Role of the EHD Family of Endocytic Recycling Regulators for TCR Recycling and T Cell Function

This information is current as Fany M. Iseka, Benjamin T. Goetz, Insha Mushtaq, Wei An, of September 27, 2021. Luke R. Cypher, Timothy A. Bielecki, Eric C. Tom, Priyanka Arya, Sohinee Bhattacharyya, Matthew D. Storck, Craig L. Semerad, James E. Talmadge, R. Lee Mosley, Vimla Band and Hamid Band

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published December 6, 2017, doi:10.4049/jimmunol.1601793 The Journal of Immunology

Role of the EHD Family of Endocytic Recycling Regulators for TCR Recycling and T Cell Function

Fany M. Iseka,*,† Benjamin T. Goetz,* Insha Mushtaq,*,‡ Wei An,* Luke R. Cypher,* Timothy A. Bielecki,* Eric C. Tom,*,x,{ Priyanka Arya,*,†,1 Sohinee Bhattacharyya,*,‡,2 Matthew D. Storck,* Craig L. Semerad,‖ James E. Talmadge,‡ R. Lee Mosley,*,# Vimla Band,*,†,‡ and Hamid Band*,†,‡,x,{,#

T cells use the endocytic pathway for key cell biological functions, including receptor turnover and maintenance of the immunological synapse. Some of the established players include the Rab GTPases, the SNARE complex proteins, and others, which function together with EPS-15 homology domain–containing (EHD) proteins in non–T cell systems. To date, the role of the EHD protein

family in T cell function remains unexplored. We generated conditional EHD1/3/4 knockout mice using CD4-Cre and crossed Downloaded from these with mice bearing a myelin oligodendrocyte glycoprotein–speciﬁc TCR transgene. We found that CD4+ T cells from these mice exhibited reduced Ag-driven proliferation and IL-2 secretion in vitro. In vivo, these mice exhibited reduced severity of experimental autoimmune encephalomyelitis. Further analyses showed that recycling of the TCR-CD3 complex was impaired, leading to increased lysosomal targeting and reduced surface levels on CD4+ T cells of EHD1/3/4 knockout mice. Our studies reveal a novel role of the EHD family of endocytic recycling regulatory proteins in TCR-mediated T cell functions. The Journal

of Immunology, 2018, 200: 000–000. http://www.jimmunol.org/

nderstanding the mechanisms that underlie proper T cell CD28 and lymphocyte function–associated 1 (LFA-1), to form an function is of great interest in basic immunology and area of intimate contact with APCs, the immunological synapse U immunopathology. T cell activation requires recognition (IS) (2, 3). The endocytic pathways supply the IS with newly of an antigenic peptide bound to MHC proteins on APCs by the synthesized and recycled receptors to replenish those that are TCR and downstream signaling mediated by cytoplasmic regions targeted to the lysosome for degradation (4–6). of the TCR-CD3 complex. Effective generation of an immune Cells use clathrin-dependent as well as clathrin-independent response requires concurrent engagement of and signaling through pathways to internalize surface receptors into early endosome/

costimulatory proteins (e.g., CD28) on T cells by their cognate sorting endosome compartments, from where they trafﬁc to late by guest on September 27, 2021 ligands on the APC surface (1). Therefore, the mechanisms that endosome/multivesicular bodies and lysosomes for degradation, or ensure optimal levels of the TCR and accessory receptors on the recycle back to the cell surface either through a Rab4+ fast T cell surface prior to Ag stimulation are fundamentally critical to recycling endosome (directly to the plasma membrane) or through generating an effective immune response. However, mechanisms a slow recycling route via the Rab11+ recycling endosome (7, 8). that regulate the surface pool of TCR and its accessory This sorting processes is known to be regulated by the Rab family receptors have been primarily investigated in the context of T cell of small GTPases and their interacting partners (9). TCRs such as activation. During T cell activation, TCR signaling elicited by the TCR-CD3 (1), CD28 (10), and LFA-1 (11, 12) are known to be APC-presented Ag, together with costimulatory signals, leads to internalized and recycled to the IS during T cell activa- spatial reorganization of the TCR and accessory receptors, such as tion. Internalized TCR-CD3 traverses the Rab5+ early endosome,

*Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Department of Defense Grants W81XWH-07-1-0351 and W81XWH-11-1-0171 (to Medical Center, Omaha, NE 68198; †Department of Genetics, Cell Biology and V.B.), a University of Nebraska System Science Collaborative grant (to H.B.), and an Anatomy, University of Nebraska Medical Center, Omaha, NE 68198; Institutional Development Award from the National Institute of General Medical ‡Department of Pathology and Microbiology, University of Nebraska Medical Center, Sciences under National Institutes of Health Grant P30 GM1063. Support for the Omaha, NE 68198; xDepartment of Biochemistry and Molecular Biology, University University of Nebraska Medical Center Confocal, Flow Cytometry, and other core of Nebraska Medical Center, Omaha, NE 68198; {Department of Pharmacology and facilities was from National Cancer Institute Cancer Center Support Grant Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE P30CA036727 to the Fred and Pamela Buffett Cancer Center, as well as from funding 68198; ‖Flow Cytometry Research Facility, University of Nebraska Medical Center, by National Institute of General Medical Sciences Grant P30 GM1063 and the Omaha, NE 68198; and #Fred and Pamela Buffet Cancer Center, University of Nebraska Research Initiative. Nebraska Medical Center, Omaha, NE 68198 Address correspondence and reprint requests to Dr. Hamid Band, Eppley Institute for 1Current address: Department of Medical and Molecular Genetics, Indiana University Research in Cancer and Allied Diseases, BCC 10.12.390, 986805 Nebraska Medical School of Medicine, Indianapolis, IN. Center, Omaha, NE 68198-6805. E-mail address: [email protected] 2Current address: Department of Cell, Developmental and Cancer Biology, Oregon The online version of this article contains supplemental material. Health Science University, Portland, OR. Abbreviations used in this article: EAE, experimental autoimmune encephalomyeli- ORCIDs: 0000-0002-4029-8943 (M.D.S.); 0000-0002-6328-6056 (J.E.T.); 0000- tis; EHD, EPS-15 homology domain–containing protein; IS, immunological synapse; 0002-4241-6149 (R.L.M.); 0000-0002-4996-9002 (H.B.). KO, knockout; LFA-1, lymphocyte function–associated 1; LN, lymph node; MFI, mean ﬂuorescence intensity; MOG, myelin oligodendrocyte glycoprotein; 4-OHT, Received for publication October 20, 2016. Accepted for publication November 1, 4-hydroxytamoxifen. 2017. This work was supported by National Institutes of Health Grants CA105489, Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 CA87986, CA99163, and CA116552 (to H.B.), as well as CA105489 (supplement; to F.M.I.), and CA96844 and CA144027 (to V.B.). This work was also supported by

