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. Furthermore, in X-linked lymphoproliferative disease patients, SAP deficiency reduces CD74 expression, resulting in the perturbation of B cell maintenance from the naive stage. Thus, naive T cells regulate B cell survival in a SLAMF6- and SAP-dependent manner.

The survival of peripheral naive mature B cells is dependent on three key cascades: 1) BCR tonic signals (e.g., Igα and Syk) (1, 2); 2) the B cell activating factor receptor, which binds the B cell activating factor, belonging to the TNF family (the B cell activating factor is also known as BLyS/TALL-1/THANK/zTNF4) (3); and 3) CD74 (invariant chain) expressed on B cells, and its cognate ligand, macrophage migration inhibitory factor (MIF), which is secreted by almost all cell types. These pathways have complementary roles in B cell survival (4, 5).

CD74 is a type II integral membrane protein that acts as a chaperone for MHC class II protein expression (6). A small proportion of CD74 is modified by the addition of chondroitin sulfate, and this form of CD74 is expressed on the surface of APCs (including monocytes and B cells) and epithelial cells (7). It was previously shown that macrophage MIF binds to the CD74 extracellular domain, a process that results in the initiation of a signaling pathway in these cells (8).

CD74 stimulation by MIF induces a signaling cascade leading to NF-κB activation, as well as transcription of genes that regulate the entry of the stimulated B cells into the S phase, an increase in DNA synthesis, cell division, and augmented expression of antiapoptotic proteins (5, 9, 10). The CD74 receptor induces a similar 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 CLL cell survival, we previously screened for CD74 target genes. One molecule, whose expression was strongly upregulated by CD74 activation, is signaling lymphocyte activation molecule (SLAM)F5 (CD84), a member of the SLAM Ig superfamily (12).

The SLAM family of receptors includes homophilic and heterophilic receptors that modulate the behavior of immune cells (1315). These receptors share a common ectodomain organization: a membrane-proximal Ig-like constant domain, and a membrane-distal Ig variable domain that is responsible for ligand recognition. SLAM receptors interact with SLAM-associated protein (SAP)–related molecules, a group of SRC homology 2 (SH2) domain adaptors. The SAP family is comprised of three members: SAP, Ewing’s sarcoma–associated transcript 2 (EAT-2), and, in rodents, EAT-2–related transducer (16, 17). SAP controls signal transduction pathways downstream of the SLAM family receptors, and it is a key regulator of normal immune function in T, NK, and NKT cells (15, 18). However, B cells do not express SAP (19), and EAT-2 was suggested to serve as its functional homolog in these cells (20, 21).

The SLAM receptors and their adaptor molecules were shown to be required for germinal center development and humoral memory (2224). However, their role in naive B cell maintenance has not been assessed in detail. Lymphocyte populations derived from SAP-deficient mice are grossly normal, although occasional mutant animals exhibit a higher percentage of T and NK cells, as well as a lower percentage of B cells in the spleen (25).

In the present study, we hypothesized that the SLAM family might be involved in the regulation of naive B cell survival in the cross-talk between naive B and naive T cells in an Ag-independent environment. Our findings demonstrate that interaction of B cells with T cells in a SLAMF6/SAP-mediated manner upregulates CD74 cell surface expression on B cells, inducing their survival in vitro and in vivo. This study highlights a crucial role of SAP expression on T cells in regulating B cell survival and provides new insights into the requirements for cell collaboration during homeostasis.

C57BL/6, CD74-deficient (26), MIF-deficient (27), SLAMF5-deficient (24), SH2D1A (SAP)-deficient (25) SLAMF6-deficient (28), and SLAMF1-deficient mice (29) were used in this study. All animal procedures were approved by the Animal Research Committee at the Weizmann Institute of Science. The animals in each experiment were aged and sex matched.

Human peripheral blood lymphocytes.

X-linked lymphoproliferative disease (XLP) patient samples were provided in compliance with the Institutional Review Board of the Hadassah Hospital. Healthy control samples were provided by the Magen David Adom in Israel blood bank.

Cells were separated as previously described (11). T lymphocytes were enriched by positive selection using anti-CD3 MACS beads (Miltenyi Biotec) according to the manufacturer’s protocol. B lymphocytes were enriched in the T cell depleted flow through.

Mouse splenic lymphocytes.

B lymphocytes were enriched by positive selection using anti-B220 beads (BD Biosciences), as previously described (9). The CD4 T cell population was enriched from the B220-negative fraction by positive selection using CD4 (L3T4) beads (Miltenyi Biotec) according to the manufacturer’s protocol. The CD8-enriched population was obtained as the negative fraction of the CD4 enrichment.

