Membrane‐bound CD95 ligand modulates CD19‐mediated B cell receptor signaling and EBV activation

Post‐transplant lymphoproliferative disorders (PTLDs) are associated with Epstein‐Barr virus (EBV) infection in transplant recipients. Most of lymphoblastoid cell lines (LCLs) derived from EBV‐immortalized B cells or PTLDs are sensitive to CD95‐mediated apoptosis and cytotoxic T cell (CTL) killing. CD95 ligand (CD95L) exists as a transmembrane ligand (mCD95L) or a soluble form (sCD95L). Using recombinant mCD95L and sCD95L, we observed that sCD95L does not affect LCLs. While high expression of mCD95L in CTLs promotes apoptosis of LCLs, low expression induces clathrin‐dependent CD19 internalization, caspase‐dependent CD19 cleavage, and proteasomal/lysosomal‐dependent CD19 degradation. The CD95L/CD95‐mediated CD19 degradation impairs B cell receptor (BCR) signaling and inhibits BCR‐mediated EBV activation. Interestingly, although inhibition of the caspase activity restores CD19 expression and CD19‐mediated BCR activation, it fails to rescue BCR‐mediated EBV lytic gene expression. EBV‐specific CTLs engineered to overexpress mCD95L exhibit a stronger killing activity against LCLs. This study highlights that engineering EBV‐specific CTLs to express a higher level of mCD95L could represent an attractive therapeutic approach to improve T cell immunotherapy for PTLDs.

from either the recipients or donor grafts can drive the latently infected B cells to undergo uncontrolled proliferation and transformation in the setting of compromised T cell immune surveillance in the transplant recipient. 5EBV+ PTLDs result from the outgrowth of EBV-infected B cells, which typically express latency III program. 6,7e incidence of EBV+ PTLDs varies with the transplanted organs, the highest incidence observed for lung graft and the lowest incidence with kidney transplantation.
CTLs kill EBV-infected cells through perforin/granzyme and CD95 (Fas/APO-1)-mediated apoptotic signal. 13,14CD95-mediated immune surveillance is also essential for the control of spontaneous B cell lymphomas 15 and lymphoblastoid cell lines (LCLs) derived from EBV+ PTLDs or EBV immortalization are sensitive to CD95-mediated apoptosis. 16,17It has been reported that stimulation of peripheral blood lymphocytes with autologous LCLs gives rise to cytotoxic EBVtargeting CD4+ and CD8+ T cells, [18][19][20][21] which inhibit the growth of EBV-transformed LCLs through a CD95/CD95L (FasL)-mediated apoptotic mechanism. 19,22e CD95-CD95L signaling pathway is mainly viewed as an apoptotic inducer for maintaining immune homeostasis and eliminating virus-infected and transformed cells.CD95 is ubiquitously expressed and often upregulated at the surface of cancer cells and virus-infected cells.[25] Upon binding of mCD95L, predominantly expressed by activated T cells and natural killer cells, CD95 assembles the death-inducing signaling complex (DISC) composed of CD95, procaspase-8, procaspase-10, FADD, and c-FLIP, subsequently triggering apoptosis.Beyond apoptotic function, CD95-CD95L signaling also evokes nonapoptotic signals, promotes inflammation, and enhances tumorigenesis.[28] We have previously demonstrated that although CD95 engagement mediates apoptosis in sensitive EBV-associated lymphoma cells, it fails to induce cell death in apoptosis-resistant LCLs but inhibits EBV lytic reactivation through a molecular mechanism that remains to be elucidated. 29In this study, we established that although sCD95L does not affect LCLs, mCD95L exerts different biological activities based on its expression level.While a high level of mCD95L expression in CTL kills LCLs, low mCD95L expression induces nonapoptotic signals, downregulates CD19 expression, and impairs CD19-mediated B cell receptor (BCR) signaling, ultimately resulting in the inhibition of BCR-mediated EBV activation.

| Cell lines
All the blood samples used for PBMC isolation were collected from healthy EBV-voluntary blood donors who provided informed consent, which was also approved by the Ethics Committee of the Children's Hospital of Fudan University.
Other reagents were described in Supporting Information: Table 2.

| Plasmids
The EBV Zta and RTA promoter sequences were amplified from the genomic DNA of B95.8 cells and cloned into the pGL2 vector using the primers as shown in Supporting Information: Table 1.To generate the Flag-CD19-HA plasmid with an N-terminal Flag tag and a C-terminal HA tag, the CD19 coding fragment was amplified from LCL cDNA and cloned into pLVX-IRES-puro vector (Clontech) using the primers and restriction enzymes as shown in Supporting Information: Table 1.The CD19 mutants D388A, D435A, and D538A were generated by overlap PCR using the primers as shown in Supporting Information: Table 1.The CD19-HA and CD95-Flag plasmids with a C-terminal HA and Flag tag, respectively, were similarly generated using the primers as shown in Supporting Information: Table 1.