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601793 2 EHD PROTEIN REGULATION OF TCR followed by transport through Rab4+ and Rab11+ endosomes, Ag-driven cell proliferation and IL-2 secretion. In vivo, these mice indicating the involvement of both fast and slow recycling path- exhibit a reduced severity of experimental autoimmune encepha- ways (13, 14). Naive and resting T cells constitutively internalize lomyelitis (EAE) elicited by immunization with the MOG peptide. and recycle their surface receptors, and the balance of these We show that surface levels of TCR, CD28, and LFA-1 are re- processes dictates their prestimulation surface levels and hence the duced and TCR recycling is impaired in CD4+ T cells of mice with levels of T cell activation (13). However, the mechanisms of a CD4-Cre–mediated EHD1/3/4 knockout. Therefore, our studies constitutive basal traffic of TCR-CD3 are not fully understood. reveal a novel role of the EHD family of endocytic recycling Recently, a new family of endocytic regulators, the EPS15 proteins in the surface display of TCR in unstimulated T cells that homology domain–containing proteins (EHD1–4), has been contributes to their positive functional role in Ag-induced T cell identified. These proteins exhibit a conserved domain architecture activation and immunopathology. with helical regions near the N terminus and the middle of the protein surrounding an ATP-binding G-domain that can hydrolyze Materials and Methods ATP slowly, and a C-terminal EH domain that binds to proteins Reagents and Abs with asparagine-proline-phenylalanine–containing or related tri- peptide motifs as a major mechanism of protein–protein interac- Reagents. BSA (catalog no. A7906-100G), paraformaldehyde (catalog no. 158127-500G), Triton X-100 (catalog no. 93418), EGTA (catalog no. tions (15, 16). Structural studies of EHD2 reveal that the helical E8145-50G), sodium orthovanadate (catalog no. S6508-50G), sodium regions form a curved membrane lipid-binding interface, and that deoxycholate (catalog no. D6750-100G), 4-hydroxtamoxifen (catalog no. the ATPase domain folds similar to the GTPase domain of T176-10MG), and brefeldin A (catalog no. B7651) were from Sigma- dynamin and mediates the formation of stable dimers (17). Other Aldrich (St. Louis, MO). Propidium iodide staining solution (catalog Downloaded from 3 structural findings support a model that dimeric EHDs organize no. 00-6990-42) was from eBioscience. [ H]thymidine (catalog no. 2407001, 2.0 Ci/mmol) was from MP Biomedicals. Bafilomycin into oligomeric rings around curved membrane structures and A1 (catalog no. BML-CM110-0100) was from Enzo Life Sciences. MOG function in membrane tubulation and vesiculation (17–21). Con- peptide (aa 35–55) (catalog no. BP001328-PRO-371) was from Syd sistent with these biochemical observations, cell biological studies Labs. OVA peptide (aa 323–336) (sequence: ISQAVHAAHAEINE) was have shown that EHD proteins regulate recycling of several synthesized from Tufts University Core Facility (Medford, MA). CFA (catalog no. 231131) and heat-inactivated Mycobacterium tuberculosis surface receptors that traffic through clathrin-dependent or were from Difco Laboratories. NaF (catalog no. S299-500), NaCl (cat- http://www.jimmunol.org/ clathrin-independent pathways, including transferrin recep- alog no. S271-10), and Tris (catalog no. BP152-5) were from Fisher tor, MHC class I, b1 integrin, 2-amino-5-hydroxy-5-methyl-4- Chemicals. Pertussis toxin was from Enzo Life Sciences. ECL devel- isoxazolepropion acid receptors, insulin-like growth factor 1 opment reagent (catalog no. 32106), BSA for bicinchoninic acid assay receptor, insulin-responsive glucose transporter 4, and others (catalog no. 23209), and PMSF (catalog no. 36978) were from Thermo Scientific. CFSE (catalog no. C34554), CellTrace Violet stain (cat- (7, 22–29). However, the physiological relevance of the many alog no. C34557), RPMI 1640 (catalog no. SH30027.02), penicillin/ in vitro–assigned roles of EHD proteins remains to be defined. streptomycin (catalog no. 15140-122), and FBS (catalog no. 10427- We used gene targeting in mice to reveal the in vivo functional 028; lot no. 1662765A120-01) were from Life Technologies. Live/Dead roles of EHD proteins and cell surface receptors whose traffic is fixable blue dead cell stain kit was from Molecular Probes. IL-2 ELISA

kit (catalog no. M2000) was from R&D Systems. by guest on September 27, 2021 regulated by EHDs (30–36). For example, EHD1 knockout (KO) Abs. Anti-CD28 (catalog no. 122007) and anti–LFA-1 (catalog no. 141011) mice exhibit strain-dependent phenotypes varying degrees of were from BioLegend. Anti-CD3ε (referred to as anti-CD3 in this study) embryonic lethality, male infertility, ocular developmental defects, (catalog nos. 553064 and 553057), anti-CD25 (catalog no. 558642), anti- and neural tube closure defects due to impairment of ciliogenesis CD4 (catalog nos. 553051 and 553047), anti-CD8a (catalog no. 560469), and SHH signaling (37, 38). anti-CD28 (catalog no. 553294), and anti-CD44 (catalog no. 553135) were ε To date, any roles of EHD family proteins in TCR traffic or T cell from BD Biosciences. Anti-B220 (catalog no. 25-0452-81) and anti-CD3 (referred to as anti-CD3 in this study) (catalog no. 56-0032-80) were from function are unknown. Given their importance in the regulation of a eBioscience. Anti-HSC70 (catalog no. sc-7298) and anti-LAMP1 1D4B variety of other cell surface receptors and the consequences of (catalog no. sc-19992) were from Santa Cruz Biotechnology. Anti-EHD1 deleting their genes, singly or in combination, on organ/cell (catalog no. ab109311) was from Abcam. Anti–b-actin (catalog no. function in vivo, we hypothesized that EHD proteins could play A5316) was from Sigma-Aldrich. Allophycocyanin-conjugated anti– annexin V (catalog no. 17-8007-72) was from eBioscience. Polyclonal an important functional role in T cells by regulating receptor traffic. rabbit Abs recognizing EHD1 plus EHD4, EHD2, or EHD3 have been Consistent with such a hypothesis, EHD proteins have been shown described previously (45). Secondary fluorochrome-conjugated Abs were to interact with Rab effectors, such as Rabenosyn-5, a dual Rab4 from Life Technologies. and Rab5 effector in the early endosome (39), and Rab11 effector, Rab11-FIP2, which regulates the exit of vesicles from the recy- Mice generation and genotyping cling endosome back to the plasma membrane (40). Moreover, Whole-body knockout mice (Ehd1-null) derived from Ehd1flox/flox mice EHD proteins (EHD1, EHD3, and EHD4) were shown to interact have been described previously (49). Ehd1-null mice were maintained on a with SNARE complex proteins such as SNAP-25, SNAP29 mixed 129, B6 background. EHD1fl/fl; EHD3fl/fl; EHD4fl/fl mice in a pre- (41, 42), Syndapin I and II (7, 43, 44), and other proteins in non– dominantly C57BL/6 background were crossed with B6.Cg-Tg (Cd4-cre) 1Cwi/BfluJ mice to generate Cd4-cre; EHD1fl/fl; EHD3fl/fl; EHD4fl/fl mice. T cell systems. EHD proteins have been shown to localize in These mice were further crossed with C57BL/6-Tg (Tcra2D2, Tcrb2D2) endosomes positive for Rab5, Rab4, Rab8, Rab11, or Rab35 (9, 1Kuch/J, 2D2 Tcr,orMOG Tcr to generate Cd4-cre; 2D2Tcr; EHD1fl/fl; 25, 29, 45), compartments that are implicated in TCR traffic EHD3fl/fl; EHD4fl/fl mice. EHD1fl/fl; EHD3fl/fl; EHD4fl/fl mice in a pre- (46–48). Thus, we posited that EHD proteins could play an im- dominantly C57BL/6 background were also crossed with tamoxifen- portant functional role in T cells. inducible CreERT2-expressing mice from The Jackson Laboratory [Gt (ROSA)26Sortm1(cre/ERT2) Tyj; strain 008463] to generate CreERT2; We found that EHD1, EHD3, and EHD4 are expressed in EHD1fl/fl; EHD3fl/fl; EHD4fl/fl mice. These mice were further crossed with + lymphoid tissues and in purified CD4 T cells. Accordingly, we C57BL/6-Tg (Tcra2D2, Tcrb2D2)1Kuch/J, 2D2 Tcr,orMOG Tcr)to generated myelin oligodendrocyte glycoprotein (MOG)–specific generate CreERT2; 2D2Tcr; EHD1fl/fl; EHD3fl/fl; EHD4fl/fl mice. Genotypes TCR transgene (2D2-TCR Tg)–bearing mice in which floxed were confirmed by subjecting tail clip DNA to PCR analysis using the KAPA mouse genotyping kit (Kapa Biosystems). Mice were treated hu- EHD1, EHD3, and EHD4 were deleted using a CD4-Cre trans- + fl/fl fl/fl fl/fl manely according to the National Institutes of Health and University of gene. In vitro, CD4 T cells of EHD1 ; EHD3 ; EHD4 ; Nebraska Medical Center guidelines. Animal studies were preapproved by Cd4-cre; 2D2Tcr mice (EHD1/3/4 knockout) exhibit reduced the Institutional Animal Care and Use Committee (no. 14-067). The Journal of Immunology 3