FACS analysis was performed using a FACSCanto flow cytometer (BD Biosciences). Abs from the following sources were used: anti-CD74, Ly108, CD40L (eBioscience), CD19, B220, CD4, CD3, CD21, CD24, CD23, SLAMF5, SLAMF1 (BioLegend), anti-MIF (Abcam), and anti–EAT-2 (Proteintech). For assessment of apoptosis, annexin V and 7-aminoactinomycin D (7-AAD) staining was performed (BD Pharmingen). Flow cytometry data analysis was performed using FlowJo software (Tree Star).

Naive wild-type (WT) B cells (5 × 106) were cultured with WT or SAP−/− T cells (5 × 106) and incubated for 24 h in 24-well plates. Conjugates were enumerated by flow cytometry after the cell mixture was stained for CD3/CD4/CD8 and B220 at 4°C.

Cells were examined by imaging flow cytometry using the ImageStreamX Mark II (Amnis [part of EMD Millipore], Seattle, WA). At least 105 cells 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 root mean square feature (30).

B220+ B/CD3+ T cell conjugates were gated according to their area and aspect ratio to include doublets of relevant size. To eliminate doublets that did not consist of viable cells, or cells that were too far apart, only cells with the correct distance (3–7 μm) were gated. All the gates were visually inspected to verify the classifications.

Human cells.

Purified T cells and B cells (5 × 106) were subjected to electroporation as previously described (12). The cells were treated with the following small interfering RNAs (siRNAs): SAP, NTB-A, CD74 (ON-TARGETplus SMARTpool) and negative control siRNA (ON-TARGET plus nontargeting pool) (Dharmacon).

Mouse B cells.

B cells (5 × 106) were subjected to electroporation as previously described (12). The cells were treated with the following siRNAs: EAT-2 (ON-TARGET plus SMART pool) and negative control siRNA (ON-TARGETplus nontargeting pool) (Dharmacon).

RNA extraction and quantitative RT-PCR were performed as previously described (31). Primers are summarized in Table I.

Table I.
Primer sets for quantitative RT-PCR
GeneDirectionPrimer Sequence
Bcl-2 Forward 5′-GCCAACTGAGCAGAGTCTC-3′ 
 Reverse 5′-GGACAACATCGCCCTGTG-3′ 
CD74 Forward 5′-GGAGTACCCGCAGCTGAAGGGG-3′ 
 Reverse 5′-GAAGATAGGTCTTCCATGTCCAGTG-3′ 
SAP Forward 5′-TCATGGGGCTTTCATTTCAGGCAGACATCAGG-3′ 
 Reverse 5′-GACGCAGTGGCTGTGTAT-3′ 
NTB-A Forward 5′-TCCTTCTCTGTCTCTGCCCA-3′ 
 Reverse 5′-GCCCTGTGTTCGCTGAGTAG-3′ 
GeneDirectionPrimer Sequence
Bcl-2 Forward 5′-GCCAACTGAGCAGAGTCTC-3′ 
 Reverse 5′-GGACAACATCGCCCTGTG-3′ 
CD74 Forward 5′-GGAGTACCCGCAGCTGAAGGGG-3′ 
 Reverse 5′-GAAGATAGGTCTTCCATGTCCAGTG-3′ 
SAP Forward 5′-TCATGGGGCTTTCATTTCAGGCAGACATCAGG-3′ 
 Reverse 5′-GACGCAGTGGCTGTGTAT-3′ 
NTB-A Forward 5′-TCCTTCTCTGTCTCTGCCCA-3′ 
 Reverse 5′-GCCCTGTGTTCGCTGAGTAG-3′ 

Activation of CD74 was performed as previously described (9).

Human B cells (1 × 107) were incubated in the presence of 20 μg/ml monoclonal anti–NTB-A Ab (clone NT-7) or mouse IgG1 κ isotype control Abs (BioLegend), as previously described (32).

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 in 24-well plates containing an insert with a 5-μm pore polycarbonate membrane. B cells were seeded in the well, and T cells were seeded in the insert.

Immunoprecipitation of endogenously expressed proteins and their detection were performed on fresh mouse lymphocytes as previously described (12). For immunoprecipitation and blotting of EAT-2, anti–EAT-2 Ab (N-14; Santa Cruz Biotechnology) was used. pTYR was detected with the anti–p-Tyr (FL-293; Santa Cruz Biotechnology) Ab. Immunoprecipitation of Ly108 was performed with anti-Ly108 Ab (13G3-19D; eBioscience).

A mixture of B and T cells at a 1:1 ratio (total of 1 × 107 cells) in 300 μl of PBS per mouse was injected i.v. into the tail vein of RAG1−/− mice.

WT mice were injected with 150 μg/ml anti-CD4 mAb GK1.5 (BE0003-1; Bio X Cell) diluted in sterile PBS to a final volume of 200 μl, i.v. into the tail vein.

