Bcl-xL Regulates CD1d-Mediated Presentation to NKT Cells by Altering CD1d Trafficking through the Endocytic Pathway

This information is current as Priyanka B. Subrahmanyam, Gregory B. Carey and Tonya J. of September 24, 2021. Webb J Immunol published online 28 July 2014 http://www.jimmunol.org/content/early/2014/07/26/jimmun ol.1400155 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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 28, 2014, doi:10.4049/jimmunol.1400155 The Journal of Immunology

Bcl-xL Regulates CD1d-Mediated Antigen Presentation to NKT Cells by Altering CD1d Trafficking through the Endocytic Pathway

Priyanka B. Subrahmanyam, Gregory B. Carey, and Tonya J. Webb

NKT cells are a unique subset of T cells that recognize glycolipid Ags presented in the context of CD1d molecules. NKT cells mount strong antitumor responses and are a major focus in developing effective cancer immunotherapy. It is known that CD1d molecules are constantly internalized from the cell surface, recycled through the endocytic compartments, and re-expressed on the cell surface. However, little is known about the regulation of CD1d-mediated Ag processing and presentation in B cell lymphoma. Prosurvival factors of the Bcl-2 family, such as Bcl-xL, are often upregulated in B cell lymphomas and are intimately linked to sphingolipid metabolism, as well as the endocytic compartments. We hypothesized that Bcl-xL can regulate CD1d-mediated Ag presentation to Downloaded from NKT cells. We found that overexpression or induction of Bcl-xL led to increased Ag presentation to NKT cells. Conversely, the inhibition or knockdown of Bcl-xL led to decreased NKT cell activation. Furthermore, knockdown of Bcl-xL resulted in the loss of CD1d trafficking to lysosome-associated membrane 1+ compartments. Rab7, a late endosomal protein, was upregulated and CD1d molecules accumulated in the Rab7+ late endosomal compartment. These results demonstrate that Bcl-xL regulates CD1d-mediated Ag processing and presentation to NKT cells by altering the late endosomal compartment and changing the intracellular localization of CD1d. The Journal of Immunology, 2014, 193: 000–000. http://www.jimmunol.org/

atural killer T cells are a unique subset of T cells that The process of CD1d-mediated Ag presentation is complex recognize lipid Ags presented by CD1d, an MHC class and begins with the synthesis of the CD1d a-chain in the en- N I–like molecule (1–3). Once activated, NKT cells can doplasmic reticulum (ER) (12). Here chaperons like calnexin, mediate direct cytotoxicity and also rapidly produce large amounts calreticulin, and Erp57 ensure that it is properly folded (13). The of such as IFN-g and IL-4. One of the most striking and AgbindinggrooveofCD1disoccupiedbyaself-lipidAg well-established functions of NKT cells is their antitumor effect, thought to be loaded by the microsomal triglyceride transfer

mediated directly by cytotoxicity, as well as indirectly by protein (14, 15). After association with b2-microglobulin, the production, leading to the recruitment and activation of other cell CD1d molecule follows the secretory pathway from the ER to the by guest on September 24, 2021 types (4–6). However, the precise mechanisms that underlie the Golgi and reaches the plasma membrane (PM). To present an recognition of tumors by NKT cells, in the absence of an exog- activating endogenous Ag to NKT cells, CD1d molecules re- enous activating Ag like the prototypical a-galactosylceramide cycle from the PM to endocytic compartments because of the (a-GalCer), remain poorly understood. presence of a tyrosine-based targeting motif (Yxxw, where Y is In contrast to the MHC restriction of classical T cells, NKT cells are tyrosine, x is any amino acid, and w is a hydrophobic amino CD1d restricted (7, 8). Mice possess CD1d1 and CD1d2 ; acid) (16, 17). This is analogous to the invariant chain for MHC however, Ag presentation to NKT cells is dependent upon CD1d1 class II molecules. In fact, invariant chain associates with CD1d, molecules (referred to as CD1d). The CD1d molecule is structurally but the Yxxw motif is necessary for the proper trafficking of the similar to MHC class I with a three-domain a-chain that associates CD1d molecules to the endocytic compartments (18). Following with b -microglobulin, but unlike the classical MHC class I molecule, 2 internalization from the PM, adaptor AP2 and AP3 direct CD1d has a hydrophobic Ag binding groove (9, 10). Also, in con- CD1d molecules to the endocytic compartment, also known as trast with the ubiquitous expression of MHC class I, CD1d is mainly MHC class II compartment, where MHC class II molecules are expressed on dendritic cells, macrophages, B cells, and T cells (11). normally loaded with peptide Ags (19, 20). Once in the endocytic recycling compartment, the stabilizing self-lipid is exchanged for Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum Cancer Center, Baltimore, MD other lipid Ags with the help of saposins (21). These loaded CD1d 21201 molecules are then re-expressed on the PM and can be recognized Received for publication January 22, 2014. Accepted for publication June 30, 2014. by canonical Va14Ja18 NKT cells. The localization of CD1d to This work was supported by National Institutes of Health, National Cancer Institute cholesterol-rich lipid rafts is important for efficient Ag presenta- Grants K01 CA131487, R21 CA162273, and R21 CA162277 and grants from the P30 tion, especially in the presence of low concentrations of Ags, and Tumor Immunology and Immunotherapy Program (to T.J.W.). the disruption of these lipid rafts leads to reduced Ag presentation Address correspondence and reprint requests to Dr. Tonya J. Webb, University of Maryland School of Medicine, 685 West Baltimore Street, HSF I-Room 380, Balti- (22, 23). The complex multistep process of CD1d-mediated Ag more, MD 21201. E-mail address: [email protected] processing and presentation has several potential levels of control, The online version of this article contains supplemental material. yet very few endogenous regulatory factors have been identified. Abbreviations used in this article: BH3, Bcl-2 homology 3; ER, endoplasmic reticulum; Prominent among these are the MAPKs, protein kinase Cd, and a-GalCer, a-galactosylceramide; HSA, serum albumin; LAMP1, lysosome- Rho kinases (24–26). In this study, we sought to identify a target associated membrane protein 1; PM, plasma membrane; shRNA, short hairpin RNA. that regulates CD1d-mediated Ag presentation and is relevant to Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 tumor growth and survival.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400155 2 Bcl-xL REGULATES CD1d-MEDIATED Ag PRESENTATION