Western blotting and EHD4. Cells were washed in PBS, stained with CFSE, and restimulated for proliferation as previously described. Lymphoid tissues or isolated cells were lysed in ice-cold Triton X-100 lysis buffer (0.5% Triton X-100, 50 mM Tris [pH 7.5], 150 mM NaCl, 1 mM ELISA PMSF, 10 mM NaF, 1 mM VO ) or with RIPA lysis buffer (same as Triton 4 5 X-100 lysis buffer with an increase of Triton X-100 to 1% and an addition Isolated spleen cells (5 3 10 cells per well) were cultured in 96-well m of 5 mM EDTA, 1 mM EGTA, 1% SDS, and 0.5% sodium deoxycholate). U-bottom plates in the presence of 10 g/ml of MOG35–55 peptide at 37˚C Lysates were vortexed, centrifuged at 13,000 rpm for 30 min at 4˚C either for indicated time points. IL-2 secretion was measured in culture super- immediately or after overnight rocking in the cold room, and supernatants natants using an ELISA kit according to the manufacturer’s instructions. were collected. Protein lysates were quantified using the bicinchoninic acid assay. Forty-microgram aliquots of lysate protein per sample were resolved Cell surface TCR internalization assay by SDS-PAGE and transferred to polyvinylidene difluoride membranes Internalization of the cell surface pool of TCR-CD3 was assessed as (from Immobilon-P, catalog no. IPVH00010). In certain experiments, previously described (13, 51) with modifications. Briefly, freshly isolated lysates from equal numbers of cells were resolved by SDS-PAGE. The LN cells at 5 3 106 cells/ml in 96-well U-bottom plates (in triplicates) membranes were blocked in TBS/5% BSA, incubated with the appropriate were incubated in the presence of 10 mg/ml brefeldin A at 37˚C for the primary Abs diluted in TBS–0.1% Tween 20 for 1 h, washed in TBS–0.1% indicated times to allow the surface TCR to internalize in the absence of Tween 20 (three times for 5 min each) followed by a 45-min incubation newly synthesized TCR transport to the cell surface. At each time point, with HRP-conjugated secondary Ab in the same buffer. The membrane cells were transferred to ice and subsequently stained with PE-conjugated was then washed in TBS–0.1% Tween 20 (three times for 5 min each) and anti-CD3 followed by FACS analysis to quantify the surface TCR levels ECL-based detection was performed. remaining at each time point. The extent of the TCR remaining at the cell CD4+ T cell isolation surface was calculated by expressing the mean fluorescence intensity (MFI) of PE–anti-CD3 staining at each time point relative to time 0, which To isolate primary CD4+ T cells, a negative selection protocol was per- was set as 100%. Downloaded from formed as described (50) using magnetic beads (biotin binder kit, catalog no. 11533D; Invitrogen) and biotinylated Abs (BioLegend), and purity was Analysis of TCR recycling established to be 91–95% based on flow cytometry. The recycling of pre-existing intracellular pools of TCR-CD3 was carried FACS out by adapting a previously described protocol (52). Briefly, the acces- sibility of the cell surface CD3 on freshly isolated LN cells (at 5 3 106 T cells were incubated on ice in the dark for 15–30 min (depending on the cells/ml in 96-well U-bottom plates in triplicates) was blocked by staining experiment) with appropriate conjugated Abs at the manufacturer’s rec- with a predetermined saturating concentration of unconjugated anti-CD3 at http://www.jimmunol.org/ ommended dilution in FACS buffer (0.1% BSA in PBS). Cells were pel- 4˚C for 30 min. The cells were extensively washed and incubated at 37˚C. leted, washed twice, and suspended in 400 ml of cold FACS buffer. In other At the indicated times, the cells were transferred to 4˚C and stained with a cases, cells were fixed with 4% cold paraformaldehyde for 15 min at room PE-conjugated version of the same anti-CD3 Ab that was used to block the temperature after staining, then washed and suspended in 400 ml of cold staining of the initial cell surface cohort of CD3. The MFI of PE–anti-CD3 FACS buffer. Cells were protected from light until analyses using either an cell surface FACS staining was used as a measure of the recycling of in- LSR II Green or LSR II cytometer (BD Biosciences). FACS data were tracellular TCR-CD3. Recycling of the cell surface TCR-CD3 pool fol- analyzed using DIVA (BD Biosciences FACSDiva software), FlowJo data lowing internalization was assayed by a modified protocol (53, 54). analysis software (FlowJo, Ashland, OR) and ModFit LT software (Verity Freshly isolated LN cells were first stained with PE-conjugated anti-CD3 Software House, Topsham, ME). for 30 min on ice. After extensive washes, the cells were incubated at 37˚C for 30 min (in complete RPMI) to allow the Ab-labeled TCR-CD3 to by guest on September 27, 2021 CFSE dye dilution and CellTrace Violet cell proliferation undergo internalization. The cells were then subjected to an acid wash to assays strip any remaining Ab bound to CD3 on T cell surface. The cells were washed in cold FACS buffer and resuspended in complete RPMI followed Spleen cells (5 3 106 cells/ml) from CD4-Cre; 2D2Tcr; EHD1fl/fl; EHD3fl/fl; fl/fl fl/fl fl/fl fl/fl by incubation at 37˚C for the indicated times to allow recycling of the EHD4 mice and control (2D2Tcr; EHD1 ; EHD3 ; EHD4 )mice labeled internalized pool of TCR-CD3 back to the cell surface. The cells were stained with CFSE or CellTrace Violet according to the manufac- were spun down, acid washed, and resuspended in FACS buffer for FACS m turer’s instructions. Cells were treated with 50 g/ml MOG35–55 peptide analysis to determine the remaining intracellular fraction of the internal- for 72 h. On the indicated day, cells were stained with FITC-CD4 before ized TCR-CD3. The percentage of TCR-CD3 recycling was calculated analysis. Dilution of CFSE or CellTrace Violet fluorescence as an indicator from the equation [(T0 2 Tx)/T0] 3 100, where T0 is the MFI of cells of cell division was assessed via FACS analysis. Data were analyzed using incubated for 0 min before the second acid wash, and Tx is the MFI of cells FlowJo and ModFit LT software to delineate the percentage of cells that subjected to a second acid wash after incubation for the indicated times. had undergone increasing number of divisions and to determine the proliferation index. In some instances, the cells were not stained with the Confocal microscopy proliferation dye, but were stained with anti–annexin V and propidium iodide staining solution after 72 h of stimulation and analyzed by FACS for LN T cells were expanded as described under T cell expansion. A total of cell death analysis. 5 3 106 expanded T cells in complete RPMI medium were plated onto poly-L-lysine–coated coverslips placed in 24-well plates and allowed to 3 [ H]Thymidine incorporation assay and T cell expansion attach at 37˚C for 10 min. Cells on coverslips were fixed in 4% ice-cold paraformaldehyde for 20 min and then incubated with 0.1 M glycine 3 5 CD4-Cre 2D2Tcr EHD1fl/fl Spleen cells (5 10 cells per well) from ; ; ; (in PBS) for 3 min at room temperature. After washing, the cells were EHD3fl/fl EHD4fl/fl 2D2Tcr EHD1fl/fl EHD3fl/fl EHD4fl/fl ; or control ( ; ; ; )mice incubated with the blocking plus permeabilization buffer (2% BSA plus were seeded in 96-well U-bottom plates in 100 ml of medium in the presence 0.1% Triton X-100 in PBS) for 30 min at room temperature. Cells were of varying concentrations of MOG peptide for 72 h. Cells were pulsed 35–55 stained with anti–Lamp-1 and anti-CD3 primary Abs overnight a 4˚C. with 1 mCi of [3H]thymidine per well for the last 6 h of incubation, harvested After three washes, the cells were stained with secondary anti-rat onto filter disks, and the radioactivity (counts per minute) was counted using (for Lamp-1) and anti-Armenian hamster Abs for 45 min at room tem- a scintillation counter (Packard). For T cell expansion, spleen cells or lymph perature. After washes, the cells were mounted with Vectashield HardSet CD4-Cre 2D2Tcr EHD1fl/fl EHD3fl/fl EHD4fl/fl node (LN) cells from ; ; ; ; mice mounting medium with DAPI (catalog no. H-1500; Vector Laboratories). 2D2Tcr EHD1fl/fl EHD3fl/fl EHD4fl/fl and control ( ; ; ; ) mice were stimulated Images were acquired at room temperature using a Zeiss ELYRA S.1 with 10–20 mg/ml MOG peptide and T cells were expanded in the 35–55 or a superresolution structured illumination microscopy. The sCMOS presence of 30 U/ml IL-2 for 7–8 more days (fresh IL-2–containing media camera mounted on side port was used [objective lenses, Plan- every 2 d after 3 d of stimulation). Apochromat 363/1.40 oil differential interference contrast; for resolu- In vitro deletion of EHD (EHD1, EHD3, and EHD4) genes tion: lateral resolution (XY), 120 nm; axial resolution (Z), 300 nm (typical experimental full width a half maximum values with objective Spleen cells or LN cells from EHD1fl/fl; EHD3fl/fl; EHD4fl/fl; CreERT2; lens Plan-Apochromat 363/1.40 oil differential interference contrast, 2D2Tcr mice were prestimulated with either the MOG peptide or with anti- subresolution beads of 40 nm diameter and excitation at 488 nm)]. CD3 and anti-CD28 for 3–4 d in the presence (+) or absence (2)of4- Merged fluorescence images were generated and analyzed using ZEN hydroxytamoxifen (4-OHT) (200–300 nM) for deletion of EHD1, EHD3, 2012 software from Carl Zeiss. 4 EHD PROTEIN REGULATION OF TCR

