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D in TCR-Mediated Signaling and T Minghua Zhu, Daniel P. Foreman, Sarah A. O'Brien, Yuefei Jin and Weiguo Zhang This information is current as of September 23, 2021. J Immunol published online 31 January 2018 http://www.jimmunol.org/content/early/2018/01/31/jimmun ol.1701291 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 31, 2018, doi:10.4049/jimmunol.1701291 The Journal of Immunology

Phospholipase D in TCR-Mediated Signaling and T Cell Activation

Minghua Zhu, Daniel P. Foreman, Sarah A. O’Brien, Yuefei Jin, and Weiguo Zhang

Phospholipase D (PLD) is an that catalyzes the of , the major in the plasma , to generate an important signaling , phosphatidic . is a second messenger that regulates vesicular trafficking, cytoskeletal reorganization, and in immune cells and other cell types. Published studies, using pharmacological inhibitors or overexpression, indicate that PLD plays a positive role in TCR-mediated signaling and cell activation. In this study, we used mice deficient in PLD1, PLD2, or both to assess the function of these in T cells. Our data showed that PLD1 deficiency impaired TCR-mediated signaling, T cell expansion, and effector function during immune responses against Listeria monocytogenes; however, PLD2 deficiency had a minimal impact on T cells. Biochemical analysis indicated that u PLD1 deficiency affected Akt and PKC activation. In addition, it impaired TCR downregulation and the secondary T cell Downloaded from response. Together, our results suggested that PLD1 plays an important role in T cell activation. The Journal of Immunology, 2018, 200: 000–000.

hospholipase D (PLD) is a widely expressed enzyme that wild-type (WT) or dominant-negative (DN) forms of PLD pro- catalyzes the hydrolysis of phosphatidylcholine, the major teins, indicate that PLD1 and PLD2 play positive roles in FcεRI- P phospholipid in the plasma membrane, to produce water- mediated signaling and mast cell function (6, 8, 9). However, we http://www.jimmunol.org/ soluble and phosphatidic acid (PA). PA is a second recently generated PLD1- and PLD2-deficient mice to study their messenger that is thought to function in vesicular trafficking and function in mast cells. Our data demonstrate that PLD1 deficiency cytoskeletal reorganization (1–3). PA can also bind important impairs FcεRI-mediated F- disassembly and mast cell de- signaling , such as Raf and mTOR, to regulate their ac- granulation; however, PLD2 deficiency enhances microtubule tivation and function in different signaling pathways (4, 5). PLD is formation and degranulation. In addition, PLD deficiency affects not the only enzyme that can generate PA. PA is primarily pro- FcεRI-mediated activation of the PI3K pathway and RhoA (10). duced in two ways: the hydrolysis of phosphocholine by PLD and Our results suggest that PLD1 and PLD2, two proteins that cat- the phosphorylation of diacylglycerol (DAG) by diacylglyercol alyze the same enzymatic reaction, can regulate different steps in kinase (DGK). The PLD family consists of two closely related mast cell degranulation. by guest on September 23, 2021 members, PLD1 and PLD2. Interestingly, although these two proteins In addition to studies on PLD in macrophages, neutrophils, and share similar sequences and structural domains, they have different mast cells, it has been demonstrated that PLD proteins play an subcellular localizations. PLD1 is primarily localized to secretory important role in T cells. Early studies using the Jurkat T cell line granules; however, it redistributes to the plasma membrane after show that cross-linking of the TCR or PMA treatment increases stimulation. In contrast, PLD2 is present in the plasma membrane (6). PLD activity (11, 12). 1-Butanol can reduce TCR-mediated The function of PLD proteins in the immune system has been flux and overall tyrosine phosphorylation of proteins, extensively studied in the past decade. Published data have clearly including the TCRz chain, and block AP-1 activation. Over- indicated that PLD proteins are important in immunoreceptor expression of PLD1 or PLD2 in Jurkat cells enhances TCR- signaling and immune cell function. It has been shown that PLDs mediated NFAT activation. In contrast, overexpression of the can function in Fcg-mediated by macrophages and DN form of PLD1 or PLD2 inhibits it, indicating that PLD pro- are involved in the activation of NADPH oxidase in neutrophils teins play a positive role in TCR-mediated signaling (13, 14). (7). PLD’s function in mast cells has also been studied previously. Interestingly, overexpression of DN PLD2, but not DN PLD1, Those studies, which were done mostly by using pharmacologi- impairs TCRz phosphorylation, suggesting that PLD1 and PLD2 cal inhibitors, such as 1-butanol, or by overexpression of the function differently in this signaling pathway. Another study using the Jurkat T cell line shows that PLD2 can function upstream of Ras during T cell activation (12). The engagement of the TCR and Department of Immunology, Duke University Medical Center, Durham, NC 27710 LFA-1 leads to the activation of PLD2 and production of PA, Received for publication September 7, 2017. Accepted for publication January 3, which might be converted to DAG by phosphatidic acid phos- 2018. phatase. DAG then recruits and activates RasGRP1. It is also This work was supported by National Institutes of Health Grant AI093717. possible that PA is involved in Ras activation through binding to Address correspondence and reprint requests to Dr. Weiguo Zhang, Box 3010, De- Sos directly. Recent data showed that the PLD1-specific inhibitor, partment of Immunology, Duke University Medical Center, Jones Building, Room VU0359595, could inhibit TCR-mediated Ras–MAPK activation 112, Durham, NC 27710. E-mail address: [email protected] and block HIV infection (15), suggesting that PLD1 is required for Abbreviations used in this article: DAG, diacylglycerol; DGK, diacylglyercol kinase; DN, dominant negative; Lm-OVA, L. monocytogenes expressing OVA; MFI, mean activation of this pathway. In addition, PLD may have different fluorescence intensity; PA, phosphatidic acid; PLD, phospholipase D; PLDdKO, PLD roles in T regulatory cells (Tregs) and conventional T cells. PLD1 double knockout; PLDKO, PLD knockout; PLD1KO, PLD1 knockout; PLD2KO, expression is significantly lower in T than in conventional PLD2 knockout; T , T regulatory cell; WT, wild-type. regs reg T cells. Consequently, anti-CD3ε stimulation increases PLD ac- Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 tivity in conventional T cells but not in Tregs (13). However, it has

