Costimulatory CD70 is delivered to the immunological synapse by shared intracellular trafficking with MHC class II molecules

Anna M. Keller*, Tom A. Groothuis†, Elise A. M. Veraar*, Marije Marsman†, Lucas Maillette de Buy Wenniger*, Hans Janssen†, Jacques Neefjes†, and Jannie Borst*‡

Divisions of *Immunology and †Tumor Biology, The Netherlands Cancer Institute, 1066 CX, Amsterdam, The Netherlands

Communicated by Douglas T. Fearon, University of Cambridge, Cambridge, United Kingdom, February 1, 2007 (received for review September 6, 2006) TNF family member CD70 is the ligand of CD27, a costimulatory Signaling via CD27 must be tightly regulated to avoid excessive receptor that shapes effector and memory T cell pools. Tight effector T cell formation, which can have detrimental conse- control of CD70 expression is required to prevent lethal immuno- quences. In CD70 transgenic mice, constitutive CD27 stimula- deficiency. By selective transcription, CD70 is largely confined to tion disturbs lymphocyte homeostasis and results in progressive activated lymphocytes and dendritic cells (DC). We show here that, and ultimately lethal combined T and B cell immunodeficiency in addition, specific intracellular routing controls its plasma mem- (10, 11). Strict control of CD70 expression apparently guarantees brane deposition. In professional antigen-presenting cells, such as the optimal level of CD27 signaling. In this study, we have DC, CD70 is sorted to late endocytic vesicles, defined as MHC class examined intracellular of the CD70 . We ob- II compartments (MIIC). In cells lacking the machinery for antigen served that in cells containing the machinery for MHC-class- presentation by MHC class II, CD70 travels by default to the plasma II-restricted antigen presentation, CD70 was sorted exactly like membrane. Introduction of class II transactivator sufficed to re- MHC class II molecules to the inner vesicles of late endocytic route CD70 to MIIC. Vesicular trafficking of CD70 and MHC class II compartments [MHC class II compartments (MIIC)] (12, 13). is coordinately regulated by the microtubule-associated dynein On coculture of naı¨ve antigen-specific T cells and recently motor complex. We show that when maturing DC make contact activated DC, newly synthesized CD70 was specifically trans- IMMUNOLOGY with T cells in a cognate fashion, newly synthesized CD70 is ported via MIIC to the immunological synapse formed between specifically delivered via MIIC to the immunological synapse. these cells. Therefore, we propose that routing of CD70 to MIIC Therefore, we propose that routing of CD70 to MIIC serves to serves to coordinate delivery of the costimulatory signal with the coordinate delivery of the T cell costimulatory signal in time and T cell antigen receptor stimulus at the time of priming. space with antigen recognition. Results CD70 Has a Subcellular Distribution Similar to That of MHC Class II in costimulation ͉ dendritic cell ͉ intracellular transport DC. To determine the subcellular distribution of endogenous CD70, we used primary bone-marrow-derived DC. In immature nduction of T cell immunity requires activation of naı¨ve T DC, CD70 protein was virtually undetectable by flow cytometry Ilymphocytes by professional antigen presenting cells (APC). In of intact and permeabilized cells (Fig. 1A). Expression levels this process, the T cell antigen receptor recognizes peptides were also below detection by confocal laser scanning microscopy bound to MHC molecules and triggers T cell proliferation and (CLSM) (Fig. 1B). However, on LPS-induced maturation of DC, differentiation into effector cells. Size and quality of the effector CD70 expression was greatly increased, as described in ref. 8. pool are additionally determined by costimulatory signals. Col- CD70 was distributed between a cell surface and an intracellular lectively, signaling via the T cell antigen receptor and costimu- pool, as indicated by flow cytometry (Fig. 1A) and directly latory receptors promotes cell cycle entry and activity, survival, visualized by CLSM (Fig. 1B). MHC class II resided in intra- and functional characteristics of primed T cells. Apart from cellular vesicles in immature DC, whereas in mature DC, it was CD28, various