Data analysis was performed as previously described (33), using GraphPad Prism version 6.0f (GraphPad Software, La Jolla, CA). Data are reported as mean ± SEM. To determine significance of differences, we used either the unpaired Student t test or one-way ANOVA test for mouse group experiments, and a ratio paired Student t test for treatments of human samples.

To determine the role of SLAMs and their adaptor molecule, SAP, which is expressed in T cells, in the regulation of B cell maintenance, a coculture experiment was first performed. In this experiment, WT B220+ cells, which are mostly naive mature B cells, were cultured alone or in the presence of WT or SAP-deficient naive T cells. As shown in Fig. 1A, the presence of WT T cells significantly increased the survival of naive B cells compared with B cells cultured alone by ∼3-fold, whereas incubation with SAP-deficient T cells supported less B cell survival. No significant change was observed in WT and SAP−/− T cell survival rates in this coculture setting (Supplemental Fig. 1A). These results suggest an SAP-dependent beneficial effect of naive T cells on naive B cell survival.

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 × 106 WT or SAP−/− 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 SAP−/−-derived B cells were cultured alone or at a 1:1 ratio with 5 × 106 WT or SAP−/− 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 SAP−/− 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.

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 × 106 WT or SAP−/− 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 SAP−/−-derived B cells were cultured alone or at a 1:1 ratio with 5 × 106 WT or SAP−/− 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 SAP−/− 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.

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To examine whether cell–cell contact or soluble factors are responsible for the T cell–dependent regulation of B cell survival, similar cultures were established, but direct B cell–T cell interactions were prevented by a Transwell membrane. As shown in Fig. 1A (open bars), B and T cell contacts were required for the T cell control of the B cell live population. Abrogation of cell contacts by the Transwell membrane reduced cell survival to levels observed in B cells cocultured with SAP-deficient T cells.

It was previously shown that SAP−/− T cells show impaired ability to generate stable interactions with B cells following immunization (22). To determine whether SAP deficiency affects naive B cell/T cell conjugates, WT or SAP−/−-derived B cells were cultured with WT or SAP−/− T cells (25). After 24 h, the cells were analyzed for double-positive staining of CD3+ and B220+, as was previously described (22). Surprisingly, as seen in Fig. 1B, SAP−/− T cells formed similar numbers of T cell/B cell conjugates as WT T cells. We further validated our results and the existence of naive T cell/B cell conjugates by looking for naive T cell/B cell doublets using image-based quantification. B cells were cocultured with WT T cells for 24 h, and the cocultures were stained for CD3 and B220. As shown in Fig. 1C and 1D, T cell (CD3+)/B cell (B220+) doublets were detected in this setting, suggesting direct contact between the cells. To further resolve the role of SAP in this interaction, and to determine whether formation of doublets is inhibited in the absence of SAP in T cells, B cells were cocultured with SAP-deficient T cells, and the number of doublets was analyzed. As shown in Fig. 1D, SAP deficiency did not affect the percentage of T cell/B cell doublets, suggesting that in the absence of SAP, T cells can still interact with the B cells but their downstream cascade is altered. Because SAP deficiency in B cells did not cause any intrinsic defect on their cell survival (Supplemental Fig. 1B), these results suggest that T cells support B maintenance in an SAP-dependent manner.

We next followed the SAP-dependent molecular mechanism regulating splenic B cell survival. Our hypothesis was that because CD74 is a survival receptor expressed on B cells, its expression or function might be modulated by an SAP-dependent T cell–B cell interaction. Therefore, CD74 cell surface expression was analyzed on B cell splenic populations derived from WT and SAP-deficient mice. A significant reduction in surface CD74 expression was detected on total B cells, mature (B220+, CD21intCD24low) and transitional B cell (transitional 1, B220+, CD21lowCD24high; transitional 2, CD21highCD24highCD23high) populations derived from SAP-deficient mice (Fig. 2A).

FIGURE 2.

SAP-dependent B and T cell interactions regulate CD74 expression on the B cell surface. (A) WT and SAP−/− 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 SAP−/− derived B cells and WT or SAP−/− T splenocytes were isolated. B cells were cultured alone or at a 1:1 ratio with 5 × 106 WT or SAP−/− T cells in 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 SAP−/− B cells were cultured for 24 h alone or at a 1:1 ratio with 5 × 106 WT or SAP−/−. 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).

FIGURE 2.

SAP-dependent B and T cell interactions regulate CD74 expression on the B cell surface. (A) WT and SAP−/− 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 SAP−/− derived B cells and WT or SAP−/− T splenocytes were isolated. B cells were cultured alone or at a 1:1 ratio with 5 × 106 WT or SAP−/− T cells in 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 SAP−/− B cells were cultured for 24 h alone or at a 1:1 ratio with 5 × 106 WT or SAP−/−. 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).