| Generation of lentiviruses and transduction
The mCD95L-expressing plasmid with a C-terminal Flag tag was generated by cloning the mCD95L fragment into the pLVX-tdtomato vector (Clontech) using the primers as shown in Supporting Information: Table 1.To generate lentivirus, pLVX-mCD95L-IRES-tdTomato or pLVX-IRES-tdtomato vector, together with psPAX2 packaging and pMD2.G envelop plasmids were transfected into HEK293T cells using Tenfect DNA transfection reagent (TEYE Biotechnology, FT19301) following the manufacturer's instruction.
After 48 h transfection, lentiviral supernatants were centrifuged at 2000 rpm for 10 min and filtered through 0.45-micron filter to remove cell debris.EBV-CTLs were transduced with lentiviruses by spinoculation at 2000 rpm for 2 h, followed by incubation for 48 h at 37°C for further analysis.

| Luciferase assay
DG75 cells were transfected with EBV RTA or Zta promoter luciferase plasmid, along with the Renilla reporter plasmids by using Amaxa Nucleofector II system (Lonza) according to the manufacturer's instruction.At 24 h postnucleofection, the transfected cells were stimulated with 100 ng/mL mCD95L or sCD95L in the presence or absence of 10 μg/mL anti-human Ig(G + M) for 24 h.Luciferase activity was measured using the Dual-Luciferase Reporter Gene Assay kit (Beyotime Biotechnology) according to the manufacturer's instructions and read using Hybrid Multi-Mode Microplate Reader Synergy H1 (Biotek).The luciferase value was normalized to renilla activity.Each sample was done in triplicate, each experiment was repeated three times.

| Apoptosis, proliferation, and CTL cytotoxicity assays
For the apoptosis assay, LCLs (1 × 10 6 cells/mL) were cultured in the presence of 0, 20, 100, or 500 ng/mL mCD95L (Ig-CD95L) or sCD95L for 24 h.Each sample had three replicates and the experiment was done three times.The treated cells were collected for Annexin V and propidium iodide staining with the Annexin V Apoptosis Detection kit APC (88 − 8007; eBioscience) following the manufacturer's instruction.
The stained cells were analyzed on an LSR Fortessa™ (Becton Dickinson) running FACSDiva Software version 6.
For proliferation assays, LCLs were stained with 1 μΜ carboxyfluorescein succinimidyl ester (CFSE) at 37°C for 15 min and washed three times with 2% FBS in PBS.CFSE-labeled cells were subsequently cultured in the presence of 100 ng/mL mCD95L or sCD95L and subjected to FACS analyses using LSR Fortessa™ (Becton Dickinson) running FACSDiva Software version 6. at Day 1, 2, and 3 post-treatment.
For the CTL cytotoxicity assay, lactate dehydrogenase (LDH) release was measured using the CytoTox 96 ® Non-Radioactive Cytotoxicity Assay kit (Promega G1780) according to the manufacturer's instructions.Briefly, 5 × 10 3 LCL target cells were cocultured with autologous EBV-CTLs transduced with lentiviruses expressing mCD95L or control in triplicate at various effector-target ratios in 96well plates for 24 h at 37°C.The absorbance value at 490 nm was read using Hybrid Multi-Mode Microplate Reader Synergy H1 (Biotek) for statistical analysis.Percent-specific lysis was calculated as follows: % Cytotoxicity = (Experimental value−Spontaneous value)/(Maximum value−Spontaneous value) × 100.

| Co-immunoprecipitation, Western blot, and flow cytometry
For co-immunoprecipitation, DG75 cells were nucleofected with CD19-HA and CD95-Flag expression plasmids using the Amaxa Nucleofector II system (Lonza) according to the manufacturer's instructions.After 48 h postnucleofection, the cells were treated with or without 100 ng/mL mCD95L for 3 h and lysed by sonication with NP40 buffer (50 mM Tris-HCl, pH7.4,150 mM NaCl, 1% NP-40, 5 mM EDTA) containing a complete protease inhibitor cocktail (Roche, #11836153001).Cell lysates were subjected to immunoprecipitation using anti-Flag affinity gel.The immunoprecipitated proteins were detected by immunoblotting with HA antibody.For endogenous immunoprecipitation, LCLs were stimulated with 100 ng/mL mCD95L (Ig-CD95L) or control for 1.5 h.Cell lysates were prepared and subjected to immunoprecipitation using CD95 (4C3) antibody, followed by immunoblot detection with CD19 and CD95 antibodies.
Cell surface staining and immunofluorescence staining were carried out as previously described. 29Flow cytometry was performed using FACS Celesta (BD Biosciences) and data were analyzed using Flowjo (Treestar Inc).For immunofluorescence, DG75 cells were nucleofected with CD95-Flag and CD19-HA expression plasmids.
After 48 h post-nucleofection, the cells were stimulated with mCD95L (100 ng/mL) or control medium for 3 h and fixed with 4% paraformaldehyde for 1 h at room temperature, followed by immunofluorescence staining.Images were acquired using an OLYMPUS Laser Scanning Microscope (FV1200).All experiments were at least repeated twice or three times.