TNF receptor family members deliver costimu- also found at the plasma membrane, consistent with its known latory signals to the T cell when triggered by their respective distribution (14, 15). In 45% of the mature DC that were ligands on APC or neighboring T cells (1). Among these, CD27 analyzed (n ϭ 40), MHC class II was partially intracellularly makes a key contribution to formation of effector and memory retained. Interestingly, in those cells, localization of CD70 and T cell populations by promoting the survival of primed T cells MHC class II strongly overlapped, both in intracellular vesicles (2–4). and at the cell surface (Fig. 1B). In the remaining 55% of cells, Timing of CD27 signaling is determined by availability of its MHC class II was found exclusively at the plasma membrane, ligand CD70, which is a homotrimeric, type II TNF-related transmembrane molecule (5–7). CD70 is a unique marker for Author contributions: A.M.K., J.N., and J.B. designed research; A.M.K., T.A.G., E.A.M.V., immune activation. On antigenic challenge, dendritic cells (DC), M.M., L.M.d.B.W., and H.J. performed research; A.M.K., T.A.G., J.N., and J.B. analyzed data; as well as B and T lymphocytes at priming and effector sites, and A.M.K., J.N., and J.B. wrote the paper. acquire CD70. Its expression is transient, paralleling the pres- The authors declare no conflict of interest. ϩ ence of antigen-specific CD8 T cells (3, 8). CD70 gene tran- Abbreviations: APC, antigen-presenting cells; CLSM, confocal laser scanning microscopy; scription is induced by Toll-like receptor and antigen receptor CIITA, class II transactivator; DC, dendritic cells; DGE, density gradient electrophoresis; signaling in DC and lymphocytes (8). Patterns of receptor and ICAM-1, intracellular adhesion molecule 1; mCD70, murine CD70; MIIC, MHC class II com- ligand expression suggest that CD27–CD70 interactions play a partment; OVA, ovalbumin; RILP, Rab-interacting lysosomal protein. ‡To whom correspondence should be addressed at: Division of Immunology, The Nether- role throughout the priming, expansion, effector, and contrac- lands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands. E-mail: tion phases of the T cell response (4). CD70 is also constitutively [email protected]. present on epithelial cells in the medulla of the thymus (8) and This article contains supporting information online at www.pnas.org/cgi/content/full/ on certain nonhematopoietic cells in the intestine, which have 0700946104/DC1. antigen-presenting capacity (9). © 2007 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0700946104 PNAS ͉ April 3, 2007 ͉ vol. 104 ͉ no. 14 ͉ 5989–5994 Downloaded by guest on September 24, 2021 Fig. 2. Expression and subcellular localization of exogenous CD70 in Mel Fig. 1. Subcellular localization of CD70 in immature and mature DC. (A)DC JuSo cells. (A) Immunoblotting of total lysates of mock-transduced Mel JuSo were fixed, stained with fluorescent anti-CD70 mAb, permeabilized, and cells (MJ) and CD70-transduced Mel JuSo cells (MJ CD70) with anti-CD70 mAb. stained again with anti-CD70 or control mAb. Mean fluorescence intensity (B) Flow cytometric analysis of control and CD70-transduced cells after surface (MFI) as determined by flow cytometry is indicated for surface (not perme- or total staining with anti-CD70 mAb. (C) CLSM of CD70-transduced cells after abilized) and total (permeabilized) staining. (B)DC(n ϭ 40) were analyzed by combined staining with anti-CD70 mAb followed by Alexa Fluor 488 goat CLSM after staining with anti-CD70 mAb followed by Alexa Fluor 488 goat anti-rat Ig (green) and anti-CD63 mAb followed by Alexa Fluor 568 goat anti-rat Ig (green) and rabbit anti-MHC class II followed by Alexa Fluor 568 anti-mouse Ig (red), or rabbit anti-MHC class II followed by Alexa Fluor 568 goat anti-rabbit Ig (red). T, transmission image. (Scale bars: 5 ␮m.) goat anti-rabbit Ig (red), or anti-early endosomal antigen (EEA)-1 mAb fol- lowed by Alexa Fluor 568 goat anti-mouse Ig (red). (Scale bars: 15 ␮m.) Pixel analysis indicates colocalization. Zooms represent ϫ5 enlargements of the whereas CD70 resided both at the plasma membrane and within framed areas in the merged images. the cell [supporting information (SI) Fig. 