Cutting Edge: DNAX Accessory Molecule 1− Deficient CD8+ T Cells Display Immunological Synapse Defects That Impair Antitumor Immunity This information is current as of October 4, 2021. Kelly M. Ramsbottom, Edwin D. Hawkins, Raz Shimoni, Mairi McGrath, Christopher J. Chan, Sarah M. Russell, Mark J. Smyth and Jane Oliaro J Immunol 2014; 192:553-557; Prepublished online 13

December 2013; Downloaded from doi: 10.4049/jimmunol.1302197 http://www.jimmunol.org/content/192/2/553

Supplementary http://www.jimmunol.org/content/suppl/2013/12/13/jimmunol.130219 http://www.jimmunol.org/ Material 7.DC1 References This article cites 25 articles, 10 of which you can access for free at: http://www.jimmunol.org/content/192/2/553.full#ref-list-1

Why The JI? Submit online.

by guest on October 4, 2021 • Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

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. Th eJournal of Cutting Edge Immunology

Cutting Edge: DNAX Accessory Molecule 1–Deficient CD8+ T Cells Display Immunological Synapse Defects That Impair Antitumor Immunity Kelly M. Ramsbottom,* Edwin D. Hawkins,* Raz Shimoni,† Mairi McGrath,* ,‡ ,†,x ,‡,{ Christopher J.x Chan,* Sarah M. Russell,* Mark J. Smyth,* and Jane Oliaro*, DNAX accessory molecule 1 (DNAM-1) is expressed on receptors. These receptors bind to nectin and nectin-like pro- all CD8+ T cells and promotes their activation and teins, and include DNAX accessory molecule 1 (DNAM-1; effector function. DNAM-1 interacts with LFA-1, a crit- CD226, PTA-1) and others (5). DNAM-1 interacts with the ical molecule for immunological synapse formation be- polarity protein, Discs Large, and the actin binding protein, tween T cells and APCs, and for cytotoxic killing of 4.1G, to mediate cytoskeletal changes important for cell ad- Downloaded from target cells. Mice that lack DNAM-1 display abnormal hesion (6). DNAM-1 also interacts with the integrin LFA-1 T cell responses and antitumor activity; however, the (7–9), an important mediator of stable conjugation and syn- apse formation between T cells and APCs or target cells (10). mechanism involved is unclear. In this article, we show 2/2 that DNAM-1 deficiency results in reduced proliferation Studies using DNAM-1 gene–deficient mice (DNAM-1 ) + have highlighted the importance of DNAM-1 in both pro-

of CD8 T cells after Ag presentation and impaired http://www.jimmunol.org/ tection against infectious agents and control of tumor growth. cytotoxic activity. We also demonstrate that DNAM- 2 2 DNAM-1 / mice take longer to clear lymphocytic chorio- 1–deficient T cells show reduced conjugations with tu- meningitis virus infection (11). DNAM-1 expression on NK mor cells and decreased recruitment of both LFA-1 and cells promotes cytotoxic activity against DNAM-1 ligand- 2 2 lipid rafts to the immunological synapse, which corre- expressing tumor cells (5, 12), whereas DNAM-1 / mice lates with reduced tumor cell killing in vitro. This syn- show impaired antitumor responses in vivo (13). DNAM-1 apse defect may explain why DNAM-1–deficient mice ligands are frequently overexpressed by tumor cells (12, 14), cannot clear tumors in vivo, and highlights the impor- and polymorphisms in the DNAM-1 gene are associated with