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Next, WT or SAP−/−-derived B cells were cultured alone or with naive WT or SAP-deficient T cells for 24 h, and expression of surface CD74 was analyzed. B cells (WT or SAP−/−) cultured alone or with SAP-deficient T cells exhibited significantly lower cell surface expression levels of surface CD74 compared with B cells cocultured with WT T cells (Fig. 2B). Furthermore, lower levels of CD74 were observed in the Transwell setting, in which direct interaction between WT B and T cells was inhibited (Fig. 2B, open bars).

The lower cell surface expression of CD74 seen in the absence of SAP-expressing T cells might result from an upregulation of its levels on B cells in the presence of T cells or by downregulation of its levels due to the lack of a proper signal from the SAP−/− T cells. To address this point, the levels of cell surface CD74 on freshly isolated splenic B cells were compared with its levels detected on B cells cocultured for 24 h with T cells. As seen in Fig. 2C, following coculture with WT T cells, elevated levels of cell surface CD74 were detected on the B cells, whereas incubation with SAP−/− T cells resulted in a milder elevation. These results suggest a mechanism whereby T cells upregulate CD74 cell surface levels on B cells in an SAP-dependent manner.

SAP transmits signals induced by the SLAM family of receptors (16). To determine which SLAM member regulates the T and B cell contact that results in survival of B cells, the effects of three SLAM members, SLAMF6 (Ly108 or NTB-A in humans), SLAMF5 (CD84), and SLAMF1 (CD150), were analyzed. These receptors were shown to work in synergy in the regulation of Ab responses and were identified as mediators of the B cell–T cell contact in germinal centers (24, 34). We first analyzed the expression of these receptors on splenic B cells. As shown in Fig. 3A, high levels of SLAMF6 were detected on the surface of B cells, whereas SLAMF1 and SLAMF5 levels were significantly lower. Additionally, SLAMF6 expression levels on B cells derived from SAP-deficient mice were slightly reduced (Supplemental Fig. 1C) whereas almost no differences were detected in SLAMF5 (Supplemental Fig. 1D) and SLAMF1 (Supplemental Fig. 1E) cell surface levels. These results suggest that SLAMF6 may have a more important role in support of B cells by naive T cells.

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 × 106 WT, SLAMF6−/−, SLAMF5−/−, or SLAMF1−/− 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 SAP−/− (B cells before co-culture) and purified naive WT B cells were cultured for 24 h alone at a 1:1 ratio with 5 × 106 WT or SAP−/− 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 SAP−/−-derived B cells and WT or SAP−/− T splenocytes were cultured alone or at a 1:1 ratio with 5 × 106 WT or SAP−/− 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 × 106 WT or SLAMF6−/− 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 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).

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 × 106 WT, SLAMF6−/−, SLAMF5−/−, or SLAMF1−/− 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 SAP−/− (B cells before co-culture) and purified naive WT B cells were cultured for 24 h alone at a 1:1 ratio with 5 × 106 WT or SAP−/− 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 SAP−/−-derived B cells and WT or SAP−/− T splenocytes were cultured alone or at a 1:1 ratio with 5 × 106 WT or SAP−/− 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 × 106 WT or SLAMF6−/− 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 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).

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We then analyzed the role of these receptors expressed on T cells in the regulation of naive B cell survival. Naive B cells were cocultured with naive WT or SLAMF6/SLAMF5/SLAMF1-deficient T cells (24, 28, 29) for 24 h, and B cell survival was then analyzed. Although deficiency of each of these receptors interfered with the support induced by T cells, SLAMF6 deficiency caused the most significant reduction in B cell survival (Fig. 3B). Additionally, the abrogation of T cell support to B cells following disruption of the interaction was independent of whether the B cells or the T cells were SLAMF6 deficient (Supplemental Fig. 1F), SLAMF5 deficient (Supplemental Fig. 1G), or SLAMF1 deficient (Supplemental Fig. 1H). Based on these results, we decided to further focus on the role of SLAMF6 in the T cell–induced survival support.

Next, to determine the role of SLAMF6 expression in T cell–dependent B cell survival, murine splenic WT or SAP−/−-derived B cells were cultured alone or with naive WT or SAP-deficient T cells for 24 h, and expression of SLAMF6 was analyzed. SLAMF6 expression was significantly upregulated on B cells cultured with T cells, compared with its levels on freshly isolated B cells (Fig. 3C). This elevation did not occur in the presence of SAP−/− T cells (Fig. 3C, 3D). Moreover, no change in SLAMF6 expression levels on T cells was detected under these conditions (Supplemental Fig. 1I). These results suggest a T cell–dependent elevation of SLAMF6 expression on B cells mediated through SAP. When B cell–T cell interactions were prevented by a Transwell membrane, CD74 cell surface levels on B cells remained at the low levels observed in B cells incubated with SAP-deficient T cells (Fig. 3D, open bars).