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701291 2 PLD PROTEINS IN T CELLS

also been shown that of CTLA-4 in Tregs is dependent cytotoxicity assays on PLD activity, because 1-butanol inhibits the surface expression CD8+ T cells were purified from PLD1KO, PLD2KO, PLDdKO, and WT of CTLA-4 (16). Although these data strongly suggest that PLD1 OT-1+ splenocytes using MojoSort Streptavidin Nanobeads (BioLegend). and PLD2 may play an important role in TCR-mediated signaling These cells were primed with 1 mMOVA257–264 peptide for 2 d. EL4 cells and T cell activation, most of these studies were done using loaded with 10 mM OVA peptide for 1 h were labeled with 10 mM Cell- high pharmacological inhibitors or by overexpression of PLD; there- Tracker Orange (Invitrogen) as target cells (CellTracker Orange ). EL4 cells without OVA peptide loading were labeled with 0.5 mM CellTracker fore, the function of PLDs in TCR signaling and T cell activation Orange as controls (CellTracker Orangelow). These two populations of EL4 has yet to be fully delineated. cells were mixed at a 1:1 ratio. CTLs were mixed with 1 3 105 EL4 cell In this study, we used PLD-deficient mice to study the function of mixture at E:T ratios of 1:1, 2:1, and 4:1 in a 96-well round-bottom plate at PLD proteins in TCR-mediated signaling and T cell function 37˚C for 3 h. Specific killing was determined using the following formula: 100 2 [100 3 (% of peptide-loaded targets/% of control targets in the in vitro and during immune responses against Listeria mono- presence of CTLs)/(% of peptide-loaded targets/% of control targets in the cytogenes infection. Our data showed that, of the two members of absence of CTLs)]. the PLD family, PLD1 plays a more dominant role in T cells than PLD2. PLD1 deficiency impairs TCR-mediated signaling and Western blotting T cell expansion during primary and memory responses, whereas For detection of phosphorylated proteins, CD4+ and CD8+ T cells were PLD2 deficiency had a minimal impact on T cell function. purified by negative selection using MojoSort Streptavidin Nanobeads (BioLegend). These T cells were stimulated with biotin–anti-CD3ε and biotin–anti-CD28, followed by streptavidin, for the indicated times. Cell Materials and Methods lysates were analyzed by Western blotting with the following Abs: p-Tyr Mice (4G10), p-AKT (Ser473), p-AKT (Thr308), p-ERK1/2 (Thr202/Tyr204), Downloaded from p-PKCu (Thr538), p-PKCd (Thr505 and Y311), AKT, and PKCu (all from PLD1-knockout (PLD1KO; PLD12/2), PLD2-knockout (PLD2KO; 2/2 2/2 2/2 Cell Signaling Technology). For Western blotting, samples were separated PLD2 ), and PLD–double-knockout (PLDdKO; PLD1 PLD2 ) by SDS-PAGE and transferred to nitrocellulose . After incu- mice were generated as described previously (10). They were crossed with bation with primary Abs, nitrocellulose membranes were washed three the OT-I transgenic mice (The Jackson Laboratory, Bar Harbor, ME) to times and probed with anti-mouse or anti-rabbit Ig conjugated to Alexa generate PLD1KO/OT-1, PLD2KO/OT-1, and PLDdKO/OT-1 mice. All Fluor 680 (Molecular Probes) or IRDye 800 (Rockland). Membranes were mice were used in accordance with the National Institutes of Health