| In vitro caspase cleavage assay
The Flag-CD19-HA plasmid, D338A, D435A, or D538A mutants were transfected into HEK293T cells using Tenfect DNA transfection reagent (TEYE Biotechnology, Cat: FT19301).After 48 h, the transfected cells were harvested and lysed with NP40 buffer by sonication.After incubation on ice for 15 min, the cell lysates were centrifuged at 12,000 rpm at 4°C to eliminate insoluble materials.
CD19 and mutant proteins were immunoprecipitated using anti-Flag M2 affinity gel (Sigma-Aldrich).The precipitated CD19 and mutants were incubated with the individual active caspases (Active recombinant caspases set, BioVision, #K230-4 − 25) in assay buffer following the manufacturer's instruction and subsequently analyzed by immunoblotting with Flag and HA antibodies.

| Statistical analyses
Statistical analyses were performed in Prism (Graph Pad Software).
The data were reported as means ± SD.Differences between groups of research subjects were analyzed for statistical significance with two-tailed unpaired t-tests.A p value of 0.05 was considered significant.

| High concentration of mCD95L mediates apoptosis in LCLs
We previously observed that some LCLs are sensitive and others resistant to the CD95-mediated apoptotic response. 29Because CD95L physiologically exists as a transmembrane ligand at the cell surface and a metalloprotease-cleaved soluble form in the peripheral blood, we investigated the effect of each form on LCLs.We used a recombinant dodecameric Ig-CD95L that mimics the biological effects of the membrane-bound cytotoxic ligand (mCD95L) 28 to avoid the mix of effector and target cells and thereby facilitate the biochemical analysis of the CD95 stimulation in EBV-infected B cells.
We also produced the metalloprotease-cleaved CD95L (sCD95L) as previously described 28 (Supporting Information: Figure 1A).In agreement with previous reports, 33,34 mCD95L induced apoptosis in LCLs at high concentration (500 ng/mL), whereas sCD95L did not trigger any cell death in LCLs regardless of the concentration (Figure 1A,B).We next examined the cell proliferation effect of a nonapoptotic dose of mCD95L and sCD95L (100 ng/mL) on the LCLs using CFSE.Flow cytometry analyses showed that both mCD95L and sCD95L did not influence the proliferative capacity of LCLs (Figure 1C).

| Nonapoptotic concentration of mCD95L inhibits BCR-mediated EBV reactivation in LCLs
Because CD95L can also inhibit BCR-mediated EBV lytic reactivation in some LCLs resistant to CD95-mediated apoptosis, 29 we investigated the impact of mCD95L and sCD95L on EBV activation.LCLs were treated with nonapoptotic concentrations of mCD95L (i.e., 10, 50, and 100 ng/mL) or sCD95L.Immunoblot analyses showed that mCD95L treatment reduced the expression of EBV immediate-early transactivator Zta and early D antigen (EAD) upon anti-Ig-induced activation.In contrast, LCLs treated with sCD95L retained the expression level of Zta and EAD similar to that detected in cells treated with a control medium (Figure 2A).Overall, these findings suggested that although low concentrations of mCD95L did not trigger apoptosis in LCLs, they inhibited the BCR-mediated EBV activation in LCLs.
6][37] To test whether lowdose mCD95L acted on Zta and RTA transcription, we performed a reporter assay with Zta and RTA promoters in EBV-negative DG75 B cells in the presence or absence of mCD95L or sCD95L.Alone, mCD95L and sCD95L stimuli did not affect the activation of Zta and RTA promoters (Figure 2B).While BCR activation in DG75 B cells did not activate the RTA promoter in DG75 B cells, same treatment stimulated transcription through the Zta promoter (Figure 2C).Interestingly, nontoxic concentrations of mCD95L (100 ng/mL) impaired the BCR-induced activation of Zta promoter in DG75 B cells (Figure 2C).Overall, these findings suggested that inhibition of the BCR-mediated EBV reactivation by low-dose mCD95L could partially occur by inhibition of the Zta-driven transcription.