Induction and monitoring of EAE family members, we reasoned that EHD1, EHD3, and EHD4 Six to eight-week-old female CD4-Cre; 2D2Tcr; EHD1fl/fl; EHD3fl/fl; might function redundantly in T cells, and we therefore used a EHD4fl/fl or control (2D2Tcr; EHD1fl/fl; EHD3fl/fl; EHD4fl/fl) mice were s.c. combinatorial knockout strategy to further explore their role in immunized in the flank with 200 mg of MOG35–55 peptide emulsified (1:1) T cells. in CFA containing 0.5 mg of heat-killed Mycobacterium tuberculosis m CD4-Cre–mediated conditional deletion of EHD1/3/4 in H37RA in a total of 200 l of emulsion per mouse, together with an i.p. + injection of pertussis toxin (200 ng per mouse) in 200 ml of PBS followed T cells leads to a reduced proportion of CD4 T cells in by a second dose of pertussis toxin 2 d later. Mice were monitored daily secondary lymphoid tissues and scored for clinical signs of EAE on a scale of 0–5 as follows: 0, no disease; 1, decrease tail tone; 2, hindlimb weakness or partial paralysis; Given the likelihood of redundant EHD function, we generated 3, complete hindlimb paralysis; 4, forelimb and hindlimb paralysis; and mice with a CD4-Cre–dependent, T cell–directed, conditional 5, moribund state or dead (55). Composite scores of each cohort were used deletion of floxed Ehd1, Ehd3,andEhd4 genes. To examine the to determine statistical significance. impact of EHD1/3/4 deletions in T cells in the context of Ag- Statistical analyses specific responses, we also incorporated the MOG peptide- specific 2D2-Tcr transgene (55) into our conditional knockout An unpaired Student t test was used to calculate the p values. Data are model (referred to as CD4-Cre+ mice). Littermates without a presented as mean 6 SEM, and a p value ,0.05 served as the threshold for statistical significance. CD4-Cre transgene were used as controls (referred to as CD4- Cre2 mice). The presence of floxed EHD1/3/4 gene alleles targeted for CD4-Cre–dependent deletion was detected by tail Results

PCR using specific primers for each floxed allele (Supplemental Downloaded from Multiple EHD family members are expressed in lymphoid + + Fig. 2A). Western blot analysis of CD4 T cells isolated from the organs and purified CD4 T cells 2D2-Tcr transgenic mice showed that the presence of the trans- Our previous Western blotting studies of EHD protein expression in genic TCR by itself did not have any significant impact on the levels the mouse thymus and spleen (49, 56, 57), which we extended here of EHD proteins (Supplemental Fig. 2B). CD4-Cre+ mice were to LN (Fig. 1A), showed that multiple EHD family members are developmentally indistinguishable from CD4-Cre2 mice, and no expressed, albeit unequally, in lymphoid organs. This suggested gross phenotypic changes were seen in adult mice. Western http://www.jimmunol.org/ that multiple EHD family members are likely to be expressed in blot analysis confirmed the CD4-dependent deletion of EHD1/3/4 individual immune cell types, consistent with our recent findings (Fig. 3A). that bone marrow–derived macrophages express both EHD1 and Next, to examine whether EHD1/3/4 deletion impacted T cell EHD4 (36). With the focus of this study on functional analyses in development, we isolated single cells from lymphoid organs of the context of CD4+ T cells, our additional analyses showed that 4-wk-old mice and analyzed these for changes in T cell numbers EHD1, EHD3, and EHD4 are expressed in primary murine CD4+ using cell counting combined with FACS. We found that total T cells (Fig. 1B) whereas EHD2 protein was undetectable to numbers of thymocytes, splenocytes, and LN cells were compa- barely detectable (Fig. 1C). Analyses of mRNA expression data- rable in mice from both groups (Fig. 3B). Similarly, comparable