visualized with an Odyssey system (LI-COR Biosciences). http://www.jimmunol.org/ guidelines. The procedures in this study were approved by the Duke University Institutional Care and Use Committee. Mice were housed in a specific pathogen–free facility. Results PLD1 is required for optimal expansion of CD8+ T cells Listeria infection and adoptive transfer We recently generated PLD1KO, PLD2KO, and PLDdKO mice 3 5 + For adoptive transfer of naive OT-I cells, 1 10 CD45.1 WT and ε CD45.2+ PLD1KO, PLD2KO, and PLDdKO OT-I CD8+ T cells were and used them to study PLD function in Fc RI-mediated signaling adoptively transferred into nonirradiated naive recipient mice (CD45.1+ and mast cell function (10). In this study, we used these mice to CD45.2+) at day 0. One day after OT-I cell transfer, recipient mice were assess PLD function in T cells during immune responses. To this injected i.v. with 1.5 3 104 CFU L. monocytogenes expressing OVA (Lm- end, we infected WT, PLD1KO, PLD2KO, and PLDdKO mice 3 5 by guest on September 23, 2021 OVA). To induce a secondary response, mice were injected with 1.5 10 with a low dose (1.5 3 104 CFU per mouse) of Lm-OVA by i.v. CFU Lm-OVA. injection. Seven days postinfection, T cells from these mice were FACS analysis analyzed by flow cytometry for their activation and expansion. b Most of the Abs used for flow cytometry were purchased from BioLegend, To identify Ag-specific T cells, we loaded H-2K DimerX with unless otherwise indicated. Single-cell suspensions were prepared from the the OVA 257–264 peptide and used it in combination with other spleens, lymph nodes, and blood from different mice after lysis of RBCs with cell surface and activation markers in FACS analysis. As shown in ammonium–chloride–potassium lysis buffer. To detect OVA-specific CD8 Fig. 1A, there was a clear difference in the percentages of OVA- T cells, cells were stained with DimerX (H-2Kb-Ig recombinant fusion pro- specific T cells (CD8+DimerX+) in these Lm-OVA–infected mice. tein; BD Biosciences) loaded with the OVA257–264 peptide (SIINFEKL). To + determine the activation status of T cells, cells were stained with PE- or Close to 15% of CD8 T cells were present in WT and PLD2KO allophycocyanin-conjugated Abs, such as PE–anti-CD62L and PE–anti- mice; however, the percentages of OVA-specific T cells in CD11a, allophycocyanin–anti-CD44, and anti-TCRb. To detect the PLD1KO and PLDdKO mice were reduced to ∼12.9 and 7.1%, + percentage of OT-I T cells among total CD8 T cells after adoptive transfer, respectively. We also enumerated the total numbers of OVA- cells were stained with PE–anti-Va2, allophycocyanin–Cy7–anti-CD8, PE– Cy7–anti-CD45.1, FITC–anti-CD45.2, and Pacific blue–LIVE/DEAD (Invi- specific T cells in the spleens of these mice. Corresponding to trogen). To detect granzyme and Ki-67 expression, cells were stained with the reduced percentages of these T cells, PLD1KO and PLDdKO surface markers, fixed and permeabilized using a Cytofix/Cytoperm kit mice had an apparent reduction in the number of OVA-specific (eBioscience), and stained with allophycocyanin–anti-granzyme A and PE– T cells (Fig. 1B). In addition, there was a significant difference in anti-granzyme B or anti–Ki-67. For intracellular staining of cytokines, the numbers of Ag-specific T cells between PLD1KO and splenocytes were restimulated with 1 mMOVA257–264 peptide for 5 h in the presence of monensin (BioLegend), stained with anti-CD8, fixed, per- PLDdKO mice (p = 0.035, Fig. 1B). These data suggested that meabilized, and stained again with allophycocyanin–anti–IFN-g. Samples PLD1 may play a more dominant role in T cells than PLD2; were analyzed on a FACSCanto II (BD Biosciences). Flow plots shown were however, in the absence of PLD1, PLD2 may also contribute to analyzed with FlowJo (TreeStar, Ashland, OR). Ag-driven T cell expansion. TCR downregulation To further determine whether PLD deficiency affected T , we performed intracellular staining of these cells Downregulation of the TCR on CD4+ and CD8+ T cells was performed as described previously (17). Briefly, splenocytes were treated with biotin– with Abs against the Ki-67 protein, which marks proliferating + anti-CD3ε (2C11, 10 mg/ml) and anti-CD28 (2 mg/ml) on ice for 30 min, cells. At day 7 after Listeria infection, WT and PLD2KO CD8 followed by washing with cold RPMI 1640. Cells were moved to 37˚C at T cells had similar percentages of Ki-67+ cells (67 and 65%, re- the indicated time points. The level of the surface TCR remaining was spectively), whereas PLD1KO T cells had a slightly reduced quantitated by FACS analysis after staining with streptavidin-conjugated percentage (53%). In contrast, PLDdKO T cells had the lowest allophycocyanin. The percentage of TCR downregulation was calculated + based on the mean fluorescence intensity (MFI) of CD3 surface expression percentage of Ki-67 cells (∼37%) (Fig. 1C). These data sug- using the following formula: 100 3 [MFI(0) 2 MFI(t)]/MFI(0). gested that PLD deficiency impaired T cell proliferation. The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 1. CD8+ T cell response to Lm-OVA infection in PLD-deficient mice. PLD1KO, PLD2KO, PLDdKO, and WT mice were infected with Lm- OVA. At day 7 postinfection, splenocytes from these mice were analyzed by flow cytometry. (A) OVA-specific CD8+ T cells in the spleen of infected mice. (B) The number of OVA-specific CD8+ T cells in the spleen. Data are shown as mean 6 SEM. (C) Ki-67 expression in CD8+ T cells. (D) Representative plots of CD11a and CD62L/CD44 on CD8+ T cells. (E) IFN-g production. Splenocytes were restimulated with 1 mM OVA peptide for 5 h in the presence of monensin before intracellular staining. (F) Intracellular staining of granzymes A and B. Data are representative of three independent experiments.

Upon T cell activation, CD8+ T cells upregulate CD11a, which granzyme A. A similar reduction in the expression of granzyme B is involved in CD8+ T cell expansion and differentiation (18). As was seen in PLD1KO and PLDdKO T cells (Fig. 1F), indicating shown in Fig. 1D, CD11a upregulation on CD8+ T cells was that PLD deficiency affected the expression of these effector impaired by deficiency in both PLD1 and PLD2; however, it was in CD8+ effector T cells. not affected by PLD1 deficiency alone. T cell activation also leads We further tested whether CD8 T cell function was affected by to downregulation of CD62L and upregulation of CD44. At day 7 PLD deficiency in vitro. WT, PLD1KO, PLD2KO, and PLDdKO postinfection, the percentage of activated CD8+ T cells (CD44+ OT-1 T cells were activated by APCs in the presence of the OVA CD62L2) in WT and PLD2KO mice was ∼51.5 and 47%, re- peptide and differentiated into CTLs. To test whether their cyto- spectively; however, it was reduced to 40.9% in PLD1KO mice toxicity was affected, we loaded EL-4 cells with the OVA peptide and further reduced to 31.2% in PLDdKO mice. and 10 mM CellTracker Orange as target cells (CellTracker Upon activation and differentiation into effector cells, CD8+ Orangehigh). EL-4 cells without the OVA peptide were loaded with T cells produce IFN-g and granzymes, which are important ef- a lower concentration (0.5 mM) of CellTracker Orange as control fector molecules in T cell function. We next examined the ability cells (CellTracker Orangelow). CTLs were incubated at 1:1, 2:1, of these CD8+ T cells to produce IFN-g. Splenocytes from these and 4:1 ratios with these EL-4 cells that were premixed at a 1:1 mice were stimulated in vitro in the presence of the OVA 257–264 ratio. After incubation for 3 h, these cell mixtures were analyzed peptide. The percentage of IFN-g–producing cells was analyzed by flow cytometry. As shown in Fig. 2A, in the presence of WT by intracellular staining, followed by flow cytometry. As shown in CTLs, the percentage of CellTracker Orangehigh cells was grad- Fig. 1E, IFN-g production by CD8+ T cells was impaired by de- ually reduced with the increased ratio of CTLs/EL-4 cells. At a ficiency in PLD1, PLD1, or both. We further analyzed expression 4:1 ratio, the percentage of EL-4 cells loaded with the OVA of granzyme A and B by intracellular staining. As seen in Fig. 1F, peptide was reduced from 50 to 19%. PLD2KO cells killed sim- WT and PLD2KO CD8+ T cells expressed the highest percentages ilarly as WT CTLs; however, PLD1KO and PLDdKO CTLs killed of granzyme A (47 and 46%, respectively). In contrast, only 28% less efficiently than WT and PLD2KO cells. At a 4:1 ratio, 34 and of PLD1KO T cells and 23% of PLDdKO T cells expressed 37% of cells were still CellTracker Orangehigh. Together, these 4 PLD PROTEINS IN T CELLS Downloaded from http://www.jimmunol.org/