| Nonapoptotic concentration of mCD95L impairs CD19-mediated BCR signaling in LCLs
BCR stimulation using anti-IgG/M antibodies induces lytic replication in some EBV-positive lymphoma cell lines including LCLs. 38,39It has been reported that inhibition of the PI3K signaling pathway renders these cells unresponsive to BCR-mediated EBV activation. 40Accordingly, we wondered whether low concentrations of mCD95L inhibited the BCR-mediated PI3K signaling pathway.LCLs were treated with anti-Ig in the presence of 100 ng/mL mCD95L or sCD95L for the indicated time points, and the phosphorylation status of Akt and ERK were analyzed by immunoblotting (Figure 3A,B).
Strikingly, anti-Ig-induced phosphorylation of Akt and ERK was reduced at an early point (1 h) in the presence of mCD95L and remained low at later time points, 3 and 6 h (Figure 3A and Supporting Information: Figure 1A).On the other hand, sCD95L did not affect the intensity of Akt and ERK phosphorylation (Figure 3B).These results highlighted that nonapoptotic concentration of mCD95L blocked BCR-mediated PI3K/Akt and ERK activations.Of note, the phosphorylation level of the NF-κB transcriptomic activator p65 was higher in the LCLs treated with mCD95L as compared to the control-treated LCLs at 0.5 and 1 h poststimulation but was reduced at 3 and 6 h post-treatment (Figure 3A and Supporting Information: Figure 2A).The p65 activation at earlier time points might be attributed to a CD95-driven activation of NF-κB, as previously reported, 41,42 which was subsequently suppressed by the mCD95L-mediated inhibition of BCR signaling.
Because CD19 is a proximal key regulator of the PI3K/Akt response following BCR activation, 43 we evaluated whether the nonapoptotic concentrations of mCD95L inhibited the PI3K/Akt signal by regulating CD19.FACS analyses showed that mCD95L stimulation not only induced the downregulation of surface CD95 but also led to the decrease in surface CD19 and its associated partners CD21 and CD81 (Figure 3C), whereas the surface expression of BCR (i.e., IgG), CD20, and HLA-DR was not significantly affected in mCD95L-treated cells (Figure 3C and Supporting Information: -Figure 2B).Of note, we observed two B-cell populations in the EBVinfected LCL population exposed to mCD95L and while the expression level of CD19, CD21 and CD81 was not altered in some B-cells, a second population underwent a robust downregulation of this complex (Figure 3C).This latter observation could be explained by either incomplete saturation of the CD95 binding sites, a delayed internalization process in some LCLs, or the presence of a mCD95L insensitive LCL population.sCD95L treatment did not affect the expression level of these factors (Figure 3C), indicating that mCD95L specifically downregulated a B-cell complex including CD95/CD19/ CD21/CD81, which could in turn account for the mCD95L-driven inhibition of the PI3K/Akt and ERK signaling pathways.

| CD95L-CD95 interaction induces CD19 internalization in LCLs
To evaluate whether nonapoptotic concentrations of mCD95L reduced the CD19 surface expression through its internalization, LCLs were treated with mCD95L or sCD95L in the presence or absence of 0.1 M sucrose, an inhibitor of surface receptor internalization. 44FACS analyses showed that sucrose treatment blocked mCD95L-induced CD19 surface downregulation (Figure 4A and Supporting Information: Figure 3A,B), suggesting that nonapoptotic concentrations of mCD95L triggered CD19 internalization.Importantly, sucrose treatment restored both the BCRmediated Akt and ERK phosphorylation and the expression of EBV Zta and EAD after mCD95L stimulation (Figure 4B and Supporting Information: Figure 3C).Of note, cells were harvested at 24 h to detect the induction of EBV lytic genes.Dynasore is an inhibitor of the dynamin-and clathrin-mediated endocytosis. 45Like sucrose, dynasore treatment inhibited mCD95L-mediated CD19 surface downregulation (Figure 4C), pointing out that a nontoxic concentration of mCD95L triggered clathrin-dependent CD19 internalization.
To strengthen that CD95L-CD95 interaction was responsible for the inhibition of the BCR signaling, LCLs were stimulated with F I G U R E 2 Low-dose mCD95L inhibits BCR-mediated EBV reactivation in LCLs.(A) LCLs were stimulated with the indicated concentration of mCD95L, sCD95L, or control (Ctrl) for 24 h, in the presence (+) or absence (−) of 10 μg/mL anti-human Ig(G + M).The expression of EBV EAD and Zta was detected by immunoblot analyses using the specific antibodies.The relative amount of EAD and Zta expression was quantified using imageJ software and indicated under the bands.Immunoblot is representative of three independent experiments.(B) DG75 cells were transfected with Zta or RTA promoter luciferase plasmids (Zta-Luc or Rta-Luc), along with Renilla plasmid for 24 h, followed by the treatment with 100 ng/mL mCD95L, sCD95L, or control (Ctrl) for 24 h.Luciferase assays were carried out in triplicate and normalized to renilla activity.The cells transfected with the luciferase vector alone were used as an empty control (Con) for the luciferase plasmids.(C) DG75 cells were transfected with Zta-Luc or Rta-Luc vector with a Renilla plasmid for 24 h, followed by the treatment with 100 ng/mL mCD95L in the presence (+) or absence (−) of 10 μg/mL anti-human Ig(G + M).Luciferase assays were carried out in triplicate and normalized to renilla activity.The histogram represents mean ± SD of triplicate samples (three experiments).A p Value of <0.05 was considered significant.