We further investigated the role of SLAMF6 in T cell–dependent regulation of CD74 expression and B cell survival. First, CD74 expression levels were analyzed on the SLAMF6−/− cells. Similar surface levels of CD74 were detected on freshly isolated WT and SLAMF6−/− B cell populations (Supplemental Fig. 1J). Next, splenic WT or SLAMF6−/− B cells were cocultured with splenic WT or SLAMF6−/− T cells and levels of CD74 and B cell survival were analyzed (Fig. 3E, Supplemental Fig. 1K). Abolishing SLAMF6-mediated interaction between T and B cells led to a significant decrease in CD74 surface expression on B cells, accompanied by a downregulation in B cell survival (Supplemental Fig. 1F). SLAMF5 deficiency also resulted in a reduction in CD74 upregulation (Supplemental Fig. 1L), whereas SLAMF1 deficiency showed no effect at all (Supplemental Fig. 1M).

CD74 activation induces B cell survival (9, 10) (Supplemental Fig. 2A) and upregulates SLAMF6 expression on B cells (Fig. 3F). SAP−/− mice exhibit lower levels of CD74 on B cells (Fig. 2A) and a lower response of these B cells to CD74 Ab stimulation (data not shown). These results suggest that the SLAMF6 B cell–T cell interaction elevates CD74 expression on B cells, which results in B cell survival. Additionally, CD74 induces SLAMF6 levels, which can further induce/enhance the process.

B cells do not express SAP; instead, they express the mRNA of the adaptor molecule, EAT-2 (16, 35). To characterize the SLAMF6-induced cascade in B cells, we wanted to determine whether EAT-2 plays a role in the B cell side of this cascade. To this end, we first established the expression of EAT-2 protein in splenic B cells. Using a flow cytometry analysis, EAT-2 levels in B cells were compared with its levels in NK cells, which are known to express EAT-2, and in T cells, which express low levels of the adaptor (15, 18). As shown in Fig. 3G, B cells express the EAT-2 protein. Next, to determine whether EAT-2 is involved in the interaction via SLAMF6, a coimmunoprecipitation experiment was performed, in which SLAMF6 was pulled down by anti-SLAMF6 Ab from B cell lysates and EAT-2 levels were analyzed in the immunoprecipitated proteins. As seen in Fig. 3H (top panel), SLAMF6 recruited EAT-2 in B cells, suggesting that EAT-2 transmits the SLAMF6 signal in B cells. Following the recruitment of EAT-2 to the receptor, it is phosphorylated on tyrosine residues, which activates it to transmit downstream signaling (14). We next wanted to identify the presence of phosphorylated EAT-2 in naive B cells. EAT-2 was pulled down from naive B cell lysate, and the immunoprecipitated proteins were analyzed for p-Tyr. As demonstrated in Supplemental Fig. 2B, phosphorylated EAT-2 was detected in the lysates of naive B cells. To directly demonstrate recruitment of phosphorylated EAT-2 to SLAMF6, SLAMF6 was pulled down and the immunoprecipitated proteins were analyzed. Phosphorylated EAT-2 was pulled down with SLAMF6 (Fig. 3H, bottom panel), suggesting that in B cells, EAT-2 can be recruited to SLAMF6 and is activated. To directly show the involvement of EAT-2 in B cell survival, EAT-2 levels were knocked down in using siRNA against EAT-2. siEAT-2– or siControl-treated B cells were cultured with naive T cells for 48 h. A reduction of ∼40% in EAT-2 protein levels was detected in siEAT-2–treated B cells (Supplemental Fig. 2C) (Table I). In this setting, SLAMF6 (Fig. 3I) and live B cell (Fig. 3J) levels showed a significant reduction of ∼20%. This further suggests that EAT-2 regulates SLAMF6 expression and B cell survival.

Next, we wanted to identify which T cell subpopulation, CD4+ or CD8+, is involved in the maintenance of naive B cells. First, as seen in Supplemental Fig. 2D, CD4 T cells express higher levels of SLAMF6 than do CD8 T cells. Next, WT, SAP−/−, or SLAMF6−/− CD4+ or CD8+ T cells were purified (Supplemental Fig. 2E) and cocultured with B cells. As shown in Fig. 4A, a greater number of conjugates of B cells with CD4+ T cells compared with CD8+ T cells were detected. Additionally, only WT CD4+ T cells managed to support B cell survival (Fig. 4B) and SLAMF6 expression on B cells (Fig. 4C). Thus, CD4+ T cells regulate B cell survival in an SAP- and SLAMF6-dependent manner.

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 × 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 × 106 WT or SAP−/− 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 MIF−/− splenocytes (background). (E) Naive WT/SAP−/−/SLAMF6−/− T splenocytes were cultured alone or at a 1:1 ratio with 5 × 106 WT B cells. After 24 h, expression of MIF in CD3+CD4+ was analyzed by flow cytometry. n = 4 (FH) Naive WT B splenocytes were cultured alone or at a 1:1 ratio with 5 × 106 WT or MIF−/− 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).