8]. are positioned in distinct fractions, as indicated in Fig. 3 A and CD70 Resides in the MIIC. To study intracellular transport of CD70, B. were precipitated from the DGE fractions and we used the human melanoma cell line Mel JuSo as a model analyzed by Western blotting (Fig. 3B). The majority of MHC system. This cell type has all of the components required for class I was detected in the plasma membrane and endoplasmic antigen presentation by MHC class II molecules (16, 17). Be- reticulum fractions. A small pool was also present in the late cause Mel JuSo does not express CD70, it was introduced by endosomal/lysosomal fractions because MHC class I complexes retroviral transduction, as confirmed by Western blotting (Fig. undergo endocytosis and are directed to lysosomes for degra- 2A). Analysis by flow cytometry revealed that a large proportion dation or loading for cross-presentation (19). MHC class II of CD70 molecules were retained within the cell, reminiscent of ␤-chain was detected in both plasma membrane and late endo- the situation in DC (Fig. 2B). To define the intracellular somal/lysosomal fractions, consistent with its known subcellular compartment in which CD70 localized, cells were fixed and distribution (17). CD70 closely followed the distribution of MHC stained with antibodies to CD70 and late endosomal/lysosomal class II throughout fractions representing plasma membrane and protein CD63 or MHC class II and examined by CLSM. CD70 MIIC, consistent with the CLSM data. CD70 protein was also colocalized strongly with endogenous CD63 and MHC class II detected in fractions 28–32, which include Golgi and early (Fig. 2C) (Pearson’s coefficients 0.886 and 0.838, respectively). endosomes (Fig. 3B). Because early endosomes hardly labeled This finding indicated that the majority of intracellular CD70 for CD70 (Fig. 2C), CD70 is probably also present in the (trans-) resided in late endocytic structures defined as MIIC (12, 13). A Golgi. small fraction of CD70 molecules was found in intracellular To study the distribution of CD70 within MIIC, we performed vesicles that did not contain CD63 or MHC class II and did not immuno-EM. Ultrathin sections of CD70-expressing Mel JuSo represent early endosomes as marked by EEA-1 (Fig. 2C) cells were labeled with antibodies to CD70 and MHC class II and (Pearson’s coefficient 0.138). decorated with small and large gold particles, respectively (Fig. 3C). CD70 was clearly enriched in MIIC but also was visible at Dissection of the Subcellular Localization of CD70. To determine the the plasma membrane and in vesicles proximal to Golgi stacks, distribution of CD70 over distinct intracellular compartments, as shown in Fig. 3C. Because CD70 was also seen in Golgi stacks we fractionated CD70-expressing Mel JuSo cells by density (not shown), these vesicles probably represent the trans-Golgi gradient electrophoresis (DGE). This technique, which is par- network. Examination of MIIC with its characteristic multive- ticularly well established for Mel JuSo cells, separates membrane sicular morphology (13) confirmed that CD70 and MHC class II vesicles by their surface charge. These are then identified by molecules colocalized in this compartment. Quantification in- certain markers (17, 18). Late endosomes/lysosomes, early en- dicated that within MIIC, 87% of CD70 molecules localized to dosomes, Golgi, endoplasmic reticulum, and plasma membrane the internal vesicles whereas 13% resided at the limiting mem-

5990 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0700946104 Keller et al. Downloaded by guest on September 24, 2021 IMMUNOLOGY