tance of DNAM-1 and the immunological synapse in the development of autoimmune diseases (15, 16). However, by guest on October 4, 2021 T cell–mediated antitumor immunity. The Journal of the mechanism by which DNAM-1 contributes to T cell Immunology, 2014, 192: 553–557. activation remains unclear. DNAM-1 may be involved in direct T cell signaling pathways or the cell–cell interactions + required for optimal T cell activation and effector function. ytotoxic CD8 T cells form an integral part of the + immune response against pathogens (1) and in the We therefore investigated the response of CD8 T cells from immunosurveillance of cancer (2). The activation of DNAM-1–deficient mice to Ag presentation and their inter- C + action with tumor cells in vitro. Our results demonstrate that naive CD8 T cells is dependent on TCR–MHC class I + interactions with APCs, during which sustained contact be- DNAM-1 is required for optimal activation of naive CD8 T cells, and for efficient conjugation and synapse formation tween the T cell and APC leads to formation of an immu- + nological synapse. The IS is a polarized structure that brings between effector CD8 T cells and tumor cells. together the receptors, adhesion molecules, and adaptor proteins necessary for optimal signaling and downstream ef- Materials and Methods fector T cell functions such as cytokine secretion and cyto- Mice and Abs 2 2 toxic activity (3). The activation and cytotoxic activity of The C57BL/6 DNAM-1 / mice were kindly supplied by Marco Colonna CD8+ T cells is fine-tuned by a range of cosignaling mole- (Washington State University School of Medicine, St. Louis, MO) and back- crossed onto the T cell transgenic OT-1 mouse background (Peter MacCallum cules, such as members of the CD28 family (4). Another less 2 2 Cancer Centre). OT-1 DNAM-1+/+ and OT-I DNAM-1 / littermates were characterized group of costimulatory molecules important for used for all experiments. The Abs for flow cytometry were rat anti-Va2, -CD62L, + CD8 T cell function are those of the Ig superfamily of -CD25, -CD69, -CD44, -CD127, -CD107a, mouse anti–granzyme B, –IFN-g