F I G U R E 3 (See caption on next page).
LIU ET AL.
| 7 of 15 100 ng/mL of mCD95L or sCD95L in the presence or absence of the neutralizing anti-CD95 antibody ZB4.Inhibition of CD95/CD95L interaction blocked the surface downregulation of CD19, CD21, and CD81 (Figure 4D and Supporting Information: Figure 4A), restored Akt and ERK phosphorylation, and expression of EBV Zta and EAD (Figure 4E and Supporting Information: Figure 4B).Of note, the neutralizing anti-CD95 antibody also prevented the mCD95Lmediated caspase-3 activation (cleavage) (Figure 4E).These results indicated that low-dose mCD95L triggered the internalization of the CD19-containing complex, inhibited the CD19-mediated Akt and ERK phosphorylation, and blocked BCR-mediated EBV activation in LCLs.

| CD95 engagement triggers a caspase-driven CD19 degradation in LCLs
We further investigated the CD95-driven mechanisms responsible for CD19 modulation in LCLs.When these cells were treated with anti-Ig in the presence or absence of mCD95L, we observed that CD19 mRNA expression was not significantly affected (Supporting Information: Figure 5), but CD19 protein expression diminished at 3 h post-treatment and this reduction was maintained up to 24 h post-treatment in mCD95L-treated cells (Figure 5A,B).To evaluate whether the lysosome, proteasome, and/or caspases were responsible for the CD19 degradation, LCLs exposed to mCD95L were treated with either the lysosomal inhibitor bafilomycin A1, the proteasome inhibitor MG132, or the pan-caspase inhibitor Z-VAD-FMK (zVAD).Interestingly, the decreased expression of CD19 protein by mCD95L was only restored by zVAD treatment (Figure 5C,D).In addition, caspase inhibition similarly restored the CD19-mediated activation of PI3K/Akt and ERK signaling pathways in mCD95L-treated LCLs (Figure 5E).These data suggested that the CD95-mediated caspase activation contributed to the reduction of CD19 protein expression and the downstream inhibition of PI3K/Akt and ERK phosphorylation.By contrast, zVAD treatment amplified the CD95-mediated inhibition of Zta and EAD expression (Figure 5E).This effect was in agreement with a previous report showing that caspase activity is required for EBV lytic replication upon BCR activation. 46ese results pushed us to evaluate whether CD19 was a target of the mCD95L-activated caspases.Based on the cleavage site characteristics for caspases, the caspase cleavage site has a general motif (DXXD-A/G/S/T). 47In silico analysis (http://caspdb.sanfordburnham.org/)predicted that three caspase cleavage sites, D388, D435, and D538 could exist in the CD19 sequence.We, therefore, generated a recombinant CD19 containing an N-terminal Flag and a C-terminal HA tag (Figure 6A), and three mutants D388A, D435A, and D538A in which the aspartic acid (D) of the potential cleavage sites was replaced by alanine (A).The constructs were expressed into HEK/293 T cells, then cells were lyzed and tagged CD19 was immunoprecipitated to perform cell-free cleavage assay using individual recombinant caspases.Interestingly, caspase-6, −7, and −8 efficiently cleaved CD19, while caspase-3 did not.Caspase-6, −7, and −8-mediated cleavage yielded a similar pattern of fragments with one N-terminal fragment of about 48 kDa detected by Flag antibody and one C-terminal fragment of about 30 kDa revealed by HA antibody (Figure 6B).The size of the fragments suggested that CD19 was processed at the cleavage site D388.In agreement with this, both D435A and D538A mutants still displayed the abovementioned two fragments, whereas these cleaved fragments were absent when the CD19 mutant D388A was incubated with caspases −6, −7, or −8 (Figure 6C).Overall, these data revealed that CD19 was a novel substrate for caspases −6, −7, and −8 and this transmembrane protein was cleaved at position D388.Besides, we also observed a weak fragment of about 55 kDa detected by Flag antibody after caspase-6 and −8 cleavage, which was still present in the mutants D435A and D538A, suggesting that another cleavage site likely exists in the CD19 sequence.
Next, to decipher whether the cleaved CD19 fragments subsequently underwent proteasomal or lysosomal degradation (Figure 6D), the Flag-CD19-HA plasmid was transfected into 293 T cells exposed to mCD95L in the presence of zVAD, bafilomycin A1, or MG132.In agreement with the cell-free assay, a protein fragment at 40 kDa corresponding to the D388 cleavage site was detected in the cells treated with mCD95L, but not in the cells treated with both mCD95L and zVAD (Figure 6E).This cleavage product slightly increased in cells exposed to mCD95L together with bafilomycin A1 or MG132 (Figure 6E), suggesting that the caspase-mediated cleavage products of CD19 subsequently underwent additional degradation processes through the proteasome and lysosome.
F I G U R E 3 Low-dose mCD95L inhibits CD19-mediated Akt and ERK activation in LCLs.(A) LCLs were stimulated with (+) or without (-) 10 μg/mL anti-human Ig(G + M) in the presence of 100 ng/mL mCD95L or control (mCtrl) for the indicated time.Cell lysates were prepared and subjected to immunoblot analyses with the indicated antibodies.For quantitation analyses, see Appendix FIG S1A.(B) LCLs were stimulated with (+) or without (-) 10 μg/mL anti-human Ig(G + M) in the presence of 100 ng/mL sCD95L or control (sCtrl) for the indicated time.Cell lysates were prepared and subjected to immunoblot analyses with the indicated antibodies.Immunoblot is representative of two independent experiments.(C) LCLs were stimulated with 100 ng/mL mCD95L or control (mCtrl), 100 ng/mL sCD95L or control (sCtrl) for the indicated time, followed by FACS analyses to detect surface expression of BCR (IgG), CD19, CD21, CD95, CD81, CD20, and HLA-DR.The plots are representative of two independent experiments.The MFIs of CD19, CD21, CD95, and CD81 were indicated for LCLs treated in the presence or absence (control) of mCD95L for 3 h.For MFI quantitation analyses, see Supporting Information: Appendix Figure 1B.