Fig. 3. Localization of CD70 in Mel JuSo cells by subcellular fractionation and Fig. 4. Localization of exogenous CD70 in HeLa cells with and without MHC EM. (A) CD70-transduced cells were fractionated by DGE. Protein concentra- class II antigen-presenting system, imposed by CIITA expression. Cells were tion and ␤-hexosaminidase activity were detected in the odd-numbered frac- transfected with CD70 cDNA, labeled to detect CD70 and CD63 or MHC class tions. (B) Total protein precipitated from the even-numbered fractions was II as outlined for Fig. 2, and examined by CLSM. (A) Control cells. (B) Cells stably analyzed by Western blotting with antibodies to CD70 and MHC class I and II. expressing CIITA. (Scale bars: 10 ␮m.) Localization of plasma membrane (PM), endoplasmic reticulum (ER), early endosomes (EE), Golgi (G), and late endosomes (LE)/lysosomes (L) are indi- cated. (C) EM of CD70-transduced cells. (Left) A cell fragment with organelles indicated (M, mitochondrion). Frame indicates the area with two MIIC shown activity of CIITA. Moreover, in these cells, CD70 was routed at higher magnification (Right). CD70 is decorated with small (10 nm) gold toward compartments that contained CD63 as well as MHC class particles and MHC class II with large (15 nm) gold particles. The small arrow II, classifying them as MIIC (Fig. 4B). These results indicate that indicates an internal vesicle and the large arrow indicates the limiting mem- trafficking of CD70 toward CD63-marked late endocytic struc- brane of MIIC. For quantification of the relative distribution of CD70 within tures is actively regulated and occurs only in cells equipped with MIIC, a total of 400 small gold particles were counted and their distribution the machinery for MHC-class-II-mediated antigen presentation. was determined: 87% localized to internal vesicles; 13% localized to the limiting membrane. (Scale bar: 100 nm.) Vesicular Trafficking of CD70 Is Regulated by the Microtubule- Associated Dynein Motor Complex. We next investigated whether trafficking of CD70 after delivery to MIIC is controlled by the brane, indicating that CD70 has a distribution in these compart- same mechanism that regulates intracellular transport of MHC ments similar to that of MHC class II (18). class II. Late endosomal/lysosomal compartments (including MIIC) move along microtubules in a bidirectional manner The Class II Transactivator (CIITA) Master Regulator of Transcription because of the alternating activities of a plus-end-directed Controls Transport of CD70 to the MIIC. MIIC is a compartment with kinesin motor and a minus-end-directed dynein–dynactin motor features of late endosomes and lysosomes, where antigenic (21). The active Rab7 GTPase associates with late endosomes/ peptides are loaded onto MHC class II molecules for presenta- lysosomes and recruits the dynein–dynactin motor via the Rab- tion (12, 13). To study which route CD70 travels in cells that lack interacting lysosomal protein (RILP) effector (22). RILP there- the attributes for antigen presentation by MHC class II, HeLa fore directs movement of Rab7-containing vesicles toward the cervix carcinoma cells were transfected with CD70 and its minus-end of microtubules. Its overexpression leads to clustering intracellular localization was examined by CLSM. Apart from of these vesicles around the microtubule-organizing center. The occasional weak perinuclear staining, CD70 was not found ⌬N mutant of RILP (C-terminal half of RILP) also binds to within these cells and colocalization with the late endosomal/ active Rab7, but fails to recruit the motor protein complex. As lysosomal marker CD63 was absent. Rather, CD70 was effi- a result, expression of ⌬N RILP disperses Rab7-associated ciently transported by default to the plasma membrane (Fig. 4A). vesicles throughout the cytoplasm due to the remaining kinesin We next tested whether introduction of the MHC class II motor activity (22). Mel JuSo cells stably expressing a GFP-Rab7 antigen-presentation pathway in these cells could influence chimera and CD70 were used to monitor trafficking of CD70- CD70 trafficking. Because CIITA functions as a transcriptional containing vesicles. When these cells were transfected to over- master regulator of genes involved in antigen presentation (20), express wild-type RILP, the majority of intracellular CD70 HeLa cells stably transduced with CIITA were used for this colocalized with Rab7 and RILP in the perinuclear region (Fig. purpose. HeLa cells acquired MHC class II protein on CIITA 5 Upper). This localization is consistent with RILP enforcing expression (compare Fig. 4 A and B), confirming the functional unidirectional dynein motor-driven transport of CD70-