bases (ImmGen and BioGPS) further supported this expression cell numbers were observed when spleens of older (6–8 wk old) by guest on September 27, 2021 pattern (Supplemental Fig. 1). Notably, Western blotting of puri- mice of the two genotypes were analyzed (Fig. 3C). We carried fied LN T cells at various time points of anti-CD3/CD28 stimu- out FACS analyses on the live/B2202 T cell population and lation showed that levels of EHD proteins vary as a function of assessed the proportion of cells at various developmental stages CD4+ T cell activation, with a time-dependent increase in EHD1 based on staining with Abs against CD4, CD8, CD25, and CD44 and EHD4 levels and a reduction in EHD3 levels (Fig. 1B). These (58–60). Single-positive thymocytes in 2D2-Tcr Tg mice are findings suggested that EHD proteins may play important roles in known to be skewed toward the CD4+ T cell compartment; this is T cell function. also seen in spleen and LN (55). CD4-Cre–mediated deletion of Our previous knockout studies have revealed relatively unique as EHD1/3/4 did not affect the relative percentages or total cell well as redundant functional roles of EHD proteins, with the numbers within various thymic developmental stages when ana- knockout of EHD1 alone producing by far the most obvious de- lyzed in 4-wk-old mice (Fig. 3D, 3E). velopmental and adult organ functional aberrations, depending on In contrast, CD4-Cre–mediated EHD1/3/4 deletion led to a the strain background (27, 37, 38, 49). Therefore, we assessed decrease in the percentage of T cells in the periphery. This phe- whether EHD1 deficiency had any impact on T cell activation. notype was seen in both the LN and spleen (Fig. 3F, 3G), although Notably, the initial analysis of EHD12/2 splenic T cells did not only the splenocyte reduction was statistically significant. How- reveal any detectable abnormality in anti-CD3/CD28–induced cell ever, the absolute number of CD4+ T cells in these organs did not proliferation (Fig. 2A). These findings supported a redundant differ significantly between the CD4-Cre–positive and control functional role of EHD proteins, at least in the context of T cell mice. Additionally, an apparent increase in peripheral B cell proliferation. Consistent with this, a slight increase in EHD4 numbers and their percentages were seen (Fig. 3F, 3G); the ba- levels was noted in LN from EHD12/2 mice (Fig. 2A). Initial sis of this phenotype is unknown but is likely to reflect altered analysis of EHD32/2 CD4+ T cells showed no difference in anti- T cell/B cell interactions due to T cell deficiency of EHD1/3/4. We CD3/CD28–induced proliferation compared with CD4+ T cells also saw a decrease in the percentage of CD8+ T cells in spleens of from control mice (Fig. 2B). We observed that deletion of CD4-Cre–positive mice, but this did not reflect an increase in EHD3 did not cause an increase in the level of either EHD1 or CD8+ T cell numbers in these organs. From these results, we EHD4 in LN cells (Fig. 2B). However, EHD42/2 CD4+ T cells conclude that expression of EHD proteins is dispensable during showed reduced proliferation compared with control CD4+ thymic T cell development, but may play a role in peripheral T cells, although the decrease was not statistically significant T cell function. To exclude the possibility that the lack of impact (Fig. 2C). We also noted that the deletion of EHD4 did not cause on T cell development and changes in peripheral lymphocyte an increase in the level of either EHD1 or EHD3 in LN cells. numbers might reflect the presence of an autoreactive TCR Given the lack of impact or a mild impact of single EHD gene transgene, we also analyzed the non–TCR-transgenic mice for the knockouts on T cell proliferation and the expression of multiple effect of CD4-Cre–mediated EHD1/3/4 deficiency during thymic The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 1. Expression of EHDs in lymphoid tissues and isolated CD4+ T cells. (A) Lymphoid tissues express all EHD proteins. One membrane (left) was probed with an Ab recognizing both EHD1 and EHD4, and a second one (right) was probed for EHD3 followed by reprobing for EHD2. (B) EHD protein expression in CD4+ T cells and changes in expression following activation are shown. Shown is a representative blot of more than three independent experiments. CD4+ T cells were isolated from pooled LN cells of at least ﬁve mice per group for each experiment (however, quantiﬁcation shown is of EHD protein levels for the time points used in all the repeats, normalized to b-actin, and plotted as the mean 6 SEM of n = 2). (C) Lysate of CD4+ T cells from wild-type (WT) spleen was loaded together with lysates from WT spleen cells, WT lung, and EHD22/2 lung as controls. The membrane was probed for EHD1 and EHD4 and reprobed for EHD2 level; b-actin was used as loading control (n = 3). development and in spleen cells. We observed a lack of impact of proteins are dispensable for thymic T cell development beyond the T cell development similar to that seen in the TCR-transgenic CD4/8+ stage. The analysis of spleen cells showed a slight in- mice (Fig. 4A–C), further supporting the conclusion that EHD crease in the percentage of B220+ cells (B cells) and a slight 6 EHD PROTEIN REGULATION OF TCR Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. Potential redundancy among EHD proteins. (A) Left, freshly isolated CD4+ T cells from EHD12/2 and wild-type (WT) littermate mice were stimulated with anti-CD3ε and anti-CD28 for 72 h, and [3H]thymidine was added for the last 6 h of incubation. A representative figure of three independent experiments is shown. Middle, expression of EHD proteins in lymphoid organs from one WT and two EHD12/2 mice. Tissue lysates from the shown organs were resolved on a 10% SDS-PAGE gel and immunoblotted for EHD proteins. Right, quantification of each blot. (B) Left, LN cells from WT and EHD32/2 mice were stained with CellTrace Violet and then stimulated with anti-CD3ε/CD28–coated beads for 3 d. Cells were stained with anti-CD4 to assess the proliferation profile of CD4+ T cells. FACS data modeled using ModFit software are shown. PI, proliferation index (6SEM of n = 2). Right, Western blot of LN lysates showing deletion of EHD3 in EHD32/2 mice and lack of impact on EHD1/4 levels. (C) Left, LN cells from WT and EHD42/2 mice were stained with CFSE and stimulated with anti-CD3ε (1 or 2.5 mg/ml) together with 1 mg/ml anti-CD28 for 3 d. Cells were stained with anti-CD4 to assess the proliferation profile of CD4+ T cells. FACS data modeled using ModFit software are shown. PI, proliferation index (6SEM of n = 3). Right, Western blot of LN lysates showing deletion of EHD4 in EHD42/2 mice. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 3. CD4-Cre–mediated conditional deletion of EHD1, EHD3, and EHD4 does not impact thymic T cell development but leads to a relative decrease in CD4+ T cells in secondary lymphoid tissues. (A) Western blot verification of EHD1/3/4 deletions is shown. Lysates of freshly isolated splenocytes from the same mice before T cell expansion were used as controls. A representative blot of n = 6 is shown. (B and C) There was no change in the absolute number of cells in the different lymphoid tissues of CD4-Cre+ mice. In (B), cell numbers were counted in single-cell preparations of the indicated lymphoid organs of 4-wk-old CD4-Cre+ and CD4-Cre2 (control) mice; n =5.In(C), the total spleen cell numbers from adult (6–8 wk old) mice are shown; n =5.(D and E) Analysis of thymic developmental stages in CD4-Cre+ and CD4-Cre2 mice are shown. (D) Left, representative FACS plots with gating strategies used for analysis of cell subsets and developmental stages in thymocytes of 4-wk-old mice. The numbers in quadrants represent the mean percentage of total thymocytes. Right, quantification of left panels. (E) Left panel shows FACS plots with gating strategies and right panel shows quantification of relative percentages of cell subsets or cell numbers as in (D); n =4.(F and G) Reduced percentages of T cells in the spleen and increased percentages of B cells in the spleen and LN are shown. Spleen (F) and LN cells (G) from 4-wk-old CD4-Cre+ and CD4-Cre2 mice were analyzed by FACS for markers of B cells (B220+), CD8+ T cells, and CD4+ T cells (n = 4). Total cell numbers were calculated by multiplying the total live cell counts with the percentage of each subset. All values are plotted as the mean 6 SEM for the indicated sample size. *p , 0.05, **p , 0.01. DN, double-negative; DP, double-positive. 8 EHD PROTEIN REGULATION OF TCR decrease in the percentages of CD4+ and CD8+ T cells, but these These results further support our conclusion that the expression of differences were not statistically significant (Fig. 4D). EHD proteins is important for full CD4+ T cell responses to Ag. Collectively, these in vitro and in vivo analyses established, to our Expression of EHD proteins is required for optimal Ag-induced knowledge for the first time, that EHD proteins play an important T cell activation in vitro and for EAE in vivo positive role in Ag-driven T cell activation and immune response. Incorporation of an MOG Ag-specific T cell transgene in our EHD1/ 3/4 deletion models allowed analyses of CD4+ Tcellresponsetoa EHD proteins regulate the traffic of the TCR-CD3 complex to specific Ag, both in vitro and in vivo, with the latter in the context of the cell surface the development of EAE. We asked whether the mature T cells in At a cellular level, EHD proteins have been characterized as these mice exhibit any functional deficits. Initial experiments using regulators of the endocytic traffic of different cell surface receptors spleen cells from the parental 2D2-TCR transgenic and control mice as mentioned in the Introduction. Endocytic traffic of these re- with MOG or OVA peptides established the optimal concentrations ceptors is also key to their targeting to the IS (1, 10–13, 63–66). of MOG peptide to induce T cell proliferation (data not shown). To assess whether EHD proteins may indeed regulate the traffic of Analysis of CD4+ T cells from spleen using the CellTrace Violet TCR-CD3 or the accessory receptors, we first examined the cell stain and [3H]thymidine incorporation showed a significant decrease surface levels of TCR-CD3, CD28, CD4, CD25, CTLA-4, and in proliferation of T cells from CD4-Cre+ mice compared with LFA-1 by FACS analysis of unstimulated T cells (Fig. 6A). As 2 control CD4-Cre mice (Fig. 5A, 5B). Visualization of progressive assessed by comparing the MFI values, T cells from CD4-Cre+ cell divisions using ModFit LT software showed that whereas CD4- mice expressed significantly lower levels of TCR-CD3 on the cell + 2 + Cre and CD4-Cre CD4 T cells progress through a comparable surface compared with T cells from control mice (Fig. 6A, 6C). Downloaded from number of divisions, EHD1/3/4-null T cells exhibit a significant These analyses also showed that in vitro–activated CD4+ T cells underrepresentation at late divisions and an increased proportion of from CD4-Cre+ mice express lower levels, although not statisti- cells at earlier divisions (Fig. 5A). The EHD1/3/4-null T cells also cally significant, of TCR-CD3, CD28, CD4, CD25, CTLA-4, and exhibited a significantly lower overall proliferation index (Fig. 5A), LFA-1 compared with those from control mice (Fig. 6B, 6C). further supporting the conclusion that these cells proliferate slower Because the TCR-CD3 complex is the primary determinant of