*Cancer Immunology, Peter MacCallum Cancer Centre, East Melbourne 3002, Victo- Address correspondence and reprint requests to Dr. Jane Oliaro, Peter MacCallum Cancer ria, Australia; †Centre for Micro-Photonics, Swinburne University, Hawthorn 3122, Centre, East Melbourne 3002, VIC, Australia. E-mail address: [email protected] Victoria, Australia; ‡Department of Immunology, Monash University, Clayton 3168, x The online version of this article contains supplemental material. Victoria, Australia; Sir Peter MacCallum Department of Oncology, The University of { Melbourne, Parkville 3052, Victoria, Australia; and Immunology in Cancer and Infec- Abbreviations used in this article: D, distal; DC, dendritic cell; DNAM-1, DNAX tion Laboratory, Queensland Institute of Medical Research, Herston 4006, Queensland, accessory molecule 1; MTOC, microtubule-organizing center; P, proximal. Australia Ó Received for publication August 22, 2013. Accepted for publication November 15, Copyright 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 2013.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302197 554 CUTTING EDGE: DNAM-1–DEFICIENT T CELLS SHOW IMMUNE SYNAPSE DEFECTS and –TNF-a (BD Biosciences), and anti-DNAM-1 (Biolegend); for microscopy, using a Fluoview FV1000 confocal microscope (Olympus, NY) mounted with they were purified rat anti–LFA-1 and -CD43 (BD Biosciences), phalloidin- a603 oil immersion objective (NA 1.42). T cells selected for protein scoring rhodamine (Molecular Probes), rabbit anti-GM1 (Calbiochem, MERK Milli- had a single contact site with one tumor cell and polarized microtubule- pore, Darmstadt, Germany), and rabbit and rat anti-tubulin (Rockland). organizing center (MTOC). Polarization to the synapse was determined by Secondary Abs conjugated to Alexa Fluorophores and ProLong antifade with quantitating the amount of nonnuclear fluorescence in the proximal (P) re- DAPI were purchased from Molecular Probes. gion (first third of T cell closest to tumor cell) and distal (D) region (rest of T cell), and the ratio was determined by P-D/P+D where values approaching In vitro T cell assays 1 represent strong polarization to the interface of the two cells (Supplemental Fig. 2C). Data were acquired using Matlab and TACTICS software (19). Naive T cells were purified from splenocytes isolated from OT-1 DNAM-1+/+ or 2 2 DNAM-1 / mice by negative selection using an EasySep magnetic separation Statistical analyses system (Stemcell Technologies, Vancouver, BC, Canada). Immature dendritic cells (DCs) were generated as previously described (17). For proliferation assays, Statistical significance was determined using an unpaired Student t test. isolated naive T cells were labeledwithCFSEandincubatedwith1mM SIINKEL-pulsed DCs. The cells were harvested and analyzed for proliferation Results and Discussion peaks by flow cytometry or for the concentration of cytokines in the supernatant DNAM-1 is required for optimal expansion of naive CD8+ T cells in using a Mouse Inflammation Cytometric Bead Array kit (BD Biosciences) after 72 h. Intracellular staining was performed using Cytofix/Cytoperm reagents response to Ag presentation (BD Biosciences). Cytotoxic activity of activated T cells was measured by a Because DNAM-1 has been implicated in impaired T cell re- standard chromium release assay (18) using chromium-labeled MC38-OVA tumor cells or labeled/SIINFEKL-pulsed DCs as targets. For the conjugation sponses to infection in vivo (11), we first determined whether assay, activated T cells (day 5) were labeled with CellTrace Violet Dye (Mo- naive T cells deficient for DNAM-1 responded normally to lecular Probes) and incubated with CFSE-labeled, SIINFEKL-pulsed EL4 tu- Ag presentation. We used naive CD8+ T cells from OT-I mor cells at 37˚C. The cells were vortexed to separate any non-Ag-specific DNAM-1 wild-type (DNAM-1+/+) or deficient (DNAM- Downloaded from conjugates, and the cells were fixed in 4% paraformaldehyde and analyzed by 2/2 flow cytometry for the presence of double-positive conjugates. 1 ) mice to assess proliferation and cytokine production in an Ag-specific response to SIINFEKL-pulsed syngeneic DCs 2 2 Immunofluorescent microscopy in vitro. Although naive DNAM-1 / T cells displayed a wild- hi med 2 2 MC38-OVA tumor cells were adhered overnight onto eight-well chamber type phenotype (CD62L CD44 CD25 CD69 ; Supple- slides (Nalge Nunc, Rochester, NY). Activated T cells were overlaid for 1–2 h mental Fig. 1A), they showed reduced proliferation, as measured and nonadherent cells washed off. Cells were fixed and permeabilized and by dilution of the division tracking dye CFSE, compared with http://www.jimmunol.org/ then labeled with primary Abs, followed by detection with Alexa Fluor– +/+ conjugated secondary Abs and mounted in Prolong antifade containing DAPI DNAM-1 T cells when stimulated with peptide-pulsed DCs + (Molecular Probes), as previously described (17). The slides were examined (Fig.1A).WealsodetectedloweramountsoftwokeyCD8 by guest on October 4, 2021

FIGURE 1. DNAM-1–deficient T cells show reduced proliferation, cytokine production, and cytotoxic activity, but normal polarity. (A) CFSE dilution assay 2 2 of naive OT-1 DNAM-1+/+ or DNAM-1 / T cells in response to DCs pulsed with 1 mM SIINFEKL peptide. FACS plots represent T cells at 72 h after incubation with DCs. (B) Naive T cells were cocultured with DCs pulsed with 1 mM SIINFEKL, and the concentration of IFN-g and TNF-a in the supernatant at 72 h was measured by cytometric bead array. Data are presented as mean and SD of triplicate samples. (C) Activated T cells were cultured in an eight-well chamber slide overnight, then fixed and permeabilized. The cells were stained for actin, tubulin (red), LFA-1, or CD43 (green) and analyzed by confocal mi- croscopy. The protein of interest is costained with tubulin and DAPI (blue), except for tubulin, which is costained with CD8 (green). Images shown are representative of localization of each protein over at least three independent experiments. Scale bar, 10 mM. (D) Activated T cells were tested in a chromium release killing assay with peptide-pulsed DCs as target cells. Data are presented as mean and SD of triplicate samples. All data in (A)–(C) are representative of at least three independent experiments. *p , 0.05. The Journal of Immunology 555