Keller et al. PNAS ͉ April 3, 2007 ͉ vol. 104 ͉ no. 14 ͉ 5991 Downloaded by guest on September 24, 2021 Fig. 5. CD70 traffics along microtubules with the aid of the dynein motor complex. Mel JuSo cells stably expressing a Rab7-GFP chimera (green) and murine CD70 (mCD70) were transfected with cDNA encoding wild-type RILP (Upper)orthe⌬N RILP mutant (Lower). Cells were double labeled with anti-CD70 mAb followed by Alexa Fluor 568 goat anti-rat Ig (red) and rabbit anti-RILP followed by Alexa Fluor 633 goat anti-rabbit Ig (blue) and analyzed Fig. 7. CD70 localization in the immunological synapse. DC–T cell conjugates by CLSM. Cells transfected with RILP are outlined with solid white lines, were generated as outlined for Fig. 6. (A) Cells were fixed at 120 min, stained ␮ untransfected cells with dashed white lines. (Scale bars: 15 m.) The zooms are with anti-ICAM-1 mAb followed by Alexa Fluor 568 goat anti-rat Ig (red), fixed ϫ 5 enlargements of the framed areas in the merged images. again, and stained with FITC-conjugated anti-CD70 mAb (green). (Scale bar: 5 ␮m.) (B) Serial CLSM images of DC–T cell conjugates fixed at 120 min and labeled with antibodies to CD70 (green) and MHC class II (red) as outlined in containing vesicles, resulting in their clustering around the Fig. 6 were processed for three-dimensional imaging. The lines reveal the microtubule-organizing center, as defined by immuno-EM for position of the x–z and y–z stack as represented at the bottom or right side, tubulin in a previous study (22). Conversely, CD70-containing respectively. All images are representative of 60–70% of conjugates analyzed ϭ vesicles were dispersed by expression of the ⌬N RILP mutant (n 30). T, transmission image. Data are representative of three independent experiments. (Fig. 5 Lower). We conclude that trafficking of the intracellular pool of CD70 molecules takes place along microtubules and is governed by dynein–dynactin motor protein activity, as shown were allowed to endocytose intact ovalbumin (OVA) protein for previously for MHC class II (21). 4 h, which suffices for display of OVA peptide-loaded MHC class II molecules on the cell surface (24). LPS was added simulta- CD70 Is Specifically Recruited Toward the Immunological Synapse. neously, to induce DC maturation and expression of CD70. Next, Because CD70 followed the same intracellular route as MHC DC were allowed to interact with naïve OVA-specific CD4ϩ T class II, we surmised that both molecules might have the same cells (OT-II) and after 30, 60, and 120 min, subcellular distri- destination when an APC contacts a T cell. During antigen- bution of CD70 and MHC class II was analyzed by CLSM. At 30 specific interaction of a mature DC with a naïve T cell, MHC min, polarization of DC toward the T cell interaction site was class II molecules are directed toward the contact region be- apparent and CD70 colocalized with MHC class II in internal tween these two cells, the so-called immunological synapse (23). vesicles (Fig. 6). In time, both CD70 and MHC class II were To test whether CD70 follows the same route, we studied CD70 progressively recruited toward the contact site (60 min) which distribution in DC–T cell conjugates. To this end, primary DC was followed by dispersion of both molecules over the plasma membrane of DC at the synapse area (120 min) (Fig. 6). Staining of DC–T cell conjugates for the synapse marker intracellular adhesion molecule 1 (ICAM-1) (23) at 120 min confirmed that CD70 was transported specifically toward the immunological synapse (Fig. 7A). Detailed analysis of the T cell–DC interaction site by three-dimensional reconstruction of CLSM images highlighted the synchronized recruitment of CD70 and MHC class II toward the immunological synapse (SI Movie 1). A cross-section of this image revealed that in an apparently multifocal synapse between DC and T cells, CD70 and MHC class II were segregated into discrete microdomains (Fig. 7B). Collectively, these data indicate that in maturing DC, newly synthesized CD70 is specifically directed to MIIC. On cognate T cell contact, this allows for synchronized transport of CD70 and MHC class II via the polarized microtubular network toward the immunological synapse. Discussion We studied intracellular transport of CD70 to elucidate the mechanism by which costimulation is delivered to T cells during priming. In mature DC, CD70 protein