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 × 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 × 106 WT or SAP−/− 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 MIF−/− splenocytes (background). (E) Naive WT/SAP−/−/SLAMF6−/− T splenocytes were cultured alone or at a 1:1 ratio with 5 × 106 WT B cells. After 24 h, expression of MIF in CD3+CD4+ was analyzed by flow cytometry. n = 4 (FH) Naive WT B splenocytes were cultured alone or at a 1:1 ratio with 5 × 106 WT or MIF−/− 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).

Close modal

Because CD74 expression is regulated by the SLAMF6-mediated T cell–B cell interaction, we wanted to determine whether the SLAMF6/SAP-dependent B and T cell interaction regulates the expression of CD74 ligand, MIF, in T cells. As shown in Fig. 4D, CD4+ T cells expressed higher levels of intracellular MIF compared with its levels in CD8+ T cells and B cells. Next, regulation of MIF expression levels in T cells by splenic B cells was determined. Whereas B cells elevated MIF levels in WT CD4+ T cells in the coculture setting, MIF levels in CD4+ T cells lacking SAP or SLAMF6 were not upregulated by B cells (Fig. 4E). This elevation was specific to the CD4+ T cells and was not detected in the CD8+ population (Supplemental Fig. 2F).

To directly determine whether MIF plays a role in this crosstalk, murine WT splenic B cells were cultured alone or with naive WT or MIF-deficient T cells (27) for 24 h. FACS analysis revealed a reduced B cell survival (Fig. 4F), CD74 cell surface expression (Fig. 4G), and SLAMF6 (Fig. 4H) on B cells incubated with MIF-deficient T cells. These results imply that MIF secreted from T cells is essential for B cell survival in an SLAMF6- and CD74-dependent manner. CD74 expression on B cells cultured with T cells deficient in SAP, SLAMF6, or MIF did not induce an upregulation of CD74 expression on B cells, suggesting that all of these molecules contribute to the T cell support of B cell survival (Supplemental Fig. 2G).

Following immunization or infection, binding of CD40L on the T cells to CD40 on B cells promotes B cell proliferation, germinal center development, and the differentiation of B cells into Ab-secreting plasma cells (36). It was previously shown that SAP downregulates the expression of CD40L on T cells, as SAP−/− T cells exhibit higher levels of CD40L (23). We therefore determined CD40L expression on naive T cells in the unstimulated coculture setting. WT/SLAMF6−/−/SAP−/− CD4+ T cells were cultured alone or with B cells, and CD40L was analyzed on the T cells. As described previously, SAP−/− CD4+ T cells exhibited elevated levels of CD40L compared with WT cells (Supplemental Fig. 2H). However, the presence of B cells in the coculture had no effect on CD40L cell surface expression. Furthermore, comparable levels of CD40L expression were observed on WT CD4+/CD8+ or SLAMF6−/− CD4+ T cells (Supplemental Fig. 2I). Thus, CD40L does not play a part in the naive T cell–B cell interaction.

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 WT splenic B cells were adoptively transferred together with purified WT or SAP−/− splenic T cells into lymphocyte-deficient RAG1−/− recipients, which lack mature B and T cells. The mice were sacrificed 24 h after the cell transfer. CD74 (Fig. 5A) and SLAMF6 (Fig. 5B) cell surface expression levels were significantly lower on B cells cotransferred with SAP-deficient naive T cells, compared with their levels in the presence of WT T cells. Additionally, the percentage of the live B cell population was downregulated when B cells were transferred together with SAP-deficient T cells (Fig. 5C). Moreover, to directly show the role of CD4+ T cells in vivo, WT naive B cells were adoptively transferred into RAG1−/− alone or with WT CD8+ T cells, and WT or SAP−/− CD4+ T cells. As seen in Fig. 5D, only WT CD4+ T cells supported B cell survival.

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. (AD) Naive WT B cells were injected into the tail vein of RAG1−/− mice together with WT or SAP−/− 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 RAG1−/− mice together with 5 × 106 WT or SAP−/− CD4+ or CD8+ T cells. After 24 h, splenocytes of RAG1−/− 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 μg 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.

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. (AD) Naive WT B cells were injected into the tail vein of RAG1−/− mice together with WT or SAP−/− 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 RAG1−/− mice together with 5 × 106 WT or SAP−/− CD4+ or CD8+ T cells. After 24 h, splenocytes of RAG1−/− 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 μg 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.