T cell effector cytokines, IFN-g and TNF-a, in the super- DNAM-1–deficient CD8+ effector T cells demonstrate normal 2 2 natant of DNAM-1 / T cell–DC cocultures (Fig. 1B). This phenotype and morphology in vitro but reduced cytotoxic activity was a consequence of reduced T cell numbers, because Despite a reduction in proliferation and differentiation ki- 2/2 +/+ 2 2 DNAM-1 and DNAM-1 T cells produced equivalent netics, a proportion of DNAM-1 / T cells were activated levels of intracellular IFN-g and TNF-a upon stimulation and proliferated in vitro. To determine whether this change with peptide-pulsed DCs (Supplemental Fig. 1C). This is in activation kinetics affected the phenotype or polarity of consistent with previous data demonstrating that during acute responding T cells, we first used flow cytometry to assess the + lymphocytic choriomeningitis virus priming, CD8 T cells cell-surface profile of the activated T cells. Activated DNAM- 2/2 2 2 from DNAM-1 mice make equivalent levels of IFN-g 1 / T cells upregulated CD44, CD69, and CD25, and +/+ compared with DNAM T cells (11). The reduced prolifera- downregulated CD62L to equivalent levels as DNAM-1+/+ 2 2 tion was not a result of TCR signaling defects, because anti-CD3/ T cells (Supplemental Fig. 1B). The activated DNAM-1 / CD28 Ab stimulation resulted in proliferation and cytokine levels T cells also displayed no detectable defects in polarization of 2 2 equivalent between DNAM-1+/+ and DNAM-1 / T cells (Sup- actin and LFA-1 to the leading edge, CD43 to the uropod, plemental Fig. 1D, 1E). These results indicate that expression of and localization of the MTOC to the base of the uropod, as DNAM-1 on CD8+ T cells contributes to optimal T cell activation assessed by confocal microscopy (Fig. 1C). However, activated 2 2 and proliferation in response to Ag presentation by DCs. DNAM-1 / T cells did show impaired cytotoxic activity Downloaded from http://www.jimmunol.org/ by guest on October 4, 2021

FIGURE 2. Activated DNAM-1–deficient T cells show reduced conjugation, cytotoxic activity, and synapse formation with tumor cells. (A) Activated OT-I 2 2 DNAM-1+/+ or DNAM-1 / T cells were labeled with Cell Trace and cocultured with CFSE-labeled SIINFEKL-pulsed EL4 tumor cells and analyzed by flow cytometry after 60 min. The graph is of percentage of total T cells conjugated to tumor cells at 60 min and is data pooled from three independent experiments. Data are presented as mean and SD. (B) Activated T cells were tested in a chromium release killing assay with MC38-OVA or empty vector (EV) tumor cells as target cells. Data are representative of at least four independent experiments with mean and SD of triplicate samples from one representative experiment shown. (C) Activated T cells were cocultured with adhered MC38-OVA tumor cells in an eight-well chamber slide. After 1–2 h, the conjugates were fixed, permeabilized, and stained for tubulin (MTOC), actin, LFA-1, and GM1 (lipid rafts). The protein of interest is shown in green, and the merge includes DAPI/nucleus (blue) and tubulin (red), except for tubulin/MTOC, which is costained with actin (red) (D) Quantification of polarization of proteins at the synapse (LFA-1, n = 68; GM1, n = 48; actin, n = 61). (C and D) Images and quantification are pooled from three independent experiments. Scale bar, 10 mM. *p , 0.05. 556 CUTTING EDGE: DNAM-1–DEFICIENT T CELLS SHOW IMMUNE SYNAPSE DEFECTS