Antiapoptotic Bcl-2 family members are known to be expressed from NeoBioLab (Cambridge, MA) as per the manufacturer’s directions. at high levels in lymphomas and other malignancies, and allow Stable transfectants were then cultured in dual selection medium containing cells to evade apoptotic signals and attain a neoplastic state (27). G418 sulfate (500 mg/ml) and Zeocin (200 mg/ml), and passaged every 3 d. Overexpression of Bcl-xL was confirmed by Western blotting. Bcl-xL is a potent antiapoptotic factor and exerts its antiapoptotic Bcl-xL knockdown was attained using lentiviral vectors purchased from function by heterodimerizing with other Bcl-2 family members Santa Cruz Biotechnology. These replication incompetent lentiviral par- and preventing the permeabilization of the mitochondrial outer ticles contained a pool of three to four short hairpin sequences targeted to membrane. Bcl-xL can also mediate its prosurvival function by the Bcl2l1 that encodes Bcl-xL, or the Bcl-2 gene and polybrene- based transduction was carried out as per the manufacturer’s instructions. causing the retrotranslocation of the proapoptotic factor Bax from Stable transductants were cultured in dual-selection medium containing the mitochondria to the cytosol (28). Bcl-xL also prevents the for- G418 sulfate (500 mg/ml) and puromycin (5 mg/ml), and were passaged mation of channels, which contribute to the release of every 3 d. Bcl-xL knockdown was confirmed by Western blotting. cytochrome c from the mitochondria (29). Although Bcl-xL was traditionally considered a mitochondrial apoptosis regulator, recent Mice evidence shows that this protein is also present in other compart- Female C57BL/6 mice were purchased from The Jackson Laboratory (Bar ments such as the ER (30). Bcl-xL can also affect the intracellular Harbor, ME) and used at ∼6 wk of age. All performed procedures were endosomal compartments, which are critical for CD1d-mediated Ag approved by the University of Maryland Institutional Animal Care and Use Committee. After euthanasia, spleens were removed and a single-cell processing and presentation (31). Furthermore, Bcl-xL has been suspension of splenocytes was prepared using a 70-mm nylon mesh from reported to interact with various trafficking-related proteins such as Fisher Scientific (Waltham, MA). B cells were isolated using a mouse calnexin, Rab7, ER Golgi intermediate compartment 1, and vesicle- B cell enrichment from Stem Cell Technologies (Vancouver, BC, associated membrane protein 3, underscoring the multifaceted Canada) as per the manufacturer’s instructions. Purity was .95% as de- Downloaded from effects of Bcl-xL (32). termined by flow cytometry after staining with FITC-conjugated Ab to B220 (CD45RO) from Biolegend (San Diego, CA). Enriched splenic To examine the role of Bcl-xL on CD1d-mediated Ag process- B cells were suspended in RPMI 1640 with 10% FBS and other supple- ing and presentation, we conducted a series of overexpression, in- ments as described earlier. The liver was processed and liver mononuclear duction, and knockdown studies. In addition, we examined the effect cells were obtained as previously described (35). NKT cell percentage of pharmacological agents known to disrupt Bcl-xL function and was determined by flow cytometry after staining with a FITC-conjugated Ab to the TCR b-chain from Biolegend (San Diego, CA) and allophyco- assessed intracellular trafficking to endosomal compartments. In- cyanin-conjugated CD1d tetramers loaded with a-GalCer, graciously http://www.jimmunol.org/ terestingly, we found that the mechanism of regulation is inde- provided by the National Institutes of Health Tetramer Core Facility at pendent of surface CD1d expression, costimulatory molecules, and Emory University (Atlanta, GA). CD1d recycling. After Bcl-xL knockdown, alterations in intracellular trafficking of CD1d were observed. In addition, the expression of Flow cytometry Rab7, a late endosomal protein, was increased and there was ex- Cells were stained in PBS containing 0.5% BSA and 2 mM EDTA for pansion of the late endosomal compartment. CD1d molecules were 30 min at 4˚C with a PE-conjugated Ab to CD1d (clone 1B1) from BD lost from the early endosomes and lysosomes, and increased in the Biosciences (San Jose, CA). Data were collected on an LSR II from BD Biosciences (San Jose, CA) and analyzed using FCS Express Version 3 late endosomal compartment. These data show that in the absence of from De Novo Software (Los Angeles, CA). Bcl-xL, the expanded late endosomal compartment acts as a CD1d by guest on September 24, 2021 depot and alters Ag presentation, ultimately resulting in loss of NKT cell hybridoma assay NKT cell activation. L-CD1d or LMTK-CD1d cells (5 3 105/well) were cocultured with NKT cells (5 3 104/well)for20hat37˚CinIMDMsupplemented Materials and Methods with 5% FBS and L-glutamine. Primary splenic B cells were treated with Cell lines, Ags, and other reagents 10 mg/ml anti-CD40 Ab (clone 1C10) from eBioscience (San Diego, CA), washed with complete medium, and cocultured (106 B cells/well) with 5 L-CD1d, L-CD1d-DR4, and LMTK-CD1d are mouse fibroblast cell lines liver mononuclear cells (2 3 10 /well) for 24 h at 37˚C in complete transfected with mCD1d1 or mCD1d1 and HLA-DR4 (kindly provided medium. For Bcl-xL/Bcl-2 inhibition studies, L-CD1d-DR4 cells were by Dr. Randy Brutkiewicz, Indiana University School of Medicine, In- treated with ABT-263 or ABT-199 for 4 h, washed twice with PBS and dianapolis, IN) and were cultured in DMEM with 10% FBS and 2 mM once with complete medium, and cocultured with NKT cells. For the L-glutamine. In addition, selection agents G418 sulfate (500 mg/ml) from peptide Ag presentation assay, L-CD1d-DR4 cells were loaded with HSA Mediatech (Manassas, VA), and Zeocin (200 mg/ml) and puromycin (5 mg/ml) overnight, treated with ABT-263 or ABT-199 for 4 h, washed exten- from Santa Cruz Biotechnology (Santa Cruz, CA) were added as required. sively, and cocultured with 17.9 hybridomas. After the coculture, Unless otherwise specified, all materials for cell culture were purchased supernatants were harvested and IL-2, IFN-g, or IL-4 levels were mea- from Life Technologies (Carlsbad, CA). WEHI-231 and WEHI-231/Bcl- sured by ELISA. Mouse IL-2 ELISA kit from BD Biosciences (San Jose, xL cells were kindly provided by Dr. Gregory Carey (University of CA) and mouse IFN-g and IL-4 ELISA kits from Biolegend (San Diego, Maryland) and cultured in RPMI 1640 with 10% FBS, 100 mM sodium CA) were used. Data were graphed using Prism 5.02 from GraphPad (La pyruvate, 10 mM nonessential amino acid solution, 13 vitamin solution, Jolla, CA). and 50 mM 2-ME. The mouse type I NKT cell hybridomas DN32.D3 and N38-3C3, as well as the type II NKT cell hybridomas N37-1A12, have Western blotting been described and were cultured in IMDM supplemented with 5% FBS + Cells were lysed using lysis buffer containing NaCl (150 mM), Tris-Cl and 2 mM L-glutamine (33, 34). 17.9 CD4 T cell hybridomas were kindly provided by Dr. Janice Blum (Indiana University School of Medicine) and (50 mM), EDTA (8.5 mM), sodium azide (0.02%), nonylphenoxypolyethoxy- cultured in RPMI 1640 supplemented with 10% FBS, 50 mM 2-ME, and ethanol (0.1%), and complete mini protease inhibitor cocktail tablet from Roche Applied Science (Indianapolis, IN) as directed by the manufacturer, 2mML-glutamine. a-GalCer purchased from Enzo Life Sciences (New York, NY) was used at a final concentration of 100 ng/ml. Human serum prepared in water. Proteins were resolved by electrophoresis on a 4–12% albumin (HSA) was purchased from Sigma-Aldrich (St. Louis, MO) and gradient polyacrylamide gel and transferred to a polyvinylidene difluoride used at 10 mM. ABT-263 and ABT-199 used for the inhibition of Bcl-xL membrane using the iBlot transfer system. All gels, equipment, buffers, and and/or Bcl-2 were a kind gift from Dr. Gregory Carey (University of other materials were from Life Technologies (Carlsbad, CA) and were used Maryland). as per the manufacturer’s instructions. Membranes were probed with Abs to Bcl-xL (clone 54H6) from Cell Signaling Technology (Beverly, MA) or Overexpression and knockdown of Bcl-xL Rab7 from Santa Cruz Biotechnology. GAPDH levels were detected on the same blot as the test protein using an Ab (clone 14C10) from Cell Signaling LMTK-CD1d cells were transfected with vector alone or vector encoding Technology. Dylight-800–conjugated anti-rabbit secondary Ab was pur- Bcl-xL, kindly provided by Dr. Mark Williams (University of Maryland). chased from Thermo Scientific (Waltham, MA). Membranes were scanned using Transfection was carried out using Neofectin one-step transfection reagent the Odyssey Imaging System from Li-COR Biosciences (Lincoln, NE). The Journal of Immunology 3

