Formation of a Central Supramolecular Activation Cluster Is Not Required for Activation of Naive CD8؉ T Cells

Formation of a central supramolecular activation cluster is not required for activation of naive CD8؉ T cells James P. O’Keefe*†, Kelly Blaine‡, Maria-Luisa Alegre‡§, and Thomas F. Gajewski*†‡§¶ Committees on *Cancer Biology and §Immunology and Departments of †Pathology and ‡Medicine, University of Chicago, Chicago, IL 60637 Edited by Arthur Weiss, University of California School of Medicine, San Francisco, CA, and approved May 5, 2004 (received for review September 16, 2003) Although both naive and effector T lymphocytes interact with facilitates directional release of cytokines and other effector antigen-expressing cells, the functional outcome of these interac- molecules toward antigen-expressing targets (6, 7). This model tions is distinct. Naive CD8؉ T cells are activated to proliferate and might predict distinct properties of the immune synapse between differentiate into effector cytolytic T lymphocytes (CTL), whereas naive T cells, which lack effector function, and primed effector CTL interact with specific targets, such as tumor cells, to induce T cells, which produce effector cytokines and can possess apoptotic death. We recently observed that several molecules cytolytic activity. linked to actin cytoskeleton dynamics were up-regulated in effec- Using Affymetrix (Santa Clara, CA) gene arrays, we recently tor vs. naive CD8؉ T cells, leading us to investigate whether T cell observed that several molecules linked to actin cytoskeletal differentiation is accompanied by changes in actin-dependent dynamics were up-regulated in effector T cells compared with ,processes. We observed that both naive and effector CD8؉ T cells naive CD8ϩ TCR transgenic T cells (8). This observation underwent T cell receptor capping and formed stable conjugates coupled with the fact that most analyses of the immunologic with antigen-specific antigen-presenting cells. However, the char- synapse have been done with primed CD4ϩ T cells, prompted a acteristics of the immunological synapse were distinct. Whereas careful comparison of the T cell͞APC interface in naive vs. accumulation of signaling molecules at the T cell͞antigen-present- effector CD8ϩ TCR transgenic T cells. We observed that ,ing cell contact site was detectable in both naive and effector CD8؉ effector T cells, but not naive 2C TCR transgenic CD8ϩ T cells T cells, only effector cells developed a central supramolecular formed a cSMAC, thus correlating this structure with the activation cluster as defined by punctate focusing of PKC␪, phos- acquisition of effector function. Despite the lack of detectable pho-PKC␪, and phospho-ZAP70. Extended kinetics, CD28 costimu- cSMAC formation in naive CD8ϩ T cells, microtubule organiz- lation, and high-affinity antigenic peptide did not promote PKC␪ ing center (MTOC) polarization, IL-2 production, and subse- focusing in naive cells. Nonetheless, naive CD8؉ T cells polarized quent proliferation and differentiation occurred, suggesting that the microtubule organizing center, produced IL-2, proliferated, and cSMAC formation is not required for naive CD8ϩ T cell differentiated into effector cells. Our results suggest that the activation. formation of a central supramolecular activation cluster is not required for activation of naive CD8؉ T cells and support the notion Materials and Methods that one role of an organized immune synapse is directed delivery T Cell Purification and Differentiation. All mice were housed in the of effector function. University of Chicago Animal Facility under specific pathogen- free conditions. 2C͞recombination activating gene (RAG) 2Ϫ͞Ϫ ϩ D8ϩ T cells play a critical role in the clearance of viral mice have been described in ref. 9. Naive and effector CD8 T Cinfections and the eradication of tumors. However, the cells were purified and generated as described in ref. 8. Briefly, acquisition of lytic activity occurs only after the differentiation naive T cells were purified by negative selection from the spleens Ϫ͞Ϫ of naive CD8ϩ T cells to the effector state. Activation of naive of 2C͞RAG2 mice. Effector cells were generated by cocul- CD8ϩ T cells requires direct ligation of cell surface receptors on ture of naive cells in vitro with mitomycin C-treated P815.B71 the T cell by cognate ligands on an antigen-presenting cell cells over two 4-day stimulations. (APC). Subsequent to T cell–APC contact, large-scale rear- rangement of the cytoskeleton and reorganization of cell surface Immunofluorescence Antibodies. The following reagents were and cytoplasmic molecules result in the formation of an ‘‘im- used: polyclonal rabbit anti-PKC␪, rabbit antiphospho-Zap-70, munological synapse’’ (1, 2). Spatial segregation of accumulated and goat antitalin from Santa Cruz Biotechnology; mAb anti- molecules has been reported to occur at the interface, resulting CD3 (2C11, hamster IgG) from Pharmingen; mAb antiphos- in the formation of a central and peripheral supramolecular photyrosine (4G10, murine IgG2b) from Upstate Biotechnology activation cluster (cSMAC and pSMAC, respectively) (3). The (Lake Placid, NY); mAb tubulin DM1A from NeoMarkers (Lab cSMAC has been characterized by punctate localization of Vision, Fremont, CA); polyclonal rabbit antiphospho-PKC␪ PKC␪, whereas the pSMAC can be defined by a ring of accu- from Cell Signaling Technology (Beverly, MA); FITC- mulated talin (3). A dynamic actin cytoskeleton is required, at conjugated donkey anti-rabbit IgG or donkey anti-mouse IgG; least in part, for these molecular rearrangements to occur (4). and Texas red-conjugated donkey anti-goat IgG from Jackson The functional significance of cSMAC͞pSMAC segregation is ImmunoResearch. not clear. Early studies indicated that agonist peptides induced IMMUNOLOGY cSMAC formation, whereas partial agonist or antagonist peptides, which failed to induce cytokine production, did not (1). This paper was submitted directly (Track II) to the PNAS office. These observations implied by correlation that cSMAC forma- Abbreviations: APC, antigen-presenting cell; CMAC, 7-amino-4-chloromethylcoumarin; cSMAC, central supramolecular activation cluster; CTL, cytolytic T lymphocytes; MTOC, tion might be necessary for full T cell activation. However, microtubule organizing center; pSMAC, peripheral supramolecular activation cluster; kinetic studies have not supported this model, as T cell receptor QL9, superagonist peptide QLSPFPFDL; RAG, recombination-activating gene; TCR, T cell (TCR)-mediated tyrosine kinase signaling seems to occur at the receptor. periphery of the immunological synapse and before cSMAC ¶To whom correspondence should be addressed at: 5841 South Maryland Avenue, MC2115, formation (5). An alternative hypothesis is that immune synapse Chicago, IL 60637. E-mail: [email protected]. formation is less important for T cell activation but rather © 2004 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0305965101 PNAS ͉ June 22, 2004 ͉ vol. 101 ͉ no. 25 ͉ 9351–9356 Downloaded by guest on September 25, 2021 TCR Capping. T cells (1 ϫ 106) were incubated with FITC-anti- CD3 Ab (2C11, Pharmingen) at 4°C for 30 min. Prewarmed goat anti-hamster Ab (Cappel) was added, and cells were incubated at 37°C for the indicated duration. Cold PBS was added to stop the reaction, and cells were fixed and analyzed by confocal microscopy. Conjugate Formation. This assay was performed similarly to the description in ref. 10. T cells were labeled with calcein AM (Molecular Probes), and either EL4 cells or P815 cells were labeled with PKH (Sigma). T cells (2.5 ϫ 105) and targets (5 ϫ 105) were mixed, centrifuged, lightly vortexed, and incubated at 37°C for the indicated times. Cells were then vortexed vigorously for 30 sec and immediately fixed. Two-color flow cytometric analysis was performed, and percent conjugates were determined by calculating the ratio of double-positive conjugates to the total number of T cells. Blocking of the class I MHC molecule Ld was carried out in the presence of either the anti-Ld Ab 30-5-7s or isotype control (final concentration of 50 ␮g͞ml). Immunofluorescence. To distinguish APCs from T cells, APCs were loaded with the vital dye 7-amino-4-chloromethylcoumarin (CMAC) Cell-Tracker Blue (Molecular Probes) as described in ref. 11. APCs (1.5 ϫ 105) were mixed with an equal number of Ficoll͞Hypaque purified T cells in DMEM with 10% FCS, centrifuged at 5,000 rpm for 30 sec, and incubated for the indicated time (minus 2 min) at 37°C. Supernatant was aspirated, and conjugates were gently resuspended in serum-free DMEM ␮ Fig. 1. TCR capping and conjugate formation are comparable in naive and by using a 1,000- l pipettor and plated onto poly(L)-lysine- effector CD8ϩ T cells. (A) Naive or effector T cells were preincubated with coated (molecular weight 30,000–70,000, Sigma) slides for 2 min anti-CD3␧-FITC. Prewarmed goat anti-hamster Ab was added, and cells were before fixation. Slides were fixed in 3% (wt͞vol) paraformalde- incubated at 37°C for indicated durations of time, fixed, and analyzed by hyde in PBS for 15 min. Samples were permeabilized in 0.3% confocal microscopy. (B) Naive and effector CD8ϩ T cells and either EL4 cells or (vol͞vol) Triton X-100 (Sigma) in PBS for 10 min, rinsed in PBS, P815 cells were conjugated for the indicated times. Two-color flow cytometric and blocked in DMEM containing 10% FCS for 5 min. All analysis was performed, and percent conjugates were determined by calcu- subsequent Ab incubations were performed in calcium͞ lating the ratio of double-positive cells to the total T cell population. Data are representative of three independent experiments. magnesium-free DPBS containing 2% FCS. Primary and sec- ondary Abs were applied sequentially for 60 min at room temperature and washed five times after each incubation with Results DPBS. After fluorochrome labeling, specimens were mounted in Naive and Effector CD8؉ T Cells Are Capable of Redistributing TCR and Mowiol 4-88 (Hoechst Celanese, Charlotte, NC), with 10% Forming Conjugates with APCs with Similar Kinetics. To study ho- 1,4-diazobicyclo[2.2.2]octane (Sigma) added as an antifading mogeneous populations of CD8ϩ T cells in defined differenti- agent.

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