than the control T cells. To exclude the possibility that the apparent Ag-specific T cell activation and subsequent responses, we focused http://www.jimmunol.org/ decrease in the number of EHD1/3/4-null T cells at late stages of on the TCR for further analyses. We examined the kinetics of TCR- proliferation might be due to their apoptosis, we stimulated the CD3 internalization and recycling in unstimulated CD4+ T cells. CD4+ T cells as previously described and stained these with pro- Analysis of TCR-CD3 internalization using FACS showed that pidium iodide and anti–annexin V followed by FACS analysis. We there was not a significant difference in internalization of TCR in observed no increase in the percentage of apoptotic cells in Ag- EHD1/3/4-null T cells compared with control T cells (Fig. 7A). stimulated CD4+ T cells isolated from CD4-Cre+ compared with Next, we analyzed recycling to the cell surface of the pre-existing 2 CD4-Cre (control) mice (Fig. 5C). Thus, the mature peripheral intracellular pool of TCR-CD3. For this purpose, anti-CD3 Ab 2 CD4+ T cells of CD4-Cre mice exhibit a marked defect in the recognition epitope on the pre-existing surface TCR-CD3 was specific Ag-induced cell proliferation in vitro. blocked with an unconjugated Ab, and the appearance of intra- by guest on September 27, 2021 To assess whether the impact of EHD1/3/4 protein deficiency on cellular TCR-CD3 on the cell surface at various time points was CD4+ T cell proliferation reflects their abnormal development or monitored by staining with a conjugated anti-CD3. These analyses an actual importance in the events associated with T cell activa- revealed that EHD1/3/4-null CD4+ T cells exhibit a significantly tion, we used T cells from 2D2-TCR Tg–bearing mice carrying a reduced recycling of pre-existing intracellular TCR-CD3 to the tamoxifen-inducible ERT-Cre that allows in vitro deletion of cell surface at all time points analyzed compared with control EHD1/3/4. EHD1/3/4 deletions in vitro led to a significant de- T cells (Fig. 7B). Next, we assessed the recycling of the pre- crease in Ag-induced proliferation compared with their control existing cell surface pool of TCR-CD3 by prelabeling it with a T cells (Fig. 5D–F). These results support a T cell–intrinsic pos- PE-conjugated anti-CD3 and allowing it to internalize. After acid itive role of EHD proteins in sustaining Ag-driven T cell responses wash (to strip any remaining Ab bound to TCR-CD3 on the T cell in mature peripheral T cells. surface), the internalized PE-labeled TCR-CD3 was allowed to Among the cytokines that initiate Ag-activated T cells into pro- reappear at the cell surface and restripped to remove the labeled liferation is the activation-induced secretion of IL-2 and the induced Ab. Even under these assay conditions, we observed a significant expression of the corresponding receptor on the T cell surface decrease in the recycling of TCR-CD3 in EHD1/3/4-null CD4+ (61, 62). Therefore, we assessed the impact of EHD1/3/4 deletions T cells compared with control cells (Fig. 7C). Overall, these re- on IL-2 secretion. We observed a significantly lower level of Ag- sults show a key requirement of EHD1, EHD3, and EHD4 in basal induced IL-2 secretion by EHD1/3/4-null CD4+ T cells (Fig. 5G) recycling of TCR-CD3 from intracellular endocytic recycling with a difference detectable at the earliest time point (12 h) ana- compartments back to the cell surface. lyzed, making it unlikely that the reduction is due to a smaller pool of T cells caused by reduced proliferation. The significant reduction Deletion of EHD1, EHD3, and EHD4 promotes the lysosomal in IL-2 secretion likely represents one mechanism for the defective degradation of TCR-CD3 T cell proliferation upon deletion of EHD1/3/4. Given the impairment of the endocytic recycling (Fig. 7B, 7C) The impact of EHD1/3/4 deficiencies was not restricted to together with the reduced steady-state surface TCR-CD3 levels in in vitro T cell responses alone. Using immunization with the freshly isolated EHD1/3/4-null T cells (Fig. 6), we considered the MOG35–55, we could test the impact of EHD1/3/4 deficiencies on possibility that the pool of TCR-CD3 that is retained in intracel- the development of EAE in vivo. We immunized cohorts of con- lular compartments may be aberrantly targeted for lysosomal trol (CD4-Cre2) versus experimental (CD4-Cre+) mice with degradation. To address this, EHD1/3/4-null T cells (from CD4- MOG35–55 and assessed onset and severity of EAE over time. Cre–mediated and 4OHT-induced ERT-Cre deletion) were exam- Interestingly, although no significant difference in the disease ined by Western blot for total levels of CD3ε. In both cases, we onset was seen, the EHD1/3/4-null group showed a decrease in the found that deletion of EHD proteins led to a significant decrease in disease severity compared with control littermates (Fig. 5H). the total levels of CD3ε (Fig. 8). As in unstimulated T cells, we The Journal of Immunology 9

FIGURE 4. CD4-Cre–mediated conditional deletion of EHD1, EHD3, and EHD4 does not impact thymic T cell development in non–TCR-transgenic mice.

(A) Cell numbers were counted in single- Downloaded from cell preparations of the indicated lymphoid organs of 3- to 4-wk-old CD4-Cre+ and CD4-Cre2 (control) mice; n =4. (B and C) Analyses of thymic developmental stages in CD4-Cre+ and CD4-Cre2 mice are shown. (B) The left panels show representative FACS plots with gating strat- http://www.jimmunol.org/ egies used for analysis of cell subsets and developmental stages in thymocytes of 3- to 4-wk-old mice. The numbers in quadrants represent the mean percentage of total thymocytes. The right panels show quantiﬁcation of results shown in the left panels. (C) The left panel show FACS plots with gating strategies for the double- negative stage and the right panel shows by guest on September 27, 2021 the quantiﬁcation of relative percentages of cell subsets; n =4.(D) Spleens from 3- to 4-wk-old CD4-Cre+ and CD4-Cre2 mice were analyzed by FACS for markers of B cells (B220+), CD8+ T cells, and CD4+ T cells (n =4).Allvaluesare plotted as mean 6 SEM for the indicated sample size. DN, double-negative; DP, double-positive; SP, single-positive.