| CD95 interacts with CD19
Simultaneous internalization of surface CD95 and CD19 suggested that CD95 might interact with CD19.To verify this, DG75 cells were transfected with CD95-Flag and CD19-HA plasmids in the presence or absence of mCD95L stimulation.Co-immunoprecipitation analyses revealed that CD95 interacted with CD19 without mCD95L and the intensity of this interaction increased in the presence of the ligand (Figure 7A).Microscopy analyses confirmed that CD95 and CD19 were colocalized at the plasma membrane (Pearson r = 0.95) in control cells (Figure 7B), while the mCD95L treatment triggered the re-distribution of the two factors into the cytoplasm (Pearson r = 0.90) (Figure 7B).In agreement with these data, co-immunoprecipitation experiments established that endogenous CD95 interacted with endogenous CD19 in LCLs upon mCD95L treatment (Figure 7C).

| EBV-CTLs expressing mCD95L increase killing activity against LCLs
To confirm that CD19 downregulation also occurred in LCLs during the interaction with T cells, autologous EBV-CTLs were generated and cocultured with LCLs.The CD19 surface expression of LCLs gradually decreased with stimulation time and CTLs/LCLs ratio (Figure 8A).To decipher whether the overexpression of mCD95L  CD95L-induced cytolysis is one of the major mechanisms used by CTLs to kill target cells.It has been reported that CD95Lmediated immune surveillance by T cells is essential for the control of some EBV-positive or -negative lymphoma cells. 15,22,48Consistent with previous reports showing that mCD95L mediates apoptosis, 26,27,34 we establish that mCD95L induces apoptosis in LCLs while sCD95L does not.Of note, sCD95L slightly increases the level of p-Akt and p-ERK at 6 h post-treatment.We previously established that while the binding of mCD95L to CD95 leads to the DISC formation and subsequent caspase-8 activation in LCL, sCD95L fails to do it but activates PLC γ1 49 and PI3K-Akt 31 signaling pathways.The different activities observed with the two ligands are mainly due to a difference in their stoichiometry. 32,50rikingly, the expression level of mCD95L seems to affect the induced cell signaling pathway in LCLs because low doses of this membrane-bound ligand initiate a nonapoptotic signaling pathway and inhibit EBV activation, promoting the immune escape of LCLs.
Our data raise an important point that not only the surface level of CD95 expression on tumor cells contributes to the susceptibility to T cell-mediated cytolysis, but also the expression level of surface CD95L on CTLs acts as a decisive factor for T cell-mediated cytolytic function.
The simultaneous co-ligation of BCR with a nonapoptotic dose of CD95L activates caspases, which does not result in cell death but alters the plasma membrane expression of CD19/CD21/CD81 in LCLs and prevents EBV reactivation.Interestingly, CD95 internalization has been shown to enhance the activation of caspases and cell death, while it inhibits the induction of ERK and NF-kappaB signaling pathways.In B cells, our data now reveal that CD95 internalization can occur independently of the induction of cell death and we envision that this discrepancy is linked to the co-associated complex (i.e., CD19/CD21/CD81).Indeed, we previously observed in activated T-cells, that the artificial co-ligation of CD28 with CD95 F I G U R E 7 CD95 interacts with CD19.(A) DG75 cells were nucleofected with CD95-Flag and CD19-HA expression plasmids as indicated in the presence (+) or absence of 100 ng/mL mCD95L.CD95 was immunoprecipitated and indicated immunoblots were performed.The whole cell lysates (WCL) were shown as controls.(B) Confocal analyses of DG75 cells nucleofected with CD95-Flag and CD19-HA expression plasmids in the presence of 100 ng/mL mCD95L or control (mCtrl) for 3 h.Cells were stained with anti-Flag and HA antibodies to detect CD95 (green) and CD19 (red).Nuclei were counterstained with DAPI (Blue).(C) LCLs were treated with 100 ng/mL mCD95L or control (mCtrl) for 1.5 h.Cells were lyzed and CD95 was immunoprecipitated.CD19 coimmunoprecipitation was evaluated by immunoblot.Whole-cell lysates (WCL) were depicted as controls.The WCL immunoprecipitated with mouse IgG were used as a control.enhanced cell death, while the CD59/CD95 co-aggregation inhibited it. 51In B cells, it remains to elucidate how the association of CD95 with the CD19/CD21/CD81 complex activates a nonapoptotic caspase-dependent response instead of the classical apoptotic signaling pathway.The identification of this nonapoptotic signal will be essential to further investigate its pathophysiological role.Interestingly, while inhibition of CD95/CD95L interaction restores both CD19-mediated BCR activation and lytic induction of

F
I G U R E 1 The effect of mCD95L and sCD95L on survival and proliferation of LCLs.(A) Representative plots of PI and Annexin-V staining of three individual LCLs after stimulation with the indicated concentration of mCD95L or sCD95L for 24 h.(B) Statistic analyses of LCL survival after stimulation with the indicated concentration of mCD95L or sCD95L for 24 h.The histogram represents mean ± SD (three independent experiments, each experiment was done in triplicate).(C) Representative CFSE staining plots of live LCLs after stimulation with 100 ng/mL of mCD95L or sCD95L for the indicated times.Each experiment was done in triplicate (two independent experiments).