resided in intracellular Fig. 6. Recruitment of CD70 toward the T cell contact site. Splenic DC stores as well as at the plasma membrane, which suggested the incubated with OVA and LPS for 4 h were allowed to form conjugates with existence of a mechanism regulating delivery of CD70 to the cell naïve OT-II T cells. At indicated time points, cells were labeled with anti-CD70 mAb followed by Alexa Fluor 488 goat anti-rat Ig (green) and rabbit anti-MHC surface. Transport of newly synthesized transmembrane proteins class II followed by Alexa Fluor 568 goat anti-rabbit Ig (red). Images are from the endoplasmic reticulum to the cell surface occurs by representative of 60–70% of conjugates (n ϭ 30) analyzed at the indicated default. Their delivery from the Golgi to certain intracellular time points. T, transmission image. Data are representative of three indepen- compartments such as endosomes and lysosomes is accom- dent experiments. (Scale bars: 5 ␮m.) plished by sorting the proteins into membrane microdomains,

5992 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0700946104 Keller et al. Downloaded by guest on September 24, 2021 which pinch off as vesicles (25). Sorting occurs via specific default pathway and is specifically delivered to MIIC. The sequences in the protein, such as tyrosine- and leucine-based resulting regulated trafficking of CD70 to the cell surface via the motifs (26). We have demonstrated here that CD70, which MHC class II pathway is expected to have important conse- contains no classical sorting motifs, is nevertheless specifically quences for T cell priming. transported to late endocytic structures in cells that have features of professional APC. In cells that lack such characteristics, CD70 Materials and Methods follows the default route to the plasma membrane. Cell Culture. Human melanoma cell line Mel JuSo, human cervix MHC class II also does not contain any sorting motifs. Its carcinoma cell line HeLa, and their stably transduced or transfected transport to late endocytic structures is fully dependent on associ- derivatives were grown in Iscove’s modified Dulbecco’s medium ation with the invariant chain (Ii), a chaperone that has two (GIBCO/BRL, Paisley, U.K.) supplemented with 8% FCS, peni- leucine-based motifs (27–29). Transport of CD70 most likely also cillin, and streptomycin. Mice were bred and used for experiments depends on a chaperone(s) with adequate sorting motifs. Such in accordance with institutional and national ethical guidelines. potential chaperones are apparently targets of CIITA, because its Bone marrow DC of C57BL/6 WT mice were obtained as described expression in HeLa cells directed CD70 toward MIIC. Cell surface in ref. 41. In short, bone marrow was flushed out of femurs and expression of other TNF family members, i.e., CD40 ligand, FasL, tibiae from 6- to 7-week-old mice and cultured for 6 days. Cells were and , is apparently also regulated posttranslationally (30– grown in Iscove’s modified Dulbecco’s medium supplemented with 33). The case of FasL is reminiscent of what we have found for 10% FCS, penicillin, streptomycin, 50 ␮M 2-mercaptoethanol, and CD70: it is sorted to secretory lysosomes of immune cells, but 5% X63-GM-CSF supernatant (42). DC were matured by 16- travels to the plasma membrane in HeLa cells (31). However, the h incubation with 1 ␮g/ml LPS of Escherichia coli serotype O55:B5 proline-rich motif that delivers FasL to lysosomes (33) is absent (Sigma–Aldrich, St. Louis, MO). from CD70 and other TNF family members. DC exist in immature or mature states, providing either Constructs and Gene Transfer. The retroviral LZRS-mCD70 vector tolerogenic or immunogenic contexts for naı¨ve T cells (34). was generated by ligation of the mCD70 cDNA (6) into LZRS- Immature DC are highly endocytic and express relatively few MS-IRES-Zeo/pBR, a derivative of LZRS-pBMN-lacZ (43) by MHC class II molecules at the cell surface, which are primarily using BamHI and NotI sites. Other constructs were described loaded with self peptides (35). On DC maturation, cell surface previously: pcDNA3-RILP and pcDNA3-⌬N RILP (C-terminal expression of antigenic peptide-loaded MHC class II and of amino acids 199–401 of RILP) (22) and pREP4-CIITA (44).