FIGURE 2. Effect of PLD deficiency on CTL cytotoxicity. (A) In vitro cytotoxicity assay. EL-4 cells loaded with the OVA257–264 (SIINFEKL) peptide were labeled with 10 mM CellTracker Orange (CellTracker Orangehigh). EL-4 cells without the peptide were labeled with 0.5 mM CellTracker Orange (CellTracker Orangelow). These EL-4 cells were premixed in a 1:1 ratio. CTLs were incubated with the EL-4 mixture at 1:1, 2:1, and 4:1 E:T ratios for 3 h before FACS analysis. (B) Quantification of in vitro cytotoxicity. Data are representative of three independent experiments. data indicated that PLD1 deficiency affected T cell activation, and PLDdKO mice and activated by anti-CD3ε Abs. Twenty-four expansion, cytokine production, granzyme expression, and cyto- hours after activation, expression of surface markers, such as toxicity. In the absence of PLD1, PLD2 deficiency may cause CD25 and CD62L, was analyzed on CD4+ T cells. As seen in + additional impairment of T cell function. Fig. 4A, ∼45% of WT cells and ∼47% of PLD2KO CD4 T cells by guest on September 23, 2021 upregulated CD25; however, only 36% of PLD1KO T cells and The impairment caused by PLD deficiency is T cell intrinsic 27% of PLDdKO T cells were CD25+. Similar results were also PLD proteins are ubiquitously expressed in different cell types, seen for CD62L expression. TCR-mediated downregulation of including various immune cells. To exclude the possibility that CD62L in PLD1KO and PLDdKO T cells was impaired. We also impaired T cell expansion and function in PLD-deficient mice were assayed T cell proliferation by CFSE labeling. T cell proliferation due to defects in other immune cells, such as APCs, we performed leads to dilution of CFSE. As seen in Fig. 4B, PLDdKO and an adoptive-transfer experiment (Fig. 3A). OT-1 T cells from PLD1KO T cells had slower dilution of CFSE than did WT and CD45.1+ WT mice were mixed with those from CD45.2+ PLDKO PLD2KO T cells. Similar results were also seen for CD8+ T cells mice at a 1:1 ratio and were injected i.v. into CD45.1+CD45.2+ (data not shown). WT mice. One day after transfer, these mice were infected i.v. We next performed biochemical analysis of TCR signaling in with Lm-OVA. Seven days later, splenocytes and lymph node cells these T cells. T cells were purified from the spleens and lymph were analyzed by flow cytometry. As seen in Fig. 3B, similar nodes of WT, PLD1KO, PLD2KO, and PLDdKO mice by negative percentages of WT and PLDKO T cells were mixed before the selection. These cells were then activated by cross-linking with adoptive transfer. Seven days postinfection, a similar ratio of anti-CD3ε and anti-CD28 Abs for 0, 5, and 20 min before lysis. CD45.2+/CD45.1+ cells was seen in mice that received WT and Cross-linking of the TCR leads to activation of protein tyrosine PLD2KO OT-1 T cells before and after the transfer (Fig. 2); kinases, such as Lck and ZAP-70, and subsequent phosphorylation however, the ratios of CD45.2+/CD45.1+ cells were decreased in of proteins, including LAT, SLP-76, and PLC-g1 (19). As shown mice with PLD1KO/WT and PLDdKO/WT OT-1 T cells after in Fig. 4C, overall tyrosine phosphorylation of proteins was Listeria infection. These data indicated that the defect in Ag- similar in T cells from these mice. LAT is an adaptor protein that induced T cell expansion was T cell intrinsic, although we mediates TCR calcium flux and Ras–Erk activation. LAT phos- could not exclude the possibility that deficiency in PLD proteins in phorylation was not affected by PLD deficiency. We further other cell types may also contribute to the T cell defect. assayed whether PLD deficiency affected TCR-mediated calcium flux by loading T cells with a calcium indicator, indo-1, followed PLD deficiency on TCR signaling by analysis by flow cytometry. No significant differences were The above data suggested that PLD proteins, especially PLD1, seen in calcium mobilization induced by anti-CD3ε cross-linking play an important role in T cell activation, expansion, and func- (data not shown). We further assayed activation of the Ras–Erk tion. Next, we determined the impact of PLD deficiency on and PI3K pathways by Western blotting with anti–p-ERK1/2 TCR-mediated signaling and T cell activation in vitro. First, we (Thr202/Tyr204), anti–p-AKT (Ser473), and anti–p-AKT (Thr308) examined whether PLD deficiency affected T cell activation Abs. As shown in Fig. 4D, TCR-mediated Erk activation was in vitro. Splenocytes were isolated from WT, PLD1KO, PLD2KO, similar in these T cells; however, PLD1 deficiency or PLD1 and The Journal of Immunology 5 Downloaded from

