Costimulatory ligand 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
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Communicated by Douglas T. Fearon, University of Cambridge, Cambridge, United Kingdom, February 1, 2007 (received for review September 6, 2006)
Abstract
TNF family member CD70 is the ligand of CD27, a costimulatory receptor that shapes effector and memory T cell pools. Tight control of CD70 expression is required to prevent lethal immunodeficiency. By selective transcription, CD70 is largely confined to activated lymphocytes and dendritic cells (DC). We show here that, in addition, specific intracellular routing controls its plasma membrane deposition. In professional antigen-presenting cells, such as DC, CD70 is sorted to late endocytic vesicles, defined as MHC class II compartments (MIIC). In cells lacking the machinery for antigen presentation by MHC class II, CD70 travels by default to the plasma membrane. Introduction of class II transactivator sufficed to reroute CD70 to MIIC. Vesicular trafficking of CD70 and MHC class II is coordinately regulated by the microtubule-associated dynein motor complex. We show that when maturing DC make contact with T cells in a cognate fashion, newly synthesized CD70 is specifically delivered via MIIC to the immunological synapse. Therefore, we propose that routing of CD70 to MIIC serves to coordinate delivery of the T cell costimulatory signal in time and space with antigen recognition.
Induction of T cell immunity requires activation of naïve T lymphocytes by professional antigen presenting cells (APC). In this process, the T cell antigen receptor recognizes peptides bound to MHC molecules and triggers T cell proliferation and differentiation into effector cells. Size and quality of the effector pool are additionally determined by costimulatory signals. Collectively, signaling via the T cell antigen receptor and costimulatory receptors promotes cell cycle entry and activity, survival, and functional characteristics of primed T cells. Apart from CD28, various TNF receptor family members deliver costimulatory signals to the T cell when triggered by their respective ligands on APC or neighboring T cells (1). Among these, CD27 makes a key contribution to formation of effector and memory T cell populations by promoting the survival of primed T cells (2–4).
Timing of CD27 signaling is determined by availability of its ligand CD70, which is a homotrimeric, type II TNF-related transmembrane molecule (5–7). CD70 is a unique marker for immune activation. On antigenic challenge, dendritic cells (DC), as well as B and T lymphocytes at priming and effector sites, acquire CD70. Its expression is transient, paralleling the presence of antigen-specific CD8+ T cells (3, 8). CD70 gene transcription is induced by Toll-like receptor and antigen receptor signaling in DC and lymphocytes (8). Patterns of receptor and ligand expression suggest that CD27–CD70 interactions play a role throughout the priming, expansion, effector, and contraction phases of the T cell response (4). CD70 is also constitutively present on epithelial cells in the medulla of the thymus (8) and on certain nonhematopoietic cells in the intestine, which have antigen-presenting capacity (9).
Signaling via CD27 must be tightly regulated to avoid excessive effector T cell formation, which can have detrimental consequences. In CD70 transgenic mice, constitutive CD27 stimulation disturbs lymphocyte homeostasis and results in progressive and ultimately lethal combined T and B cell immunodeficiency (10, 11). Strict control of CD70 expression apparently guarantees the optimal level of CD27 signaling. In this study, we have examined intracellular transport of the CD70 protein. We observed that in cells containing the machinery for MHC-class-II-restricted antigen presentation, CD70 was sorted exactly like MHC class II molecules to the inner vesicles of late endocytic compartments [MHC class II compartments (MIIC)] (12, 13). On coculture of naïve antigen-specific T cells and recently activated DC, newly synthesized CD70 was specifically transported via MIIC to the immunological synapse formed between these cells. Therefore, we propose that routing of CD70 to MIIC serves to coordinate delivery of the costimulatory signal with the T cell antigen receptor stimulus at the time of priming.
Results
CD70 Has a Subcellular Distribution Similar to That of MHC Class II in DC.