2 2 against peptide-pulsed DCs (Fig. 1D). Both DNAM-1 / of the T cells. Indeed, DNAM-1 is important for the LFA-1 and DNAM+/+ T cells expressed equivalent levels of the cyto- costimulatory signal necessary for naive CD4+ T cell differ- toxic molecule, Granzyme B, and showed no difference in de- entiation and proliferation (8). LFA-1–deficient mice show granulation after TCR stimulation (Supplemental Fig. 2A, 2B). normal cytotoxic T cell responses to virus but fail to reject Together, these data suggest that the DNAM-1 is critical for the immunogenic tumors (25), suggesting that LFA-1 may also cell–cell contact required for efficient killing of target cells by play a significant role in the interaction of CD8+ T cells with CD8+ T cells. tumor cells. Together, our results suggest that DNAM-1 deficiency DNAM-1 is required for optimal conjugation and killing of tumor affects T cell function in a 2-fold manner: 1) reduced activation + cells by CD8 effector T cells of responding effector T cells, and 2) reduced ability of ac- To explore potential reasons for the defect in killing shown tivated T cells to form an IS with target cells and deliver the 2 2 earlier, we next determined whether activated DNAM-1 / cytotoxic payload necessary for elimination of target cells. T cells display normal cytotoxic activity against tumor cells that Furthermore, our results demonstrate that these defects are express the ligands for DNAM-1 (CD155 and CD112) (5). manifested through suboptimal formation of a cytotoxic Mice deficient for DNAM-1 have impaired antitumor synapse involving reduced polarization of LFA-1 and lipid responses using models dependent on both NK and CD8+ rafts. These results highlight the importance of DNAM-1 in T cell activity (5, 20). Furthermore, the association of the control of optimal synapse formation required for effective DNAM-1 with LFA-1 and proteins that mediate cytoskeletal antitumor T cell responses. changes in T cells such as Dlg and 4.1G (6, 21) suggests a role Downloaded from for this accessory molecule in cell–cell interactions. We 2/2 Acknowledgments therefore assessed the ability of the DNAM-1 T cells to We thank Marco Colonna (Washington State University School of Medicine) form conjugates with tumor cells in vitro using a flow cytom- for the DNAM-1 mice and Nicole Haynes (Peter MacCallum Cancer Centre) etry–based conjugation assay. We found that activated DNAM- for the MC38-OVA cell line. 2 2 1 / effector T cells formed significantly less conjugates than DNAM-1+/+ T cells (Fig. 2A, quantitated on right), confirming http://www.jimmunol.org/ thattheabsenceofDNAM-1impairsthestableinteraction Disclosures between T cells and tumor cells. Furthermore, consistent with The authors have no financial conflicts of interest. 2 2 our previous data showing that DNAM-1 / mice do not clear 2 2 MC38-OVA tumors in vivo (22), the DNAM-1 / Tcells References showed reduced cytotoxic activity against MC38-OVA tumor 1. Zhang, N., and M. J. Bevan. 2011. CD8(+) T cells: foot soldiers of the immune system. Immunity 35: 161–168. cells in vitro, as determined by a chromium release assay (Fig. 2. Vesely, M. D., M. H. Kershaw, R. D. Schreiber, and M. J. Smyth. 2011. Natural 2B). These results demonstrate that DNAM-1 expression on innate and adaptive immunity to cancer. Annu. Rev. Immunol. 29: 235–271.