F I G U R E 4
Induction of CD19 internalization on LCLs by low-dose mCD95L is dependent on mCD95L-CD95 interaction.(A) LCLs were stimulated with 100 ng/mL of mCD95L or sCD95L in the presence of 0.1 M sucrose or control medium for 3 h.FACS analyses were performed to detect CD19 surface expression.The MFI of CD19 surface expression was indicated for LCLs treated with or without (control) mCD95L.Histogram plots are representative of two independent experiments.For MFI quantitation analyses, see Appendix FIG S2A.(B) LCLs were stimulated with 100 ng/mL mCD95L or control (mCtrl) in the presence (+) or absence (-) of 10 μg/mL anti-human Ig(G + M) for 24 h, together with or without 0.1 M sucrose as indicated.Cell lysates were prepared for the immunoblot analyses with the indicated antibodies.For quantitation analyses, see Appendix FIG S2C.(C) LCLs were stimulated with 100 ng/mL mCD95L or control (mCtrl) in the presence or absence of indicated concentrations of dynasore for 3 h.CD19 surface expression was monitored by FACS analyses.Histogram plots are representative of two independent experiments.The MFI of CD19 surface expression was indicated.(D) LCLs were stimulated with 100 ng/mL mCD95L or sCD95L in the presence of CD95 neutralizing antibody (ZB4) or control for 3 h.Cells were analyzed by FACS to detect surface expression of CD19, CD21, and CD81.The MFIs of CD19, CD21, and CD81 surface expressions were indicated in LCLs treated with or without (Ctrl) mCD95L.Histogram plots are representative of two independent experiments.For MFI quantitation analyses, see Appendix FIG S3A.(E) LCLs were pre-incubated with the neutralizing anti-CD95 antibody ZB4 or an isotypic mAb and then stimulated with 100 ng/mL mCD95L or control (mCtrl) in the presence (+) or absence (−) of 10 μg/mL anti-human Ig(G + M) for 24 h.Cell lysates were subjected to immunoblot analyses with the indicated antibodies.For quantitation analyses, see Supporting Information: Appendix Figure 3B.

F I G U R E 5
Downregulation of CD19 by low-dose mCD95L is dependent on mCD95L-mediated caspase activities in LCLs.(A) LCLs were stimulated with 100 ng/mL of mCD95L or control (mCtrl) in the presence (+) or absence (−) of 10 μg/mL anti-human Ig(G + M) for the indicated time.Cell lysates were prepared and subjected to immunoblot analyses with the indicated antibodies.(B) Densitometry analysis of CD19 relative to GAPDH based on immunoblot detection in A using the ImageJ image analysis software.(C) LCLs were stimulated with 100 ng/mL of mCD95L (+) or control (−) in the presence of DMSO, 100 nM bafilomycin A1 (BafA1), 5 μM MG132, or 20 μM zVAD for 24 h.Cell lysates were prepared and subjected to immunoblot analyses with the indicated antibodies.(D) Quantitation of CD19 amount relative to GAPDH based on immunoblot detection in C using the ImageJ analysis software.(E) LCLs were stimulated with 100 ng/mL of mCD95L or control (mCtrl) in the presence of DMSO or 20 μM zVAD for 24 h.Cell lysates were prepared and subjected to immunoblot analyses with the indicated antibodies.could enhance the elimination of LCLs, CTLs, mainly CD8+ T-cells (Figure 8B) were transduced with mCD95L-encoding lentiviruses and mixed at different ratio with autologous LCLs.CD95L-transduced EBV-CTLs exhibited a stronger cytotoxic activity against LCLs as compared to that triggered by control EBV-CTLs (Figure 8C), indicating that these T-cells can be engineered to improve the elimination of lymphomas by overexpressing mCD95L at their plasma membrane.Taken together, these data emphasized that based on the expression level of mCD95L in cytotoxic T-lymphocytes, LCLs underwent either the internalization of their CD19-containing complex or apoptosis.EBV-CTLs engineered to over-express mCD95L could favor the elimination of LCLs.