Confocal microscopy mediated Ag presentation to NKT cells with minimal confound- Cells were added to a Lab Tek II Chamber slide from Thermo Scientific ing factors. To directly test whether Bcl-xL plays a role in Ag (Waltham, MA), allowed to adhere overnight and fixed in 1% parafor- presentation to NKT cells, we transfected LMTK-CD1d cells with maldehyde in PBS for 15 min at room temperature. Nonspecific binding an empty control plasmid or a plasmid carrying the Bcl2l1 gene, sites were blocked by incubating cells in PermWash buffer from BD which encodes Bcl-xL, resulting in the overexpression of Bcl-xL Biosciences (San Jose, CA) with 5% rat serum for 1 h at 4˚C. The cells were protein as seen by Western blotting (Fig. 1G). There were no then incubated with Abs to CD1d (clone 1H6), kindly provided by Dr. Randy Brutkiewicz (Indiana University School of Medicine), CD107a apparent differences in cell morphology, growth, or proliferation (lysosome-associated membrane protein 1 [LAMP1]) from eBioscience (Supplemental Fig. 1A, 1C). Also, transfection with Bcl-xL did (San Diego, CA), EEA1 from Cell Signaling Technology (Beverly, MA), not dramatically alter surface CD1d expression as analyzed by or Rab7 from Santa Cruz Biotechnology at 4˚C overnight. After washing, flow cytometry (Fig. 1H). These LMTK-CD1d cells transfected Alexa Fluor 546–conjugated anti-mouse IgG, allophycocyanin-conjugated anti-rabbit IgG, and Alexa Fluor 647–conjugated anti-rat IgG from Life with Bcl-xL or the respective control were cocultured with a panel Technologies (Carlsbad, CA) were added and incubated for 1 h at room of three NKT cell hybridomas. Although DN32.D3 and N38-3C3 temperature in the dark. Slides were then mounted using Vectashield mo- are canonical type I NKT cells, N37-1A12 is a type II NKT cell unting medium containing DAPI from Vector Laboratories (Burlingame, CA). hybridoma (33, 34). We found that the overexpression of Bcl-xL 3 Images were acquired using a 40 lens on the LSM 510, a laser-scanning in LMTK-CD1d cells elicited significantly higher response from microscope from Carl Zeiss Microscopy (Oberkochen, Germany) using the Zen 2009 software and processed using the Zeiss LSM Image Browser all three hybridoma lines tested (Fig. 1D–F). From these data, it is Version 4.2.0.121. Colocalization was quantified using the same software evident that Bcl-xL can affect NKT cell activation because high from five different images, and Pearson’s correlation for colocalization levels of Bcl-xL elicit stronger NKT cell responses. Furthermore, was represented as percent of control. In addition, Rab7 staining was we used CD1d blocking Abs and found that IL-2 production was Downloaded from quantified by pixel number from five different images and represented as percent of control. completely lost after CD1d blockade in the control-transfected, as well as Bcl-xL–transfected LMTK-CD1d cells (Supplemental Fig. Statistical analyses 2A–C). This indicates that the regulation by Bcl-xL is specific to All experiments were repeated at least three times. Two-tailed Student t test, CD1d-dependent Ag presentation to NKT cells. one-way ANOVA, or two-way ANOVA was used as appropriate. Specific Induction of Bcl-xL by CD40 stimulation in B cells leads to experimental groups were compared with controls using the Bonferroni http://www.jimmunol.org/ posttest. A p value ,0.05 was considered significant. All analyses were increased NKT cell activation , performed using Prism 5.02 by GraphPad (La Jolla, CA); ***p 0.001, We found that the overexpression of Bcl-xL in APCs leads to en- **p , 0.01, *p , 0.05. hanced Ag presentation to NKT cells. Next, we wanted to induce Bcl-xL using a biological stimulus and determine whether Ag Results presentation to NKT cells was affected. One of the most well- Overexpression of Bcl-xL in APCs leads to increased NKT cell known aspects of B cell stimulation with anti-CD40 Ab is the responses upregulation of Bcl-xL, and it has been reported extensively in Bcl-xL is a prosurvival member of the Bcl-2 family of proteins. In WEHI-231 cells, primary mouse B cells, and also in human B cells this study, we hypothesized that Bcl-xL affects the ability of an (38–41). We treated WEHI-231 cells with anti-CD40 Ab (clone by guest on September 24, 2021 APC to present glycolipid Ags to NKT cells, independent of its 1C10) and confirmed the induction of Bcl-xL protein by Western regulation of apoptosis. To determine whether the overexpression blotting (Fig. 2A). We found that treatment with anti-CD40 Ab did of Bcl-xL has an effect on CD1d-mediated Ag presentation, we not alter surface CD1d expression (Fig. 2B). WEHI-231 cells used WEHI-231, a classical and well-characterized murine B cell treated with anti-CD40 Ab elicited higher NKT cell activation lymphoma cell line (36), and compared it with a WEHI-231–de- compared with cells treated with isotype control Ab (Fig. 2C, 2D). rived cell line stably transfected with murine Bcl-xL. Over- This shows that the induction of Bcl-xL in a B cell lymphoma cell expression of Bcl-xL protein was first confirmed by Western line leads to increased NKT cell responses. These results are blotting (Fig. 1A). Next, the WEHI-231 controls and Bcl-xL consistent with the data obtained from overexpression of Bcl-xL transfectants were cocultured with two canonical Va14Ja18 (Fig. 1B–F) and show that increased Bcl-xL levels lead to NKT cell hybridomas, DN32.D3 and N38-3C3, and IL-2 pro- a corresponding increase in CD1d-mediated Ag presentation to duction was measured in the coculture supernatant (Fig. 1B, 1C). NKT cells. In the absence of exogenously added Ags, the WEHI-231 control, as well as Bcl-xL transfected cell lines, failed to activate NKT Bcl-xL–mediated regulation of Ag presentation in primary cells. These data suggest that the endogenous Ag presented by mouse B cells CD1d molecules in these cells does not activate NKT cells. To eliminate the possibility of cell line–specific effects and validate However, when the potent NKT cell agonist, a-GalCer, was added the results in primary mouse cells, we used freshly isolated splenic to the coculture, Bcl-xL–transfected cells were able to stimulate B cells as APCs and liver mononuclear cells as a source of pri- NKT cells, unlike the untransfected controls. These data demon- mary NKT cells. Approximately 40% of T cells in the liver are strate that CD1d molecules expressed on the cell surface of NKT cells, as compared with the thymus and spleen (1–2%). Also, WEHI/Bcl-xL cells are functional, but these cells do not present liver NKT cells are the only subset known to mediate antitumor an activating endogenous ligand. Furthermore, the presentation of effector functions (42). To assess the effect of Bcl-xL induction on an exogenous Ag is enhanced following the overexpression of Ag presentation to NKT cells, we treated splenic B cells with anti- Bcl-xL (Fig. 1B, 1C). CD40 Ab for 24 h and used Western blotting to ensure the up- To characterize the potential role of Bcl-xL in CD1d-mediated regulation of Bcl-xL protein (Fig. 3A). After treatment, cells were Ag presentation, we used LMTK cells as an in vitro APC system. washed and cocultured with liver mononuclear cells in the pres- LMTK cells, transfected with CD1d (LMTK-CD1d), are known to ence or absence of a-GalCer. NKT cell activation was assessed by present an endogenous activating Ag to NKT cells (37). Further- measuring IFN-g or IL-4 in the supernatant by ELISA (Fig. 3B, more, LMTK-CD1d cells can be cocultured with a panel of mouse 3C). There was no detectable cytokine production in the absence NKT cell hybridomas, which produce high levels of IL-2 after of a-GalCer, demonstrating that the observed responses were activation, providing a straightforward system to study CD1d- NKT cell specific. In the presence of a-GalCer, anti-CD40–treated 4 Bcl-xL REGULATES CD1d-MEDIATED Ag PRESENTATION Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 1. Overexpression of Bcl-xL results in enhanced CD1d-mediated Ag presentation to NKT cells. (A) Western blot showing Bcl-xL protein levels (upper panel) in WEHI-231 or WEHI-231 following Bcl-xL overexpression. GAPDH (lower panel) is the loading control. (B and C) Medium alone, WEHI- 231 (Ctrl) or WEHI-231/Bcl-xL (Bcl-xL) cells were cocultured with (B) DN32.D3 or (C) N38-3C3 NKT cell hybridomas with or without an exogenous Ag, and IL-2 in the supernatant was measured by ELISA. (D–F) Medium alone, LMTK-CD1d cells overexpressing Bcl-xL, or the corresponding empty vector controls were cocultured with mouse NKT cell hybridomas (D) DN32.D3, (E) N37-1A12, or (F) N38-3C3 for 20 h, and IL-2 in the supernatant was measured by ELISA. (G) Western blot showing Bcl-xL protein levels (upper panel) in LMTK-CD1d cells transfected with empty vector (ctrl) or Bcl-xL. GAPDH is the loading control (lower panel). (H) Flow cytometry shows surface CD1d expression on LMTK-CD1d cells transfected with Bcl-xL (open histogram) or vector alone (shaded histogram). **p , 0.01, ***p , 0.001.