various costimulatory ligands is elevated (8, 14). MHC class II Mel JuSo cell lines stably expressing mCD70 were generated by IMMUNOLOGY molecules are recruited toward the plasma membrane from retroviral transduction. Retroviral supernatants were obtained MIIC (14, 15, 36–38) and are retained there because of de- by transfection of LZRS-mCD70 into ␾NX-Ampho packaging creased endocytosis (35). On cognate contact between a matur- cells (43). Mel JuSo stably expressing a GFP-Rab7 chimera (22) ing DC and an antigen-specific T cell, the DC is polarized and were transduced with LZRS-mCD70 and subsequently tran- positions its nucleus opposite the contact point (immunological siently transfected to express WT- or ⌬N-RILP, by using linear synapse) (23). Microtubules are directed toward the synapse with polyethylenimine (Polysciences, Warrington, PA). consequent trafficking of MIIC in this direction (37). It has been observed that in maturing DC, multivesicular MIIC dramatically Antibodies. The following antibodies were used: (i) affinity- alter their morphology, changing from vacuolar to long tubular purified, FITC- or Alexa Fluor 647-conjugated rat anti-mCD70 organelles that extend along microtubules toward the plasma clone FR70 (eBioscience, San Diego, CA) and rat anti-mouse membrane. MHC class II relocalizes from the internal mem- ICAM-1 clone YN-1; (ii) mouse mAb: anti-human EEA-1 branes of multivesicular MIIC to the limiting membrane of the (Transduction Laboratories, Lexington, KY), anti-human CD63 tubular organelles. Vesicles forming at the tip of the tubules are (NKI-C3) (45), anti-human MHC class I heavy chain (HC-10) suggested to carry MHC class II to the plasma membrane (13), and anti-HLA-DR (1B5) (12); and (iii) polyclonal rabbit (36–38). Like MHC class II, CD70 may relocalize from internal sera: anti-DR (12), anti-I-Ab␤ (JV2) (46), anti-human RILP vesicles in MIIC to the limiting membrane of the tubules and thus (22). Fluorescent secondary antibodies, conjugated to Alexa be incorporated in the plasma membrane at the synapse. An- Fluor 488 (green), Alexa Fluor 568 (red), and Alexa Fluor 633 other possible mechanism of delivery to the plasma membrane (far red, shown as blue in Fig. 5), were from Molecular Probes involves transport of intact multivesicular MIIC toward the (Leiden, The Netherlands). Antibody cross-reactivity was ex- synapse along the microtubular cytoskeleton. On exocytosis of cluded because nonspecific combinations of primary and sec- such MIIC, the internal vesicles containing CD70 and MHC class ondary antibodies did not show any staining (data not shown). II may fuse with the DC membrane (39) and thus be in the Secondary antibodies conjugated to horseradish peroxidase correct orientation to bind their respective receptors at the T cell were from DakoCytomation (Carpinteria, CA). For all stainings membrane. Lack of intrinsic lysosomal targeting sequences in of DC, cells were preincubated with Fc Block (mAb to CD16/ CD70 presumably allows for more stable surface expression as CD32, 2.4G2; BD Biosciences, Mountain View, CA). compared with other MIIC-resident proteins. Such proteins are rapidly internalized by their MIIC-targeting signals (40). CLSM. Mel JuSo cells were allowed to attach to glass coverslips for CD27–CD70 interactions are essential for efficient T cell 24 h. DC were allowed to attach for1htocoverslips coated with priming (2–4). During an in vivo response, communication poly(L-lysine) (Sigma–Aldrich). Cells were washed in PBS contain- between recently activated DC and naı¨ve T cells takes place in ing 1 mM MgCl2 and 1 mM CaCl2, fixed for 10 min with 3.7% lymphoid organs. We have demonstrated here that newly syn- paraformaldehyde in PBS, and permeabilized with 0.1% Triton thesized CD70 in such DC travels to the immunological synapse, X-100 in PBS for 3 min. Nonspecific binding sites were blocked for where it can contribute to T cell priming. In mature DC, MIIC 30 min by using 1% BSA in PBS. Incubations were performed with can travel to the plasma