+ 3. Dustin, M. L., A. K. Chakraborty, and A. S. Shaw. 2010. Understanding the structure by guest on October 4, 2021 CD8 T cells is critical for efficient conjugation and killing of and function of the immunological synapse. Cold Spring Harb. Perspect. Biol. 2: a002311. Ag-presenting tumor cells. 4. Chen, L., and D. B. Flies. 2013. Molecular mechanisms of T cell co-stimulation and co-inhibition. Nat. Rev. Immunol. 13: 227–242. 5. Chan, C. J., D. M. Andrews, and M. J. Smyth. 2012. Receptors that interact with DNAM-1 deficiency results in impaired immunological synapse nectin and nectin-like proteins in the immunosurveillance and immunotherapy of + formation between CD8 effector T cells and tumor cells cancer. Curr. Opin. Immunol. 24: 246–251. 6. Ralston, K. J., S. L. Hird, X. Zhang, J. L. Scott, B. Jin, R. F. Thorne, M. C. Berndt, To determine whether reduced cytotoxic activity of the A. W. Boyd, and G. F. Burns. 2004. The LFA-1-associated molecule PTA-1 2 2 DNAM-1 / T cells against MC38-OVA tumor cells was (CD226) on T cells forms a dynamic molecular complex with protein 4.1G and human discs large. J. Biol. Chem. 279: 33816–33828. a result of impaired cytotoxic synapse formation between the 7. Shibuya, K., L. L. Lanier, J. H. Phillips, H. D. Ochs, K. Shimizu, E. Nakayama, T cells and tumor cells, we used immunofluorescent micros- H. Nakauchi, and A. Shibuya. 1999. Physical and functional association of LFA-1 with DNAM-1 adhesion molecule. Immunity 11: 615–623. copy to dissect the polarization of synapse components during 8. Shibuya, K., J. Shirakawa, T. Kameyama, S. Honda, S. Tahara-Hanaoka, T cell–tumor cell interactions. Despite fewer conjugates being A. Miyamoto, M. Onodera, T. Sumida, H. Nakauchi, H. Miyoshi, and A. Shibuya. 2/2 2003. CD226 (DNAM-1) is involved in lymphocyte function-associated antigen 1 formed, in the DNAM-1 T cells that attached to a tumor costimulatory signal for naive T cell differentiation and proliferation. J. Exp. Med. cell, we found the MTOC was recruited to the synapse, along 198: 1829–1839. with enrichment of actin at the interface of the two cells. 9. Shirakawa, J., Y. Wang, S. Tahara-Hanaoka, S. Honda, K. Shibuya, and A. Shibuya. 2006. LFA-1-dependent lipid raft recruitment of DNAM-1 (CD226) in CD4+ However, we observed significantly impaired polarization of T cell. Int. Immunol. 18: 951–957. LFA-1 and lipid rafts (as determined by Ganglioside, GM-1, 10. Springer, T. A., and M. L. Dustin. 2012. Integrin inside-out signaling and the 2/2 immunological synapse. Curr. Opin. Cell Biol. 24: 107–115. localization) to the interface between the OT-1 DNAM-1 11. Welch, M. J., J. R. Teijaro, H. A. Lewicki, M. Colonna, and M. B. Oldstone. 2012. T cells and tumor cells (Fig. 2C and quantified in Fig. 2D). CD8 T cell defect of TNF-a and IL-2 in DNAM-1 deficient mice delays clearance in vivo of a persistent virus infection. Virology 429: 163–170. Formation of a “suboptimal” cytotoxic synapse, and sub- 12. Chan, C. J., D. M. Andrews, N. M. McLaughlin, H. Yagita, S. Gilfillan, sequent inability to secrete effector molecules in a directed M. Colonna, and M. J. Smyth. 2010. DNAM-1/CD155 interactions promote manner, may therefore account for the reduced cytotoxic cytokine and NK cell-mediated suppression of poorly immunogenic melanoma 2/2 metastases. J. Immunol. 184: 902–911. activity of DNAM-1 T cells against tumor cells in vivo. 13. Iguchi-Manaka, A., H. Kai, Y. Yamashita, K. Shibata, S. Tahara-Hanaoka, Strong interactions between T cells and target cells is de- S. Honda, T. Yasui, H. Kikutani, K. Shibuya, and A. Shibuya. 2008. Accelerated tumor growth in mice deficient in DNAM-1 receptor. J. Exp. Med. 205: 2959– pendent on cell-surface molecules such as TCR and LFA-1, 2964. but is also controlled by cytoskeletal dynamics and the in- 14. Castriconi, R., A. Dondero, F. Negri, F. Bellora, P. Nozza, B. Carnemolla, A. Raso, L. Moretta, A. Moretta, and C. Bottino. 2007. Both CD133+ and CD133- me- tegrity of lipid rafts (23). The reduced polarization of both dulloblastoma cell lines express ligands for triggering NK receptors and are sus- LFA-1 and lipid rafts containing costimulatory molecules ceptible to NK-mediated cytotoxicity. Eur. J. Immunol. 37: 3190–3196. 15. Qiu, Z. X., K. Zhang, X. S. Qiu, M. Zhou, and W. M. Li. 2013. CD226 such as DNAM-1 (9, 24) would therefore impact on conju- Gly307Ser association with multiple autoimmune diseases: a meta-analysis. Hum. gation efficiency and in the activation and cytotoxic activity Immunol. 74: 249–255. The Journal of Immunology 557