B cells led to increased IFN-g production by NKT cells as com- inhibitors. ABT-263 targets both Bcl-xL and Bcl-2, whereas ABT- pared with their untreated or isotype control–treated counterparts 199 is Bcl-2 specific. Pretreatment of L-CD1d-DR4 cells with ABT- (Fig. 3B). However, IL-4 production was unchanged after treat- 263 led to decreased CD1d-mediated Ag presentation to NKT cells ment with anti-CD40 Ab (Fig. 3C). Despite the use of total liver (Fig. 4A). In contrast, pretreatment with ABT-199 had no effect on mononuclear cells, the observed responses were NKT cell specific Ag presentation to NKT cells. These data demonstrate that BH3 because all cytokine levels were detectable only in cocultures domain-based functional inhibition of Bcl-xL, but not Bcl-2, leads to containing NKT cell Ags. In addition, we analyzed the cells by reduced CD1d-mediated Ag presentation to NKT cells. Because the flow cytometry after treatment and found no changes in the sur- Ag presentation pathway of CD1d is very similar to classical MHC face expression of MHC class I, CD1d, CD80, or CD86 (Fig. 3D– class II and involves the lysosomal MHC class II compartment or G). Together, these data show that Bcl-xL plays a role in CD1d- MIIC, we sought to determine the effect of Bcl-xL inhibition of mediated Ag processing and presentation to NKT cells in an ex peptide Ag presentation to classical CD4+ T cells (45). L-CD1d- vivo system using primary mouse B cells and NKT cells. DR4 cells loaded with HSA were treated with ABT-263 or ABT- 199, washed extensively, and cocultured with the 17.9 T cell Pharmacological inhibition of Bcl-xL leads to reduced hybridoma line (Fig. 4B). Similar to CD1d-mediated Ag presenta- NKT cell responses tion, we found that the presentation of HSA was reduced after To study the effect of Bcl-xL inhibition on CD1d-mediated Ag treatment with ABT-263 but was unaffected by treatment with presentation to NKT cells, we used small-molecule inhibitors that ABT-199. Because ABT-263 and ABT-199 can induce apoptosis at bind the Bcl-2 Homology 3 (BH3) domain of Bcl-xL. BH3–BH3 higher concentrations, cell viability was determined by Annexin V– interactions between proteins of the Bcl-2 family are critical for their PI staining, where Annexin V+ PI+ cells were considered apoptotic function. We treated L-CD1d cells with two small-molecule BH3 and the remainder of cells were considered viable (single positive mimetics, ABT-263 and ABT-199 (43, 44), which are functional cells were minimal). There were no differences in viability between The Journal of Immunology 5