membrane also in absence of T cell antibodies diluted in blocking buffer for 45 min, after which contact (14) and deposit CD70 and MHC class II there. Indeed, coverslips were washed and incubated for 30 min with the appro- mature DC contain a cell surface resident as well as an intra- priate secondary antibodies diluted in blocking buffer. Coverslips cellular pool of both molecules (Fig. 1). Whether these two pools were washed and mounted in VECTASHIELD (Vector Labora- of CD70 have distinct roles in evoking or sustaining T cell tories, Burlingame, CA) and viewed under a Leica TCS NT responses remains to be established. In summary, we have confocal laser-scanning microscope (Leica Microsystems, Heidel- uncovered that in professional APC, CD70 deviates from the berg, Germany). Images with two fluorochromes were taken by

Keller et al. PNAS ͉ April 3, 2007 ͉ vol. 104 ͉ no. 14 ͉ 5993 Downloaded by guest on September 24, 2021 simultaneous scanning, and images with three fluorochromes by Subcellular Fractionation by DGE. Samples were prepared and ap- sequential scanning. Degree of colocalization was tested by using plied to the DGE device as described in ref. 18. For details, see the Manders’ coefficients plugin (by T. Collins and W. Rasband) of SI Materials and Methods. Wright Cell Imaging Facility ImageJ software. Pearson’s coefficient was used to express degree of colocalization between the two Formation of the Immunological Synapse. DC were isolated from channels. Serial CLSM images of DC–T cell conjugates as shown in the spleen by collagenase D (Roche, Mannheim, Germany) and SI Movie 1 were processed for z-stack animation with ImageJ digestion (2.5 mg/ml in Hanks’s balanced salt solution) was software. followed by separation on an OptiPrep (Axis-Shield, Oslo, Norway) gradient. DC were purified by magnetic separation on autoMACS using anti-CD11c (N418)-labeled MACS-beads Immuno-EM. Mel JuSo cells were processed for cryosectioning as (Miltenyi Biotec, Bergisch Gladbach, Germany) and suspended described in ref. 47. For further procedures, see SI Materials and in RPMI medium 1640 supplemented with 10% FCS, penicillin, Methods. streptomycin, and 50 ␮M 2-mercaptoethanol. DC were incu- bated for4hat37°C with 40 ␮M OVA (Sigma–Aldrich) and 1 Flow Cytometry. Mel JuSo cells or DC were washed in PBS and ␮g/ml LPS and washed in medium. T cells from OT-II transgenic fixed for 10 min with 3.7% paraformaldehyde in PBS. Nonspe- mice that express a T cell antigen receptor specific for an cific binding sites were blocked for 30 min by using 1% BSA in OVA/MHC class II complex (48) were isolated from spleens by PBS. Incubations were performed with Alexa 647-conjugated using the Mouse T Lymphocyte Enrichment Set (BD Bio- anti-CD70 mAb diluted in staining buffer (PBS/1% BSA/0.01% sciences, San Jose, CA). OT-II T cells were added to DC at a 4:1 sodium azide) for 30 min on ice. For intracellular staining, cells ratio in medium in a 96-well plate and centrifuged for 1 min at were permeabilized with 0.1% Triton X-100 for 3 min at room 200 ϫ g. After incubation at 37°C, cells were allowed to adhere temperature and incubated with antibody diluted in staining briefly to poly(L-lysine)-coated (Sigma–Aldrich) coverslips, buffer for 30 min on ice. Cells were analyzed by using FACS- fixed, and processed for CLSM analysis. Calibur (Becton Dickinson, Mountain View, CA) and FlowJo software (Tree Star, Ashland, OR). We thank P. J. van den Elsen for HeLa cells expressing CIITA; E. Roos for anti-ICAM-1 antibody; L. Oomen, L. Brocks, D. Verwoerd, and J. Blitz for expert technical assistance; C. Kuijl and W. Zwart for discus- Western Blotting. Mel JuSo cell lysates and DGE fractions were sions; and T. N. M. Schumacher, R. Arens, and K. Schepers for critical immunoblotted according to standard procedures. For details, comments on the manuscript. This work was supported by grants from see the SI Materials and Methods. the Dutch Cancer Society (to J.B. and J.N.).

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