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I G U R E 6 CD19 is cleaved by caspases at amino acid D388.(A) Schematic diagram of potential caspase cleavage sites in the Flag-CD19-HA construct.(B) 293 T cells were transfected with Flag-CD19-HA for 48 h.Flag-CD19-HA proteins were immunoprecipitated with anti-Flag affinity gel and subjected to the cleavage assay with individual recombinant caspase-3, −6, −7, or −8.Incubation with buffer alone was used as a control (Con).The cleavage products of CD19 were detected with Flag and HA antibodies by immunoblot analyses and indicated with the arrows.(C) 293 T cells were transfected Flag-CD19-HA mutants D388A, D435A, or D538A for 48 h.The CD19 mutants were immunoprecipitated with anti-Flag affinity gel and subjected to the cleavage assay with individual recombinant caspase-3, −6, −7, or −8.Incubation with buffer alone was used as a control (Con).(D) Schematic diagram of Flag-CD19-HA degradation after cleavage at D388. (E) 293 T cells were transfected with vector (Vec) or Flag-CD19-HA for 48 h.The cells transfected with Flag-CD19-HA were treated with 100 ng/mL mCD95L in the absence (−) or presence (+) of 100 nM BafA1, 5 μM MG132, or 20 μM zVAD.Immunoprecipitations were carried out using anti-Flag affinity gel and immunoblot analyses were performed using an anti-Flag antibody.The cleavage products were quantified by ImageJ software and compared to the mCD95L treatment, as indicated by the number below the bands.Recipients of transplantation receive life-long immunosuppression to prevent rejection and thus increase their susceptibility to viral infections and virus-associated diseases.EBV-PTLDs are the most common diseases occurring in transplant recipients.Since no proven antiviral drugs are available, reduction of immunosuppression has been the main effective strategy of management.The infusion of autologous or third-party EBV-CTLs has been a promising approach and can augment virus-specific immune response, reduce viral load in transplant recipients, and could be safely used as prophylaxis and treatment options for EBV-PTLD patients.However, the specific cytotoxicity of EBV-CTLs exhibits poor results in some patients, the underlying mechanisms have not been fully understood.In this study, we have defined the ambivalent role of transmembrane CD95L in EBV-CTLs and illustrated the importance of its expression level in the implementation of signaling pathways in LCLs.Although high-dose mCD95L mediates apoptosis in LCLs, lower quantities of mCD95L trigger nonapoptotic signaling, surface CD19 downregulation, impaired CD19-mediated BCR signaling, and inhibition of BCRmediated EBV activation (Figure 8D).

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I G U R E 8 EBV-specific T cells expressing a high level of mCD95 increases killing activity against autologous LCLs.(A) LCLs were incubated with autologous EBV-CTLs for 1.5 h and 3 h as indicated, followed by surface staining with CD8, CD4, and CD19.CD19 expression (right panel) was evaluated in LCLs (CD8 − CD4 − cells) (left panel).LCLs only were used as control.The MFI of CD19 surface expression was indicated.Histograms are representative of three independent experiments.(B) Dot plot is representative of EBV-CTL surface staining.(C) EBV-CTLs transduced with lentiviruses expressing vector or mCD95L were incubated with autologous LCLs for 24 h.Cytotoxicity assays were performed and quantified by measuring LDH release (Bottom panel).Histograms represented the mean of three independent experiments ±SD, p value was determined by two-tailed unpaired t-test, p ≤0.05 represents significance.(D) Model of CD95L-CD95 mediated signaling in LCLs.High-dose mCD95L binding to surface CD95 of LCLs initiates apoptosis.Low-dose mCD95L triggers surface CD19 internalization, cleavage by caspase-6, −7, and/or −8, and lysosomal/ proteasomal degradation, leading to the inhibition of CD19-mediated BCR signaling pathway and the blocking of BCR-induced EBV activation.EBV Zta and EAD, the pan-caspase inhibitor zVAD rescues the CD19-mediated phosphorylation of Akt and ERK but fails to restore the lytic induction of EBV Zta and EAD upon colligation of low-dose mCD95L and anti-Ig.This observation suggests that both intact CD19-mediated BCR signaling and caspase activation are required for efficient EBV lytic replication upon BCR activation.This is in agreement with previous reports that caspase activation contributes to the effective EBV lytic replication induced by BCR ligation. 46,52As a result, both the BCR signaling pathway and caspase activation are essential for productive BCR-mediated EBV lytic replication.Our result indicates that the expression level of mCD95L at the surface of CTLs is important for T cell cytolytic function.The engineering of EBV-CTLs expressing an increased surface level of mCD95L can be proposed to improve the efficacy and efficiency of T cell immunotherapy for EBV-PTLDs.However, it is crucial to further investigate the potential undesired side effects associated with mCD95L overexpression in EBV-CTLs, including the influence on lymphocyte proliferation and the induction of inflammation.Nevertheless, our study will help in optimizing the engineering of EBV-CTLs and designing the most successful approach for the immunotherapies for EBV-PTLDs.AUTHOR CONTRIBUTIONS Mu Liu, Chenxu Huang, Congwei Jiang, Xingchen Zhou, Ying Gao, Linlin Kuang, and Zhangmengxue Lei designed the research, performed the experiment, and analyzed the data.Patrick Legembre generated the recombinant mCD95L and sCD95L, wrote and reviewed the manuscript.Shuai Liu, Ran Jia, and Jin Xu generated LCL and CTLs.Patrick Legembre provided the resources.Xiaozhen Liang and Mu Liu designed the research and analyzed the data.Mu Liu, Patrick Legembre, and Xiaozhen Liang wrote the manuscripts.