion of CD4 T cells in mice significantly decreased 8. Leng, L., C. N. Metz, Y. Fang, J. Xu, S. Donnelly, J. Baugh, T. Delohery, SLAMF6 expression. CD4+ T cells express high levels of MIF, a Y. Chen, R. A. Mitchell, and R. Bucala. 2003. MIF signal transduction initiated ligand of CD74. T cell–B cell crosstalk induces the expression of by binding to CD74. J. Exp. Med. 197: 1467–1476. + + 9. Starlets, D., Y. Gore, I. Binsky, M. Haran, N. Harpaz, L. Shvidel, S. Becker- MIF in the CD4 T population via SAP and SLAMF6. CD4 Tcells Herman, A. Berrebi, and I. Shachar. 2006. Cell-surface CD74 initiates a sig- can support the survival of B cells by secretion of MIF. Deficiency naling cascade leading to cell proliferation and survival. Blood 107: 4807–4816. 10. Gore, Y., D. Starlets, N. Maharshak, S. Becker-Herman, U. Kaneyuki, L. Leng, of MIF has an incomplete effect on B cell survival. This could be R. Bucala, and I. Shachar. 2008. Macrophage migration inhibitory factor induces explained by the existence of an additional CD74 ligand. Another B cell survival by activation of a CD74-CD44 receptor complex. J. Biol. Chem. ligand of CD74 is MIF2, also termed D-dopachrome tautomerase 283: 2784–2792. 11. Binsky, I., M. Haran, D. Starlets, Y. Gore, F. Lantner, N. Harpaz, L. Leng, (42). MIF1 (also known as MIF) and MIF2 possess overlapping D. M. Goldenberg, L. Shvidel, A. Berrebi, et al. 2007. IL-8 secreted in a mac- functions and expression patterns (43). It is possible that MIF2 rophage migration-inhibitory factor- and CD74-dependent manner regulates might play an additional role in the T cell–mediated maintenance of B cell chronic lymphocytic leukemia survival. Proc. Natl. Acad. Sci. USA 104: 13408–13413. naive B cells. MIF1 and MIF2 binding to CD74 induces a survival 12. Binsky-Ehrenreich, I., A. Marom, M. C. Sobotta, L. Shvidel, A. Berrebi, cascade involving NF-kB activation and Bcl-2 expression (5). I. Hazan-Halevy, S. Kay, A. Aloshin, I. Sagi, D. M. Goldenberg, et al. 2014. CD84 is a survival receptor for CLL cells. Oncogene 33: 1006–1016. Collectively, our results suggest a model whereby B cells can 13. Calpe, S., N. Wang, X. Romero, S. B. Berger, A. Lanyi, P. 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