FIGURE 3. T cell–intrinsic requirement of PLDs in T cell expansion. (A) Schematic diagram of the adoptive-transfer procedure. A total of 1 3 105 WT OT-I cells (CD45.1+) and 1 3 105 PLDKO OT-I cells (CD45.2+) was mixed and transferred i.v. into WT (CD45.1+CD45.2+) recipient mice. The following day, these recipients were infected i.v. with Lm-OVA. (B) FACS analysis of donor-naive OT-1 T cell mixture before adoptive transfer (left panels). FACS http://www.jimmunol.org/ analysis of OT-1 T cells at day 7 after adoptive transfer (right panels). Cells were gated on live CD8+Va2+ T cells from the spleen. (C) Percentages of WT and PLD-deficient OT-I cells among total CD8+Va2+ T cells in the spleen after Lm-OVA infection. Data are shown as mean 6 SEM. Data are repre- sentative of two independent experiments with five mice in each experiment.

PLD2 double deficiency significantly impaired phosphorylation of with anti–p-PKCu (Thr538) and anti–pan-PKCu Abs. As seen in Akt at Ser473 and Thr308, suggesting that PLD1 is important for Fig. 5C, in WT and PLD2KO T cells, phosphorylation of PKCu TCR-mediated Akt activation. was increased at 5 and 20 min after TCR cross-linking; however, PKCu phosphorylation was reduced in PLDdKO and PLD1KO

PLD deficiency on TCR downregulation by guest on September 23, 2021 T cells. As a control, phosphorylation of PKCd (Thr505) was not It has been shown that PLD proteins play an important role in affected. A similar reduction in PKCu phosphorylation was con- receptor-mediated . Epidermal growth factor endocy- sistently seen in other experiments that we performed (Fig. 5D). tosis can be accelerated by overexpression of WT PLD and re- These data indicated that, in addition to Akt activation, PLD1 tarded by overexpression of catalytically inactive forms (20). We deficiency affected TCR-mediated activation of PKCu. Impaired examined whether deficiency in PLD1 or PLD2 protein also af- PKCu activation likely led to reduced CD3g phosphorylation and fected downregulation of the TCR from the cell surface following slower downregulation of the TCR from the cell surface. T cell activation. To this end, we coated splenocytes from WT, PLD1KO, PLD2KO, and PLDdKO mice with biotinylated anti- PLD deficiency affects the secondary T cell response CD3ε Abs on ice and then activated them at 37˚C for 0, 10, 20, 30, Our data clearly indicated that TCR signaling and T cell expansion 60, 90, and 120 min in the presence of anti-CD28 Abs. The ex- in PLD1KO and PLDdKO mice were impaired during the primary pression level of the surface TCR was determined by staining T cell response against Listeria infection. Next, we examined these cells with streptavidin-allophycocyanin and analyzing by whether PLD proteins had a similar role in the secondary T cell flow cytometry. As shown in Fig. 5A and 5B, TCR was gradually response during infection. We infected WT and PLDdKO mice downregulated after cross-linking with anti-CD3ε and anti-CD28. with 1.5 3 104 CFU Lm-OVA and bled them at days 7, 16, 23, 32, At 60 min after activation, TCR surface expression was reduced and 42. The percentage and number of OVA-specific T cells in the by ∼30% for WT and PLD2KO T cells; however, maximal blood were analyzed by FACS after staining with H-2Kb DimerX downregulation only reached 20% for PLD1KO and PLDdKO with the OVA peptide. At day 37, mice were challenged again with T cells, indicating that PLD1 deficiency impairs downregulation 1.5 3 105 CFU Lm-OVA to induce memory responses. As shown of the TCR during T cell activation. in Fig. 6A, the percentage of OVA-specific CD8+ T cells in PKCs play important roles in TCR downregulation. Although PLDdKO mice was approximately three times less than in WT PKCa is involved in the downregulation of the unengaged TCR, mice at day 7 after initial infection. After the initial T cell ex- PKCu is important for the downregulation of engaged TCRs by pansion, T cells undergo contraction and differentiate into mem- phosphorylating the Ser126 residue in CD3g. Phosphorylation of ory T cells. There were no significant differences in the Ser126 makes the dileucine motif accessible to binding by AP2, percentages of OVA-specific WT and PLDdKO T cells at days 16, which leads to TCR endocytosis (21). It is possible that 23, and 32. However, upon secondary challenge with Listeria,WT PLD deficiency affects PKCu activation and subsequent CD3g T cells expand more quickly than PLDdKO T cells. phosphorylation. To determine PKCu activation, T cells were We also determined the memory responses of PLD1KO and activated with anti-CD3ε and anti-CD28 Abs for 0, 5, and 20 min. PLD2KO T cells following the same procedure. Five days after Protein lysates were prepared and analyzed by Western blotting secondary challenge (day 42), splenocytes were analyzed by flow 6 PLD PROTEINS IN T CELLS Downloaded from http://www.jimmunol.org/