FIGURE 2. Induction of Bcl-xL by anti-CD40 mAb treatment in WEHI-231 cells results in increased NKT cell responses. (A) Western blotting for Bcl-xL (upper panel) and GAPDH (loading control, lower panel) after treatment of WEHI-231 cells with anti-CD40 Ab (1C10) for 24 h (10 mg/ml) or with an isotype control Ab. (B) Cell-surface CD1d expression on WEHI-231 cells treated with anti- CD40 Ab or the corresponding controls was determined by flow cytometry. (C and D) Medium alone or WEHI-231 cells treated with anti-CD40 Ab were cocultured with mouse NKT cell hybridomas (B) DN32.D3 or (C) N38-3C3 in the presence or absence of an exogenous Ag for 20 h. IL-2 was measured in the supernatant by ELISA. ***p , 0.001. Downloaded from treated and untreated groups, indicating that our selected treatment after transduction and selection of stable integrants (Fig. 5B). concentrations were not sufficient to induce cell death and the L-CD1d cells transduced with a scrambled sequence or with role of Bcl-xL in CD1d-mediated Ag presentation is independent shRNA to knockdown Bcl-xL or Bcl-2 were cocultured with of apoptosis (Fig. 4C). Finally, there were no evident changes in NKT cell hybridomas. The knockdown of Bcl-xL or Bcl-2 led to cell-surface expression of CD1d or HLA-DR after treatment with decreased NKT cell activation as indicated by a significant re- http://www.jimmunol.org/ ABT-263 or ABT-199 (Fig. 4D). duction in IL-2 production by both DN32.D3 and N37-1A12 NKT cell hybridomas (Fig. 5C, 5D). Stable knockdown of Bcl-xL causes inhibition of CD1d-mediated Ag presentation to NKT cells Bcl-xL regulates intracellular CD1d trafficking L-CD1d cells express high levels of Bcl-xL; therefore, it is a good Bcl-xL knockdown results in a significant reduction in CD1d- system to investigate the effects of Bcl-xL knockdown on CD1d- mediated Ag presentation to NKT cells; however, the levels of mediated Ag processing and presentation. We used lentiviral surface CD1d expression are comparable with the controls. The particles carrying a pool of short hairpin RNA (shRNA) sequences overexpression or knockdown of Bcl-xL altered the activation of by guest on September 24, 2021 targeting the Bcl2l1 gene, which encodes the Bcl-xL protein. After N37-1A12, a type II NKT cell hybridoma. These NKT cells do transduction, stable cell lines were generated and Bcl-xL or Bcl-2 not require CD1d recycling for Ag presentation, suggesting that knockdown was confirmed by Western blotting (Fig. 5A). There this is not the mechanism by which Bcl-xL mediates its regu- were no apparent differences in cell morphology, growth, or lation. We therefore hypothesized that Bcl-xL alters the intra- proliferation after stable knockdown (Supplemental Fig. 1B, 1D). cellular trafficking of CD1d molecules. To determine the precise Also, there were no evident changes in surface CD1d expression mechanism of regulation, we first analyzed the intracellular