16. Song, G., S. C. Bae, S. Choi, J. Ji, and Y. Lee. 2012. Association between the A network of PDZ-containing proteins regulates T cell polarity and mor- CD226 rs763361 polymorphism and susceptibility to autoimmune diseases: a meta- phology during migration and immunological synapse formation. Immunity analysis. Lupus 21: 1522–1530. 22: 737–748. 17. Oliaro, J., V. Van Ham, F. Sacirbegovic, A. Pasam, Z. Bomzon, K. Pham, 22. Gilfillan, S., C. J. Chan, M. Cella, N. M. Haynes, A. S. Rapaport, K. S. Boles, M. J. Ludford-Menting, N. J. Waterhouse, M. Bots, E. D. Hawkins, et al. 2010. D. M. Andrews, M. J. Smyth, and M. Colonna. 2008. DNAM-1 promotes acti- Asymmetric cell division of T cells upon antigen presentation uses multiple con- vation of cytotoxic lymphocytes by nonprofessional antigen-presenting cells and served mechanisms. J. Immunol. 185: 367–375. tumors. J. Exp. Med. 205: 2965–2973. 18. Oliaro, J., A. Pasam, N. J. Waterhouse, K. A. Browne, M. J. Ludford-Menting, 23. Lim, T. S., A. Mortellaro, C. T. Lim, G. J. Ha¨mmerling, and P. Ricciardi-Cas- J. A. Trapani, and S. M. Russell. 2006. Ligation of the cell surface receptor, CD46, tagnoli. 2011. Mechanical interactions between dendritic cells and T cells correlate alters T cell polarity and response to antigen presentation. Proc. Natl. Acad. Sci. USA with T cell responsiveness. J. Immunol. 187: 258–265. 103: 18685–18690. 24. Shirakawa, J., K. Shibuya, and A. Shibuya. 2005. Requirement of the serine at 19. Shimoni,R.,K.Pham,M.Yassin,M.Gu,andS.M.Russell.2013.TACTICS,aninteractive residue 329 for lipid raft recruitment of DNAM-1 (CD226). Int. Immunol. 17: platform for customized high-content bioimaging analysis. Bioinformatics 29: 817–818. 217–223. 20. Fuchs, A., and M. Colonna. 2006. The role of NK cell recognition of nectin and 25. Schmits, R., T. M. Ku¨ndig, D. M. Baker, G. Shumaker, J. J. Simard, G. Duncan, nectin-like proteins in tumor immunosurveillance. Semin. Cancer Biol. 16: 359–366. A. Wakeham, A. Shahinian, A. van der Heiden, M. F. Bachmann, et al. 1996. LFA- 21.Ludford-Menting,M.J.,J.Oliaro,F.Sacirbegovic,E.T.Cheah,N.Pedersen, 1-deficient mice show normal CTL responses to virus but fail to reject immunogenic S. J. Thomas, A. Pasam, R. Iazzolino, L. E. Dow, N. J. Waterhouse, et al. 2005. tumor. J. Exp. Med. 183: 1415–1426. Downloaded from http://www.jimmunol.org/ by guest on October 4, 2021