also observed a reduction in the total level of CD3ε in activated and confocal microscopy (Fig. 9). Western blots of cells treated CD4+ T cells (Fig. 8C). Given our findings with the CSF-1 re- with Bafilomycin-A1 for 4 h showed a recovery of the CD3ε ceptor in macrophages (36), we hypothesized that this reduction protein level in EHD1/3/4-null T cells with levels comparable to was likely due to lysosomal targeting and degradation. To test this those in control cells (in treated cells) (Fig. 9A, 9B). We per- hypothesis, we treated the EHD1/3/4-null T cells with or without formed confocal imaging of control and EHD1/3/4-null T cells Bafilomycin-A1, an inhibitor of lysosomal degradation, for 4 h incubated with or without Bafilomycin-A1. Whereas relatively and analyzed the impact on total CD3ε levels using Western blots low CD3ε staining was colocalized with the lysosomal marker 10 EHD PROTEIN REGULATION OF TCR Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 5. Conditional deletion of EHD1, EHD3, and EHD4 in T cells leads to reduced cell proliferation and IL-2 secretion. (A) A reduced proliferation by CellTrace Violet dye dilution assay is shown. Successive cell divisions were modeled with ModFit LT software and are shown as peaks of different colors. The ﬁgure is a representative of n = 3. The mean percentages of cells at successive divisions are shown on the right as a histogram. The proliferation index (PI) shown on top is the mean 6 SEM of n =3.*p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001, *****p , 0.00001. (B) Reduced proliferation seen by [3H]thymidine incorporation assay is shown. Shown is a representative ﬁgure of n =2.(C) Cells were stimulated as previously mentioned for the proliferation assays and stained with propidium iodide (PI) and anti-annexin V (AV) Ab followed by (Figure legend continues) The Journal of Immunology 11

LAMP-1 in untreated cells (Fig. 9D), some colocalization was Additionally, an apparent increase in the peripheral B cell num- seen in Bafilomycin A1-treated control T cells (Fig. 9C, upper). bers and their percentages were seen in the EHD1/3/4-null mice in Notably, significantly more lysosome-localized CD3ε was seen in the context of 2D2 transgene; the basis of this phenotype is un- EHD1/3/4-null cells treated with bafilomycin-A1 (Fig. 9C, lower, known but is likely to reflect altered T cell/B cell interactions due 9E). These results support the conclusion that expression of to T cell deficiency of EHD1/3/4 genes. There was also a decrease EHD1/3/4 is required for efficient transport of TCR-CD3 from in the percentage of CD8+ T cells in the periphery, but this was endosomal compartments back to the cell surface and to prevent only significant in the spleens of transgenic mice. Further studies its aberrant traffic into lysosomes. are needed to precisely assess the basis of these effects. The in vitro analyses of T cells from the CD4-Cre deletion model Discussion demonstrated significantly defective Ag-elicited CD4+ Tcellpro- Cellular studies have identified members of the EHD protein family liferation and IL-2 cytokine secretion responses upon EHD1/3/4 as key new regulatory elements of endocytic trafficking of several deletion. Similar results were observed using CD4+ T cells de- cell surface receptors, but their physiological functions remain rived from an alternate model where in vitro deletion of floxed relatively unexplored and their roles in the immune system are EHD1, EHD3, and EHD4 genes was induced with 4-OHT to bypass unknown. In this study, we investigate the role of EHD proteins in any in vivo developmental abnormalities. These results support a the context of Ag-specific CD4+ T cells in vitro and in vivo. Our T cell–intrinsic positive role of EHD proteins in sustaining Ag- genetic studies, ablating genes encoding three members of the driven T cell responses in mature peripheral T cells, a role inde- EHD protein family (EHD1, EHD3, and EHD4) by CD4+ T cells pendent of any potential involvement of EHD proteins in events in the context of MOG transgenic TCR, demonstrate, to our associated with thymic T cell development. Downloaded from knowledge for the first time, an important, positive role of EHD Coordination of multiple EAE-inducing T cell functions, in- proteins in Ag-specific T cell activation in vitro and an autoim- cluding their early activation, expansion, migration to the brain mune response in vivo. Mechanistically, we show that EHD pro- across the blood–brain barrier, and effector functions to mediate teins are required for basal recycling of TCR-CD3 from demyelination in the CNS are required for full disease induction intracellular endocytic pools to the cell surface, thereby deter- (70, 71). Therefore, we analyzed the impact of EHD1/3/4 defi-

mining the subsequent level of T cell activation. Combined with ciencies on the onset and progression of EAE. Because in vitro http://www.jimmunol.org/ our recent study demonstrating a key positive role of EHD1 in the studies showed the T cell proliferation and cytokine (IL-2) se- transport of newly synthesized CSF-1R from the Golgi to the cell cretion to be impaired upon EHD1/3/4 deficiencies, we expected surface of bone marrow–derived macrophages (36), and previous to see a delayed onset and reduced severity of EAE. Although we cell biological findings that EHD1 facilitates MHC class I recy- saw no difference in the onset of disease, EHD1/3/4-null mice cling from the endocytic recycling compartment to the cell surface showed a decrease in the disease severity compared with control in a cell line system (25), our results strongly support the likeli- littermates. Therefore, further in vitro and in vivo studies using hood that EHD proteins play critical and potentially diverse roles models developed in the present study should help to compre- in the immune system. hensively reveal other aspects of T cell function that also rely on Analysis of thymic T cell development did not reveal any sig- EHD protein expression. by guest on September 27, 2021 nificant alterations in total cellularity, T cell subsets, or relative Given the reduced TCR-CD3 levels in the CD4+ T cells of our proportions of T cells at various developmental stages in mice with EHD1/3/4-null mouse model prior to deliberate T cell activation, a CD4-Cre–mediated deletion of EHD1/3/4, indicating that EHD our focus in the present study has been on the role of EHD pro- proteins are largely dispensable for thymic development beyond teins in basal (prior to antigenic stimulation) TCR-CD3 traffic, the CD4/8 double-positive stage. Whether this might reflect any which has not been mechanistically dissected in much detail. role of the remaining EHD family member, EHD2, remains a Therefore, further studies to identify partner proteins through possibility that will require future studies. It is also possible that which EHDs regulate TCR traffic will be of great interest. Nota- EHD proteins are important at earlier stages of thymic develop- bly, endocytic trafficking pathways play key roles during T cell ment, and future use of other Cre elements active at these stages activation by orchestrating the polarized transport of TCR-CD3 will be needed to address this possibility. and signaling molecules such as Lck to maintain an active im- 2 In contrast to the relatively unperturbed thymic development, munologic synapse, and Rab4 and Rab11+ endosomes are im- CD4-Cre–mediated EHD1/3/4 deletion led to a decrease in the plicated in these processes (72). percentage of T cells in the periphery. This phenotype was seen in Cell biological studies have linked EHD proteins to the regu- secondary lymphoid tissues, including the LN and the spleen, but lation of traffic in Rab11+ recycling endosomes as well as com- was only significant in the spleen. These results suggested that partments regulated by other Rabs. Thus, further studies to EHD proteins may play a more important role in mature periph- examine the potential importance of EHD proteins in regulating eral T cells. The precise basis for this phenotype remains unclear, endocytic traffic into and out of the IS during T cell activation will but lower functional responses to TCR engagement, as seen in ex be of great interest. In this regard, we and others have recently vivo analyses (discussed below), could play a role because TCR identified a key role of EHD proteins in primary cilia biogenesis signals are important in peripheral T cell maintenance (67–69). (37, 42), and recent studies have shown that the maintenance of IS