FIGURE 4. PLD deficiency on T cell activation and TCR signaling. (A) Representative flow analysis of CD25 and CD62L. Splenocytes were activated with anti-CD3ε for 20 h before analysis. (B) CFSE dilution in CD4+ T cells after anti-CD3ε stimulation for 2 d. (C and D) Biochemical analysis of TCR-mediated signaling pathway. Purified CD4+ T cells were stimulated with biotin–anti-CD3ε and anti-CD28, followed by streptavidin cross-linking for different lengths of time. Cell lysates were analyzed by Western blotting with Abs against p-Tyr, p-AKT (Ser473), p-AKT (Thr308), and p-ERK1/2 (Thr202/Tyr204). p-AKT and p-ERK proteins were normalized by reblotting with anti-AKT and anti-ERK2 Abs, respectively. The numbers shown are by guest on September 23, 2021 relative intensities for the phosphorylated form of proteins normalized to total proteins. Data are representative of three independent experiments. cytometry after staining with H-2Kb DimerX and Abs against PLD2. Although PLD2 deficiency alone had a minimal impact on CD11a, CD4, CD8, and other surface markers. As shown in T cells, PLD1 deficiency affected TCR-mediated signaling, T cell Fig. 6B, tetramer staining showed that, similar to the data in activation, and effector function. Our adoptive-transfer experiment Fig. 6A, PLDdKO T cells had reduced OVA-specific CD8+ showed that the defects in T cell activation in PLD1KO and T cell expansion upon challenge. Approximately 30% of CD8+ PLDdKO mice were T cell intrinsic, because these T cells had T cells were OVA specific (CD8+DimerX+), whereas ∼45% of reduced expansion when transferred into WT mice. We further WT CD8+ T cells and 47% of PLD2KO T cells were OVA investigated how PLD deficiency affected T cell activation by specific. PLD1 deficiency alone also affected memory T cell analyzing TCR proximal signaling events. Our data showed that expansion, because only 33% of PLD1KO T cells were OVA PLD1 deficiency did not affect overall tyrosine phosphorylation of specific. We quantitated the numbers of OVA-specific T cells in proteins and main proximal signaling events, such as Ca2+ flux and the spleens of these mice after secondary challenge. As shown in Ras–MAPK activation; however, it impaired Akt and PKCu ac- Fig. 6C, the number of OVA-specific T cells in PLD2KO and tivation. How PLD1 deficiency affects activation of these proteins WT mice was similar; however, it was reduced in PLD1KO mice is not clear. It is known that PA can bind and activate mTORC1 and further reduced in PLDdKO mice. The difference in the and mTORC2 complexes, which regulate cell growth and prolif- numbers of T cells in PLD1KO and PLDdKO mice was statis- eration (22). Although mTOR is a target protein of the PI3K/AKT tically significant (p = 0.01). In addition, PLD deficiency af- signaling pathway, interestingly, mTORC2 can also phosphorylate fected the number of IFN-g–producing CD8 T cells in PLD1KO Akt on serine 473. Thus, it is possible that, in the absence of and PLDdKO mice (Fig. 6D). Together, these data indicated PLD1, activation of mTORC complexes was impaired, further that, in addition to their roles in initial T cell activation, PLD leading to defective Akt activation. It is known that PKC can deficiency, especially PLD1 deficiency, impaired memory T cell interact with PLD1 and PLD2 and enhance their activity expansion and IFN production. (23, 24). In contrast, PA can also be converted to DAG by phos- phatidic acid . It is possible that PLD deficiency im- Discussion pairs DAG production and subsequent PKCu activation in T cells. In this study, we used mice deficient in PLD1, PLD2, or both to PLD1 deficiency affects TCR-mediated signaling and T cell assess the function of PLD in T cells in vivo and in vitro. Although activation; in contrast, PLD2 deficiency had no apparent effect. PLD1 and PLD2 catalyze the same enzymatic reaction, their de- Deficiency in both proteins had more severe effects on T cells than ficiencies had differential effects on T cell function. Our data did PLD1 deficiency alone, suggesting that, in the absence of clearly showed that PLD1 had a more dominant role in T cells than PLD1, PLD2 could compensate for the loss of PLD1 to some The Journal of Immunology 7 Downloaded from

FIGURE 5. PLD deficiency on TCR downregulation. (A) Representative plots of surface TCR expression on splenic CD4+ T cells from PLDKO and WT mice before and after anti-CD3 and anti-CD28 stimulation. The dashed vertical line represents the MFI of CD3 before stimulation. (B) Quantitated TCR + + + downregulation in CD4 and CD8 Tcells.(C and D)PKCu activation by Western blotting. Purified CD4 T cells were stimulated with biotin-anti-CD3ε and http://www.jimmunol.org/ biotin–anti-CD28, followed by streptavidin cross-linking for different lengths of time. Cell lysates were analyzed by Western blotting with p-PKCu (Thr538), PKCu, and anti–p-PKCd (Thr505). The relative intensity of p-PKCu was normalized by the intensity of pan-PKCu. Two additional experiments showed that PLD1 deficiency impaired PKCu activation (D). The numbers shown are relative intensities for the phosphorylated form of proteins normalized to total proteins. extent. It is not clear why deficiency in PLD1 or PLD2, two proteins activation. Why this result differs from ours is not clear. One with similar functions, affected T cells differently. T cells express possibility is that this PLD1 inhibitor (VU0359595) might not PLD1 and PLD2, but their relative abundance in T cells is not inhibit only PLD1. It might act nonspecifically to impair the ac- known. In addition, their intrinsic enzymatic activity or activation tivity of other proteins that function in the Ras–MAPK pathway. could be different. Moreover, PLD1 and PLD2 are known to lo- Previous studies also showed that 1-butanol could inhibit TCR- by guest on September 23, 2021 calize to different subcellular compartments. PLD1 or PLD2 de- mediated overall tyrosine phosphorylation of proteins and calcium ficiency could lead to changes in the local concentrations of PA. flux (11, 12); however, we did not observe any deficiencies Thus, the more severe impact of PLD1 deficiency on T cells could in these proximal signaling events in PLD-deficient T cells. be because there is more PLD1 protein in T cells or PLD1 has Although it is possible that primary alcohols have nonspecific higher enzymatic activity than PLD2. When T cells were only effects, it is equally possible that PLD deficiency might be com- deficient in PLD1, TCR-mediated signaling and T cell activation pensated for by other enzymes in lipid metabolism. Considering were significantly impaired. When they were only deficient in the reported importance of PLD enzymes in many cellular func- PLD2, T cells were largely unaffected because of the presence of tions, it is puzzling that deficiency in PLD1 and PLD2 did not PLD1. Deficiency in both PLD proteins led to the total absence of cause lethality or more severe . The main production of PLD activity and more severe impairment on T cells. PLD enzymatic reaction is PA, which is a secondary message that A previous study using Jurkat cells indicated that PLD2 is re- acts on other signaling proteins. PA can also be produced by hy- quired in LFA-1–mediated Ras activation (12). Interestingly, in drolysis of DAG by DGK or synthesized from lysophosphatidic Jurkat cells, PLD1 was mostly found in intracellular membranes, acid by acyltransferase. FcεRI-mediated PA whereas PLD2 was exclusively present in the plasma membrane. production is affected by DGKz deficiency, suggesting that DGKz Silencing PLD2 expression by small interfering RNA could block also contributes to FcεRI-mediated PA production (25). Thus, it is Ras activation stimulated through the TCR and LFA-1. It was possible that the activity of lysophosphatidic acid acyltransferase speculated that the engagement of TCR and LFA-1 leads to PLD2 or DGK is enhanced in PLD-deficient T cells to compensate for activation and production of PA, which may activate Ras through the loss of PLD proteins. direct binding to Sos. PA can also be converted to DAG, which Previous studies using 1-butanol as a PLD inhibitor showed that activates RasGRP1 and PKCu. However, in this published study, PLD may function differently in Tregs and conventional T cells activation of Ras was determined by imaging the localization of because of its lower expression in Tregs (13). Interestingly, it was Raf RBD, which might not accurately reflect the activation status found that 1-butanol inhibits the surface expression of CTLA-4 of Ras (12). This result needs to be confirmed by biochemical (16), which suggested that PLD might be involved in Treg func- analysis of Ras activation in T cells. Our biochemical data indi- tion. Our flow analysis of PLD-deficient mice showed that Treg cated that TCR-mediated Erk was normal in PLD1KO and development and CTLA-4 expression were not affected (data not PLDdKO T cells; however, the study by Taylor et al. (15) showed shown). Thus, it is possible that reduced CTLA-4 expression by 1- that the PLD1-specific small inhibitor VU0359595 could butanol is a consequence of the nonspecific effect of this inhibitor. inhibit Erk1/2 phosphorylation similarly to a Mek/Erik inhibitor, It has been reported that PLD proteins are involved in receptor- suggesting that PLD1 is required for TCR-mediated Ras–MAPK mediated endocytosis (20). In this study, we showed that TCR 8 PLD PROTEINS IN T CELLS Downloaded from http://www.jimmunol.org/