FIGURE 3. Induction of Bcl-xL by anti-CD40 mAb treatment in primary mouse B cells increases CD1d-mediated Ag presentation to NKT cells. (A) Splenic mouse B cells were treated with 10 mg/ml anti-CD40 Ab (1C10) or isotype control for 24 h, and Bcl-xL protein was detected by Western blotting (upper panel). GAPDH was used as the loading control (lower panel). (B and C) Treated B cells or medium alone were cocultured with primary murine liver mononuclear cells with or without a-GalCer. (B) IFN-g and (C) IL-4 were measured in the supernatant by ELISA. (D–G) Flow cytometry was performed to determine cell-surface expression of (D) MHC class I (H-2Kb), (E) CD1d, (F) CD80, or (G) CD86 after treatment of primary mouse B cells with anti-CD40 Ab. ***p , 0.001. 6 Bcl-xL REGULATES CD1d-MEDIATED Ag PRESENTATION Downloaded from http://www.jimmunol.org/ FIGURE 4. Pharmacological inhibition of Bcl-xL inhibits CD1d-mediated Ag presentation to NKT cells, as well as MHC class II–mediated Ag pre- sentation to classical CD4+ T cells. (A) L-CD1d-DR4 cells were treated with vehicle alone (DMSO), ABT-263, or ABT-199 for 4 h at a concentration of 10 or 20 mM, washed extensively, and cocultured with NKT cell hybridomas. IL-2 in the supernatant was measured by ELISA (B) L-CD1d-DR4 cells were loaded with HSA overnight and treated with vehicle alone, ABT-263, or ABT-199 as indicated earlier. After treatment, cells were washed extensively and cocultured with 17.9 T cell hybridomas. IL-2 was measured in the supernatant by ELISA. (C) Annexin V–PI staining was used to determine the percentage of viable cells (cells that were not positive for Annexin V and PI) after treatment with ABT-263 or ABT-199. (D) Flow cytometry shows surface expression or CD1d or HLA-DR after treatment of L-CD1d-DR4 cells with ABT-263 or ABT-199. ***p , 0.001.