Close modal

To further investigate the role of CD4+ T cells in the maintenance of naive B cells, CD4+ cells were depleted by injection of anti-CD4 Ab (Supplemental Fig. 2J) (37). After 4 d, spleens were harvested and analyzed for cell surface markers. SLAMF6 levels were significantly reduced on B cells derived from anti-CD4–treated mice compared with control-injected mice (Fig. 5E), suggesting a role for SLAMF6 expression on CD4+ cells in B cell maintenance. CD74 expression levels were not altered at this time point (data not shown). These results show that naive CD4+ T cells are required in vivo for the maintenance of B cells in an SAP-dependent manner.

We next wanted to determine whether a similar SAP-dependent T cell–induced survival pathway exists in human B cells; to this end, SAP expression in healthy human peripheral blood (PB) T cells was knocked down by SAP siRNA (Supplemental Fig. 3A). SAP-deficient T cells were then cocultured with untreated human PB B cells, and their SLAMF6 expression levels were analyzed. As shown in Fig. 6A and Supplemental Fig. 3B, lower SLAMF6 cell surface expression levels were detected on B cells cultured with SAP-deficient T cells compared with B cells cultured with T cells treated with control siRNA. CD74 mRNA (24 h; Fig. 6B) and protein (48 h; Fig. 6C, Supplemental Fig. 3C) levels were then analyzed in B cells incubated with T cells deficient in SAP. CD74 message and protein levels were significantly reduced in healthy human B cells cocultured with SAP-deficient T cells, compared with their levels when incubated with T cells treated with a control siRNA.

FIGURE 6.

SAP-dependent B cell–T cell crosstalk regulates the expression of CD74, SLAMF6, and B cell survival in human cells. (AD) 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.

FIGURE 6.

SAP-dependent B cell–T cell crosstalk regulates the expression of CD74, SLAMF6, and B cell survival in human cells. (AD) 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.

Close modal

Next, we determined whether reduced SAP, SLAMF6, and CD74 levels lead to cell death in human B cells. Incubation of B cells with SAP knockdown T cells significantly reduced their Bcl-2 mRNA levels (Fig. 6D) and induced their death (Fig. 6E). Collectively, these results suggest that SAP plays a dominant role in T cell induction of peripheral human B cell survival by regulating surface CD74 and SLAMF6 expression.

To further examine the role of SLAMF6 in B cells, SLAMF6 expression in healthy human PB T cells was knocked down by SLAMF6 siRNA (Supplemental Fig. 3D). SLAMF6-deficient T cells were then cocultured with human PB B cells. CD74 surface levels were analyzed after 48 h. As shown in Fig. 7A and Supplemental Fig 3E, lower levels of surface CD74 were detected on B cells incubated with SLAMF6 knockdown T cells. Additionally, healthy total PB cells (T and B cells) were incubated in the presence of either SLAMF6 neutralizing (32) or control IgG Abs. Reduced T cell–B cell interactions through SLAMF6 resulted in lower CD74 cell surface levels on B cells when compared with IgG-treated cells (Fig. 7B). Furthermore, blocking SLAMF6 reduced Bcl-2 protein levels (Fig. 7C). These results suggest that SLAMF6 regulates CD74 cell surface expression and human B cell survival.

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. (GJ) 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).

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. (GJ) 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).

Close modal

Finally, to determine whether CD74 autoregulates SLAMF6 expression, CD74 expression was downregulated in healthy human PB B cells by CD74 siRNA (Supplemental Fig. 3F). Reduced expression levels of SLAMF6 were detected on B cells with knockdown of CD74 (Fig. 7D).

XLP is a severe immunodeficiency associated with mutations of the SH2D1A gene that encodes SAP. Although, the absolute number of total B cells in XLP patients is normal (38), these patients exhibit a marked reduction in their PB memory B cell population (3941) (Fig. 7E, 7F).

To determine whether the lack of SAP in these patients regulates B cell maintenance through the control of CD74 expression, CD74 expression was analyzed in XLP patient–derived PB B cells from three patients. CD74 cell surface expression on total PB B cells (CD19+ cells; Fig. 7G) and on naive (CD19+CD24lowCD38int; Fig. 7H) and memory (CD19+CD24hiCD38low; Fig. 7I) PB B cells derived from the XLP patients was significantly reduced compared with its levels on WT human B cells. Almost no change in CD74 expression was observed on the transitional subpopulation (CD19+ CD24hiCD38hi; Fig. 7J). The reduced expression levels of CD74 in the naive state of the B cells suggest that SAP expression in T cells regulates B cell survival from the naive stage.

Peripheral B cell numbers are tightly regulated by various homeostatic mechanisms to maintain a functional and selective humoral immune response. Our present study provides evidence that naive mature B cell survival is not only an intrinsic process, but its regulation requires CD4+ T cells in an SLAM/SAP-dependent manner.