FIGURE 6. Secondary CD8 T cell response in PLD-deficient mice. Mice were infected i.v. with 1.5 3 104 Lm-OVA at day 0 and challenged with 1.5 3 105 Lm-OVA at day 37. (A) Kinetics of OVA-specific CD8+ T cell response in PLDdKO and WT mice. Representative plots of OVA-specific CD8+ T cells in the blood from infected mice. (B) OVA-specific T cells after challenge. At day 5 after reinfection, splenocytes were stained with OVA-DimerX, CD11a, by guest on September 23, 2021 and CD8 and analyzed by flow cytometry. (C) The number of OVA-specific CD8+ T cells in the spleen at day 5 after Lm-OVA reinfection. Data are shown as mean 6 SEM. (D) IFN-g production by CD8+ T cells. Splenocytes were restimulated with 1 mM OVA peptide for 5 h in the presence of monensin before intracellular staining. Data are representative of three independent infections. downregulation from the cell surface was also affected by PLD1 Acknowledgments or PLD double deficiency, likely as the result of reduced PKCu We thank the Duke University Center Flow Cytometry and Trans- activation. PKCu phosphorylates Ser126 in CD3g, leading to AP-2 genic Mouse Facilities for excellent service. binding and TCR endocytosis (21). It is also possible that PLD1 deficiency affects actin reorganization, which is required for en- Disclosures docytosis of the TCR. Our published results indicate that PLD1 The authors have no financial conflicts of interest. deficiency in mast cells impairs FcεRI-mediated F-actin disas- sembly. In contrast, PLD2 deficiency enhances microtubule for- References mation (10). It is possible that PLD proteins function differently in 1. Cockcroft, S. 2001. Signalling roles of mammalian and D2. T and mast cells, because we did not observe any significant effect Cell. Mol. Life Sci. 58: 1674–1687. of PLD2 deficiency on T cells. Still, the role of PLD proteins in 2. Lehman, N., M. Di Fulvio, N. McCray, I. Campos, F. Tabatabaian, and J. Gomez-Cambronero. 2006. Phagocyte is mediated by phos- cytoskeletal rearrangement in T cells needs to be investigated pholipases PLD1 and PLD2. Blood 108: 3564–3572. further to fully understand how they function in T cells. 3. Gomez-Cambronero, J., M. Di Fulvio, and K. Knapek. 2007. Understanding phospholipase D (PLD) using leukocytes: PLD involvement in cell adhesion and Although many previous studies suggested that PLD proteins chemotaxis. J. Leukoc. Biol. 82: 272–281. play important roles in TCR signaling, T cell activation, and im- 4. Andresen, B. T., M. A. Rizzo, K. Shome, and G. Romero. 2002. The role of mune responses, many of these experiments were done using in- phosphatidic acid in the regulation of the Ras/MEK/Erk signaling cascade. FEBS Lett. 531: 65–68. hibitors that are not highly specific or using overexpression of the 5. Zhao, C., G. Du, K. Skowronek, M. A. Frohman, and D. Bar-Sagi. 2007. WT and DN forms of PLD1 and PLD2 proteins. Our results using Phospholipase D2-generated phosphatidic acid couples EGFR stimulation to Ras T cells deficient in PLD1, PLD2, or both suggest that, of the two activation by Sos. Nat. Cell Biol. 9: 706–712. 6. Choi, W. S., Y. M. Kim, C. Combs, M. A. Frohman, and M. A. Beaven. 2002. members of the PLD family, PLD1 is required for optimal TCR- D1 and D2 regulate different phases of exocytosis in mast cells. mediated signaling, T cell expansion, and T cell function during J. Immunol. 168: 5682–5689. 7. Melendez, A. J., L. Bruetschy, R. A. Floto, M. M. Harnett, and J. M. Allen. 2001. primary and secondary immune responses. Although PLD1- and Functional coupling of FcgammaRI to nicotinamide adenine dinucleotide PLD2-specific inhibitors are currently being tested against HIV, phosphate (reduced form) oxidative burst and immune complex trafficking re- influenza, and other infections or are used to treat , our data quires the activation of phospholipase D1. Blood 98: 3421–3428. 8. Chahdi, A., W. S. Choi, Y. M. Kim, P. F. Fraundorfer, and M. A. Beaven. 2002. suggest that we must carefully consider the usefulness of these Serine/threonine protein kinases synergistically regulate phospholipase D1 and 2 inhibitors, which will likely also inhibit immune responses. and secretion in RBL-2H3 mast cells. Mol. Immunol. 38: 1269–1276. The Journal of Immunology 9