localization of CD1d in L-CD1d cells after Bcl-xL knockdown. after Bcl-xL knockdown (shBcl-xL), this colocalization was by guest on September 24, 2021 Under normal conditions, CD1d is localized in the lysosomal substantially reduced (Fig. 6A, 6B). These data show that after compartment, as seen by its colocalization with LAMP1 Bcl-xL knockdown, CD1d molecules are lost from the LAMP1+ (CD107a) (16). We used confocal microscopy to determine the compartment. Such an alteration is known to affect CD1d- intracellular location of CD1d after Bcl-xL knockdown. As mediated Ag processing and presentation, indicating that this is expected, control L-CD1d cells (scramble) showed strong at least one mechanism by which Bcl-xL regulates Ag presenta- colocalization of CD1d with LAMP1 (Fig. 6A). Interestingly, tion to NKT cells.

FIGURE 5. Knockdown of Bcl-xL or Bcl-2 leads to de- creased CD1d-mediated Ag presentation to NKT cells. (A) Western blotting showing Bcl-xL protein levels (upper panel) or Bcl-2 (middle panel) after transduction of L-CD1d cells with lentiviral vectors carrying shRNA sequences tar- geting Bcl-xL or Bcl-2. GAPDH is the loading control (lower panel). (B) Flow cytometry shows surface CD1d ex- pression on L-CD1d cells transduced with a scramble shRNA sequence or sequence targeting Bcl-xL or Bcl-2. (C and D) Medium alone or L-CD1d cells after knockdown of Bcl-xL or Bcl-2 were cocultured with (C) DN32.D3 or (D) N37-1A12 NKT cell hybridomas, and IL-2 released in the supernatant was measured by ELISA. **p , 0.01, ***p , 0.001. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ FIGURE 6. Loss of CD1d molecules from the LAMP1+ lysosomal compartment after Bcl-xL knockdown. (A) Confocal microscopy was used to de- termine the intracellular localization of CD1d. L-CD1d cells transduced with a scrambled shRNA sequence (scramble, upper row) or an shRNA sequence targeting Bcl-xL (shBcl-xL, lower row) were fixed, permeabilized, and stained with Abs to CD1d (red) and LAMP1 (green), a marker of the lysosomal compartment. DAPI was used to visualize the nucleus. The merge (rightmost panels) shows that CD1d is localized in the LAMP1+ late endosomal/ lysosomal compartment in the control (yellow), whereas this localization pattern is lost after Bcl-xL knockdown. Original magnification 3400. (B) Quantification of CD1d and LAMP1 colocalization in scramble control or Bcl-xL knockdown cells. *p , 0.05.

Bcl-xL causes expansion of the Rab7+ late endosomal molecules accumulate after Bcl-xL knockdown. We found that compartment similar to the LAMP1+ compartment, there was minimal coloc- by guest on September 24, 2021 We found that Bcl-xL knockdown leads to the loss of CD1d from alization of CD1d with EEA1, a marker for early endosomes, after the LAMP1+ compartment (Fig. 6A, 6B). We further sought to Bcl-xL knockdown (Fig. 7A, 7B). This shows that CD1d mole- determine an alternative intracellular compartment where CD1d cules are lost from the LAMP1+ lysosomes, as well as the EEA1+

FIGURE 7. Reduced expression of CD1d in early endosomes after Bcl-xL knockdown. (A)Confocal microscopy on control or Bcl-xL– deficient L-CD1d cells stained with Abs against CD1d (red) or EEA1 (green), a marker for early endo- somes. The rightmost panels show colocalization of CD1d with EEA1, which is indicative of its presence in the early endosomal compartment. As compared with the control, Bcl-xL knockdown cells show less localization of CD1d in the early endosomes (yellow). Original mag- nification 3400. (B) Quantification of CD1d and EEA1 colocalization in scramble control or Bcl-xL knock- down cells. **p , 0.01. 8 Bcl-xL REGULATES CD1d-MEDIATED Ag PRESENTATION early endosomes, indicating that CD1d molecules might have lymphoma cells have an alteration in CD1d-mediated Ag pre- accumulated in a different intracellular compartment, impairing sentation compared with healthy B cells (48), the identification of Ag presentation after Bcl-xL knockdown. such a regulatory factor is not only an advancement in our un- A previous report has shown that Bcl-xL can bind to Rab7, which derstanding of the mechanisms that form the basis of NKT cell– is a Ras-related GTP binding protein present in late endosomes mediated tumor recognition, but also have direct translational (46). Rab7 is critical to endosome biogenesis and the endocytic impact by allowing the development of more effective NKT cell– pathway. We found that there was greater Rab7 staining after Bcl- based antitumor immunotherapy. xL knockdown as compared with the scramble control (Fig. 8A, We present evidence of altered CD1d trafficking as a mechanism 8B). The increased Rab7 staining indicates an expansion of the for Bcl-xL–mediated regulation of Ag presentation. Bcl-xL was late endosomal compartment, whereas the probability of coloc- also found to regulate peptide Ag presentation by MHC class II alization of CD1d and Rab7 remained unchanged, suggesting that molecules in a similar manner, altering their ability to present Ag after Bcl-xL knockdown, CD1d molecules may be accumulating to classical T cells. This could potentially affect the antitumor im- in the late endosomes (Fig. 8A–C). We also examined Rab7 mune response and the tumor microenvironment because CD4+ protein levels by Western blotting and found that after Bcl-xL T cells are critical in orchestrating a cytokine milieu that could knockdown, Rab7 protein was increased as compared with the significantly alter the effectiveness of an antitumor response. scramble (Fig. 8D). Thus, knocking down Bcl-xL leads to up- In this study, we show that the overexpression of Bcl-xL in APCs regulation of Rab7 and an expansion of the late endosomal leads to increased activation of NKT cells. Furthermore, the compartment. Furthermore, this expanded endosomal compart- knockdown of Bcl-xL using shRNA led to decreased Ag presen- ment acts as a depot for CD1d molecules, resulting in impaired tation to NKT cells, indicating that Bcl-xL plays a role in the Downloaded from trafficking to the lysosomal compartment, which is critical for Ag regulation of CD1d-mediated Ag processing and presentation. presentation (Supplemental Fig. 3). The results indicate that under After the induction of Bcl-xL in a B cell lymphoma cell line, as well normal conditions, Bcl-xL regulates the endosomes and limits the as in primary mouse B cells by treatment with anti-CD40 Ab, there size of the late endosomal compartment. When Bcl-xL is reduced, was increased NKT cell activation. Finally, we used pharmaco- this regulation is lost, leading to upregulation of Rab7 and ex- logical inhibition using ABT-263, an inhibitor of Bcl-xL and Bcl-2,