SLAMs and SAP mediate many immunological processes, including the interaction of CD4+ T cells with B cells in the germinal centers, whereas SAP−/− CD4+ T cells are unable to form long-term conjugates with cognate B cells, leading to perturbed germinal center formation (2224). Our study followed an Ag-independent interaction between naive B and T cells. Our results reveal a novel role for SAP and SLAMF6 in regulation of mature naive B cell survival. T cells lacking SAP cannot support the SLAMF6-induced B cell survival pathway. The two cell types can interact through SLAMF6, but the absence of SAP leads to lower CD74 expression on B cells and their reduced survival.

The adaptor molecule SAP controls signal transduction pathways downstream of the SLAM family receptors and is a key regulator of normal immune function in T, NK, and NKT cells (15, 18). However, B cells do not express SAP (19), and EAT-2 was described as a functional homolog in these cells (20, 21). Our results show that EAT-2 protein is expressed in B cells. Activation of SLAMF6 induces EAT-2 binding to SLAMF6, resulting in EAT-2 phosphorylation.

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). That the steady-state levels of B cells are not dramatically altered in the SAP−/− mice might be due to redundancy and constant replenishment of the B population, and from the support provided by other cells in the environment.

The SLAMF6-induced cascade autoregulates its own expression. Abrogation of stimulation via SLAMF6 and signaling through SAP leads to downregulation of SLAMF6 expression on B cells but not on T cells. The lack of SAP in T cells results in reduced SLAMF6 expression levels on B cells due to the perturbed signaling cascade induced by the T cell–B cell interaction in B cells. SLAMF6 elevates CD74 expression levels, which increases the survival potential of naive B cells. Beyond its role in B cell survival, CD74 regulates SLAMF6 expression levels.

CD4+ T cells are the main players in supporting B cell survival. In vivo depletion of CD4+ T cells in mice significantly decreased SLAMF6 expression. CD4+ T cells express high levels of MIF, a ligand of CD74. T cell–B cell crosstalk induces the expression of MIF in the CD4+ T population via SAP and SLAMF6. CD4+ T cells can support the survival of B cells by secretion of MIF. Deficiency of MIF has an incomplete effect on B cell survival. This could be explained by the existence of an additional CD74 ligand. Another ligand of CD74 is MIF2, also termed D-dopachrome tautomerase (42). MIF1 (also known as MIF) and MIF2 possess overlapping functions and expression patterns (43). It is possible that MIF2 might play an additional role in the T cell–mediated maintenance of naive B cells. MIF1 and MIF2 binding to CD74 induces a survival cascade involving NF-κB activation and Bcl-2 expression (5).

Collectively, our results suggest a model whereby B cells can manipulate T cell signaling via SLAMF6 and its adaptor SAP to maintain their full survival potential. Additional SLAM family members have been implicated in survival of specific B cell populations: SLAMF5 (CD84) induces survival of CLL cells, which upregulate its expression in a CD74/MIF-dependent manner (12, 33). SLAMF1 (CD150) regulates apoptosis, proliferation, and differentiation and IgG synthesis of B cells (44, 45). Analysis of these SLAM members shows that although they might have some role in the T cell–B cell interaction described in this study, it is less significant than that of SLAMF6.

XLP is a severe immunodeficiency associated with mutations of the gene that encodes SAP. Although the absolute number of total B cells in XLP patients is normal (38), these patients exhibit a marked reduction in their PB memory B cell population (3941). However, previous analysis of the B cells in XLP patients revealed an increase in the circulating immature/transitional B cells (46). Our results suggest that the elevation of the immature population may result from a constant death of the mature B population that needs to be constantly replenished.

Taken together, our results suggest that T cells have the ability to regulate peripheral B cell survival through SLAMF6/SAP. This pathway, in turn, increases MIF secretion, which activates CD74. This consequently enhances cell survival and SLAMF6 expression, thereby forming a positive feedback loop supporting B cell maintenance (Supplemental Fig. 4).

We thank members of the Shachar laboratory for fruitful discussions and support. We thank Dr. Z. Shulman for suggestions and help with reagents.

This work was supported in part by the Binational Science Foundation, the Cooperation Program in Cancer Research of the Deutsches Krebsforschungszentrum and Israel’s Ministry of Science, Technology and Space, the Quinquin Foundation, and the Rubenstein Charitable Foundation. I.S. is the incumbent of the Dr. Morton and A. Kleiman Professorial Chair.

The online version of this article contains supplemental material.

Abbreviations used in this article:

7-AAD

7-aminoactinomycin D

CLL

chronic lymphocytic leukemia

EAT-2

Ewing’s sarcoma–associated transcript 2

MIF

macrophage migration inhibitory factor

PB

peripheral blood

SAP

SLAM-associated protein

SH2

SRC homology 2

siRNA

small interfering RNA

SLAM

signaling lymphocyte activation molecule

WT

wild-type

XLP

X-linked lymphoproliferative disease.

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The authors have no financial conflicts of interest.

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