FACS analysis for relative proportions of PI+/annexin V+ apoptotic cells. Values represent mean 6 SEM of n =2.(D) Spleen cells from EHD1fl/fl; EHD3fl/fl; EHD4fl/fl; CreERT2; 2D2Tcr mice were stimulated with anti-CD3/CD28 in the presence or absence of 4-OHT for 4 d to derive in vitro EHD1/3/4-deleted T cells versus their wild-type controls. Cells were washed, loaded with CFSE dye, and restimulated with anti-CD3/CD28 for 72 h. Cells were as in (A). Shown is a representative figure of n =2.(E) Western blot used to show depletion of EHD1/3/4 in tamoxifen-treated T cells. (F) Quantification of the percentage of proliferating cells from the data shown in (D). (G) Reduced IL-2 secretion is shown. Values represent mean 6 SEM. n =4.*p , 0.05, **p , 0.01. (H)There was a reduced severity of EAE in CD4-Cre+ mice. Clinical scores were recorded as described in Materials and Methods. The plotted values are the composite mean score 6 SEM. n = 5 in each group. *p , 0.05. 12 EHD PROTEIN REGULATION OF TCR Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 6. Conditional deletion of EHD1, EHD3, and EHD4 leads to impairment of basal surface level of TCR-CD3. (A) Spleen cells were stained with directly conjugated Abs for indicated receptors and were gated on CD3+ T cells to analyze the median intensity level (MFI) of each receptor. Shown is a representative histogram of the surface levels of TCR-CD3 (anti-CD3ε), CD28, CD4, CD25, CTLA-4, and LFA1 at steady-state. (B) Representative histogram of the surface levels of TCR-CD3, CD28, CD4, CD25, CTLA-4, and LFA1 after 24 h stimulation with the MOG peptide. (C) The MFI values were plotted as mean 6 SEM of n =3.*p , 0.05. The Journal of Immunology 13 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 7. Conditional deletion of EHD1, EHD3, and EHD4 leads to impairment of basal recycling of TCR-CD3 from endocytic compartments to the cell surface. (A) A relatively intact basal TCR-CD3 internalization is shown; n = 3. A representative histogram of the MFIs of TCR-CD3 internalization is shown on the left. (B) Reduced recycling of pre-existing endosomal TCR-CD3 is shown. The data represent mean 6 SEM of n =4.*p , 0.05. A representative histogram of the MFI of TCR-CD3 recycling is shown on the left. (C) Recycling of the labeled cell surface TCR-CD3 is shown. Cells were analyzed by FACS and MFI values were used to quantify the proportion of internalized receptors that were still intracellular (acid wash-resistant fraction), and the percentage of recycling was calculated as described in Materials and Methods. A representative histogram of the MFIs of TCR-CD3 recycling is shown on the left. Values plotted are mean 6 SEM of n =4.*p , 0.05, **p , 0.01. 14 EHD PROTEIN REGULATION OF TCR Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 8. Conditional deletion of EHD1, EHD3, and EHD4 leads to a reduced level of the CD3ε component of TCR-CD3. (A) Expanded T cells from LN of CD4-Cre2 or CD4-Cre+ mice probed for CD3ε and either EHD1/4 or EHD3 proteins. (B) Quantification of CD3ε signals. Values plotted are mean 6 SEM of n =5.**p , 0.01. (C) Lysates of freshly isolated CD4+ T cells from CD4-Cre–positive and CD4-Cre–negative mice before and after 72 h anti- CD3/anti-CD28 activation were Western blotted for EHD3 and CD3ε levels. b-Actin was used as loading control. (D) LN cells from EHD1fl/fl; EHD3fl/fl; EHD4fl/fl; CreERT2; 2D2-Tcr or EHD1fl/fl; EHD3fl/fl; EHD4fl/fl; 2D2Tcr (control) mice were stimulated with anti-CD3/CD28 for 4 d in the presence (+) or absence (2) of 4-OHT. Lysates from equal numbers of cells were loaded in each lane. Western blot was carried out as in (B). Shown is a representative blot run in duplicate. (E) Quantification of CD3ε signals for blot shown in (D). (F) Quantification of CD3ε signals for Cre-ERT+ T cells. Values plotted are mean 6 SEM of n =3.*p , 0.05. in cilia-less T cells involves ciliogenesis-related proteins such as T cell function in vivo (74); the latter are partially similar to the IFT20, which localize in the IS and are required for recycling of phenotype of our EHD1/3/4 KO mice. Interestingly, the require- TCR-CD3 (46, 47, 73). Although IFT20 KO under CD4-Cre led to ment of EHD1 and EHD3 were found to be important for the only minor defects in T cell development and collagen-induced recruitment of transition zone proteins and IFT20 during ciliary arthritis (an experimental model of rheumatoid arthritis), earlier vesicle formation (42). Alhough our study has been done mainly deletion using Lck-Cre led to defective thymic development and on freshly isolated and rested cells, it seems plausible that EHD The Journal of Immunology 15 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 9. Conditional deletion of EHD1, EHD3, and EHD4 leads to lysosomal degradation of TCR-CD3. (A) T cells expanded from LN of CD4-Cre2 or CD4-Cre+ mice (7–8 d) were washed and treated with (+) or without (2) 100 nM Bafilomycin-A1 for 4 h at 37˚C. Lysate from equal numbers of cells was run, probed for CD3ε and EHD1/4, and reprobed for EHD3, with b-actin as a loading control. A representative blot is shown. (B) Quantification from data exhibited in (A) are shown here. Values plotted are mean 6 SEM of n =3.*p , 0.05. (C) Expanded T cells from LN of CD4-Cre2 or CD4-Cre+ mice were treated with Bafilomycin-A1 as in (A) and stained with anti-CD3ε (red) and anti-LAMP1 (green) Abs followed by confocal imaging. Shown is a representative image of n = 3. Scale bars, 2 mm. (D) Anti-CD3/anti-CD28–expanded T cells from LN of CD4-Cre2 or CD4-Cre+ mice that were not treated with Bafilomycin-A1 were stained with anti-CD3ε (red) and anti-LAMP1 (green) Abs followed by confocal imaging. 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15.93 ± 2.71 6.87 ± 0412

C EHD3 expression in CD4+T cells

644.78 ± 72.09

D EHD4 expression in CD4+T cells

17.23 ± 1.14

S Fig. 1: BioGPS databases analyses of EHD family of proteins

mRNA expression levels in mouse CD4+ T-cells. Red box showing

mRNA level of A) EHD1 (http://biogps.org/#goto=genereport&id=13660),

B) EHD2 (http://biogps.org/#goto=genereport&id=259300), C) EHD3

(http://biogps.org/#goto=genereport&id=57440) and D) EHD4

(http://biogps.org/#goto=genereport&id=98878) in mouse CD4+ T cells.

Cropped and zoomed images are shown.

PCR Genotyping of Targeted Alleles

Primers For: EHD1 flox EHD3 flox EHD4 flox CRE CD4 Tg TCR

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

B Spleen CD4+T cells Spleen CD4+T cells

72KDa 72KDa EHD4 EHD1 EHD3

β-Actin β-Actin

- CD4 TCR

T 0.7

+ non-Tg NS

0.6 CD4 TCR

Actin -

β Tg 0.5

0.4 NS 0.3 NS 0.2

cells Normalized to to Normalized cells 0.1 EHDs Protein Level in CD4 in Level Protein EHDs 0 EHD1 EHD3 EHD4

S Fig. 2: The CD4 Transgenic TCR Mice (2D2) Express Normal Levels of EHD Proteins. A)

An example of tail PCR showing the presence of floxed alleles targetedfor conditional deletion of

EHD1/3/4 genes in CD4-Cre-bearing mouse T-cells. Shown is an example of 4 individual pups (#1,

2, 3, & 4) with or without CD4-Cre and Tg TCR, and carrying EHD (1, 3, 4)flox/flox genes. B)

Western blot of cell lysates of spleen cells and CD4+ T-cells from LN of CD4-Cre negative; Tg

TCR+ and Tg TCR- (non-transgenic) mice. Non-transgenic TCR littermate was used as control. The

membrane was stripped and re-probed for EHD3. N=3.