9. Brown, F. D., N. Thompson, K. M. Saqib, J. M. Clark, D. Powner, 17. Yang, C. W., C. D. Hojer, M. Zhou, X. Wu, A. Wuster, W. P. Lee, B. L. Yaspan, N. T. Thompson, R. Solari, and M. J. Wakelam. 1998. Phospholipase D1 lo- and A. C. Chan. 2016. Regulation of T cell receptor signaling by DENND1B in calises to secretory granules and and is plasma-membrane trans- TH2 cells and allergic . Cell 164: 141–155. located on cellular stimulation. Curr. Biol. 8: 835–838. 18. Bose, T. O., Q. M. Pham, E. R. Jellison, J. Mouries, C. M. Ballantyne, and 10. Zhu, M., J. Zou, T. Li, S. A. O’Brien, Y. Zhang, S. Ogden, and W. Zhang. 2015. L. Lefranc¸ois. 2013. CD11a regulates effector CD8 T cell differentiation and Differential roles of phospholipase D proteins in FcεRI-mediated signaling and central memory development in response to infection with Listeria mono- mast cell function. J. Immunol. 195: 4492–4502. cytogenes. Infect. Immun. 81: 1140–1151. 11. Reid, P. A., S. D. Gardner, D. M. Williams, and M. M. Harnett. 1997. The an- 19. Zhang, W., J. Sloan-Lancaster, J. Kitchen, R. P. Trible, and L. E. Samelson. tigen receptors on mature and immature T are coupled to 1998. LAT: the ZAP-70 that links T cell receptor to phosphatidylcholine-specific phospholipase D activation. Immunology 90: 250–256. cellular activation. Cell 92: 83–92. 12. Mor, A., G. Campi, G. Du, Y. Zheng, D. A. Foster, M. L. Dustin, and M. R. Philips. 20. Shen, Y., L. Xu, and D. A. Foster. 2001. Role for phospholipase D in receptor- 2007. The function-associated antigen-1 receptor costimulates plasma mediated endocytosis. Mol. Cell. Biol. 21: 595–602. membrane Ras via phospholipase D2. Nat. Cell Biol. 9: 713–719. 21. von Essen, M., M. W. Nielsen, C. M. Bonefeld, L. Boding, J. M. Larsen, 13. Singh, N., Y. Seki, M. Takami, B. Baban, P. R. Chandler, D. Khosravi, X. Zheng, M. Leitges, G. Baier, N. Odum, and C. Geisler. 2006. C (PKC) alpha M. Takezaki, J. R. Lee, A. L. Mellor, et al. 2006. Enrichment of regulatory CD4 and PKC theta are the major PKC isotypes involved in TCR down-regulation. (+)CD25(+) T cells by inhibition of phospholipase D signaling. Nat. Methods 3: J. Immunol. 176: 7502–7510. 629–636. 22. Jaafar, R., C. Zeiller, L. Pirola, A. Di Grazia, F. Naro, H. Vidal, E. Lefai, and 14. Mollinedo, F., C. Gajate, and I. Flores. 1994. Involvement of phospholipase D in G. Ne´moz. 2011. Phospholipase D regulates myogenic differentiation through the activation of transcription factor AP-1 in human T lymphoid Jurkat cells. J. the activation of both mTORC1 and mTORC2 complexes. J. Biol. Chem. 286: Immunol. 153: 2457–2469. 22609–22621. 15. Taylor, H. E., G. E. Simmons, Jr., T. P. Mathews, A. K. Khatua, W. Popik, 23. Chen, J. S., and J. H. Exton. 2004. Regulation of phospholipase D2 activity by C. W. Lindsley, R. T. D’Aquila, and H. A. Brown. 2015. Phospholipase D1 protein kinase C alpha. J. Biol. Chem. 279: 22076–22083. couples CD4+ T cell activation to c-Myc-dependent deoxyribonucleotide pool 24. Oishi, K., M. Takahashi, H. Mukai, Y. Banno, S. Nakashima, Y. Kanaho, expansion and HIV-1 replication. PLoS Pathog. 11: e1004864. Y. Nozawa, and Y. Ono. 2001. PKN regulates phospholipase D1 through direct 16. Mead, K. I., Y. Zheng, C. N. Manzotti, L. C. Perry, M. K. Liu, F. Burke, interaction. J. Biol. Chem. 276: 18096–18101. D. J. Powner, M. J. Wakelam, and D. M. Sansom. 2005. Exocytosis of CTLA-4 25. Olenchock, B. A., R. Guo, J. H. Carpenter, M. Jordan, M. K. Topham, Downloaded from is dependent on phospholipase D and ADP ribosylation factor-1 and stimulated G. A. Koretzky, and X. P. Zhong. 2006. Disruption of diacylglycerol metabolism during activation of regulatory T cells. J. Immunol. 174: 4803–4811. impairs the induction of T cell anergy. Nat. Immunol. 7: 1174–1181. http://www.jimmunol.org/ by guest on September 23, 2021