pansion of the late endosomal compartment (Supplemental Fig. 3). and found that there was decreased Ag presentation to NKT cells, http://www.jimmunol.org/ as well as classical CD4+ T cells, whereas the selective inhibition Discussion of Bcl-2 using ABT-199 had no effect. ABT-263 and ABT-199 are We have identified a novel function for Bcl-xL in CD1d-mediated BH3 mimetics, and functionally inhibit Bcl-xL and/or Bcl-2 by Ag processing and presentation through its regulation of the late binding to its BH3 domain and preventing dimerization with other endosomal compartment. Because of the current interest in using BH3-containing proteins. This type of inhibition is distinct from NKT cell–based cancer therapy (47), and with the knowledge that shRNA-mediated knockdown, which results in an overall reduc- by guest on September 24, 2021

FIGURE 8. Accumulation of CD1d molecules in the Rab7+ late endosomal compartment after Bcl-xL knockdown. (A) Confocal microscopy on control or Bcl-xL knockdown L-CD1d cells stained with Abs to CD1d (red) and Rab7 (green), a late endosomal marker. The rightmost panels show that CD1d is more heavily colocalized with Rab7 (yellow) in the Bcl-xL knockdown cells as compared with the control. (B) Quantification of Rab7 staining based on pixel number in scramble control or Bcl-xL knockdown cells. (C) Quantifi- cation of colocalization of CD1d and Rab7 in scramble control or Bcl-xL knockdown cells. (D) Western blotting shows that Rab7 protein is upregulated after Bcl-xL, but not Bcl-2, knockdown (upper panel), and GAPDH is shown as the loading control (lower panel). Original magnification 3400. The Journal of Immunology 9 tion in Bcl-xL or Bcl-2 protein expression. We have discovered Acknowledgments that although knocking down Bcl-xL or Bcl-2 led to reduced Ag We acknowledge the National Institutes of Health Tetramer Core Facility at presentation, selective BH3-dependent inhibition of Bcl-xL and Emory University for kindly providing Ag-loaded CD1d tetramers for this Bcl-2, but not Bcl-2 alone, led to reduced Ag presentation to study. NKT cells. This suggests that Bcl-xL and Bcl-2 regulate CD1d- mediated Ag presentation through different mechanisms. Our Disclosures studies demonstrate that Bcl-xL–mediated regulation is depen- The authors have no financial conflicts of interest. dent upon its BH3 domain and involves increased Rab7 and al- tered trafficking of CD1d molecules through the endocytic compartments. However, the ability of Bcl-2 to regulate CD1d- References 1. Fowlkes, B. J., A. M. Kruisbeek, H. Ton-That, M. A. Weston, J. E. Coligan, mediated Ag presentation was not dependent upon the BH3 R. H. Schwartz, and D. M. Pardoll. 1987. A novel population of T-cell receptor domain, and there was no evidence of Rab7 upregulation after alpha beta-bearing thymocytes which predominantly expresses a single V beta Bcl-2 knockdown. These findings highlight an important func- gene family. Nature 329: 251–254. 2. Makino, Y., R. Kanno, T. Ito, K. Higashino, and M. Taniguchi. 1995. Predom- tional difference between the structurally similar Bcl-xL and inant expression of invariant V alpha 14+ TCR alpha chain in NK1.1+ T cell Bcl-2 proteins. populations. Int. Immunol. 7: 1157–1161. Surface CD1d expression was unchanged after Bcl-xL over- 3. Godfrey, D. I., H. R. MacDonald, M. Kronenberg, M. J. Smyth, and L. Van Kaer. 2004. NKT cells: what’s in a name? Nat. Rev. Immunol. 4: 231–237. expression, induction, inhibition, or knockdown in WEHI-231 4. Swann, J., N. Y. Crowe, Y. Hayakawa, D. I. Godfrey, and M. J. Smyth. 2004. cells, mouse fibroblast cell lines LMTK-CD1d and L-CD1d, as Regulation of antitumour immunity by CD1d-restricted NKT cells. Immunol.

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