To determine the subcellular distribution of endogenous CD70, we used primary bone-marrow-derived DC. In immature DC, CD70 protein was virtually undetectable by flow cytometry of intact and permeabilized cells (Fig. 1 A). Expression levels were also below detection by confocal laser scanning microscopy (CLSM) (Fig. 1 B). However, on LPS-induced maturation of DC, CD70 expression was greatly increased, as described in ref. 8. CD70 was distributed between a cell surface and an intracellular pool, as indicated by flow cytometry (Fig. 1 A) and directly visualized by CLSM (Fig. 1 B). MHC class II resided in intracellular vesicles in immature DC, whereas in mature DC, it was also found at the plasma membrane, consistent with its known distribution (14, 15). In 45% of the mature DC that were analyzed (n = 40), MHC class II was partially intracellularly retained. Interestingly, in those cells, localization of CD70 and MHC class II strongly overlapped, both in intracellular vesicles and at the cell surface (Fig. 1 B). In the remaining 55% of cells, MHC class II was found exclusively at the plasma membrane, whereas CD70 resided both at the plasma membrane and within the cell [supporting information (SI) Fig. 8].
Subcellular localization of CD70 in immature and mature DC. (A) DC were fixed, stained with fluorescent anti-CD70 mAb, permeabilized, and stained again with anti-CD70 or control mAb. Mean fluorescence intensity (MFI) as determined by flow cytometry is indicated for surface (not permeabilized) and total (permeabilized) staining. (B) DC (n = 40) were analyzed by CLSM after staining with anti-CD70 mAb followed by Alexa Fluor 488 goat anti-rat Ig (green) and rabbit anti-MHC class II followed by Alexa Fluor 568 goat anti-rabbit Ig (red). T, transmission image. (Scale bars: 5 μm.)
CD70 Resides in the MIIC.
To study intracellular transport of CD70, we used the human melanoma cell line Mel JuSo as a model system. This cell type has all of the components required for antigen presentation by MHC class II molecules (16, 17). Because Mel JuSo does not express CD70, it was introduced by retroviral transduction, as confirmed by Western blotting (Fig. 2 A). Analysis by flow cytometry revealed that a large proportion of CD70 molecules were retained within the cell, reminiscent of the situation in DC (Fig. 2 B). To define the intracellular compartment in which CD70 localized, cells were fixed and stained with antibodies to CD70 and late endosomal/lysosomal protein CD63 or MHC class II and examined by CLSM. CD70 colocalized strongly with endogenous CD63 and MHC class II (Fig. 2 C) (Pearson's coefficients 0.886 and 0.838, respectively). This finding indicated that the majority of intracellular CD70 resided in late endocytic structures defined as MIIC (12, 13). A small fraction of CD70 molecules was found in intracellular vesicles that did not contain CD63 or MHC class II and did not represent early endosomes as marked by EEA-1 (Fig. 2 C) (Pearson's coefficient 0.138).
Expression and subcellular localization of exogenous CD70 in Mel JuSo cells. (A) Immunoblotting of total lysates of mock-transduced Mel JuSo cells (MJ) and CD70-transduced Mel JuSo cells (MJ CD70) with anti-CD70 mAb. (B) Flow cytometric analysis of control and CD70-transduced cells after surface or total staining with anti-CD70 mAb. (C) CLSM of CD70-transduced cells after combined 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-mouse Ig (red), or rabbit anti-MHC class II followed by Alexa Fluor 568 goat anti-rabbit Ig (red), or anti-early endosomal antigen (EEA)-1 mAb followed by Alexa Fluor 568 goat anti-mouse Ig (red). (Scale bars: 15 μm.) Pixel analysis indicates colocalization. Zooms represent ×5 enlargements of the framed areas in the merged images.
Dissection of the Subcellular Localization of CD70.
To determine the distribution of CD70 over distinct intracellular compartments, we fractionated CD70-expressing Mel JuSo cells by density gradient electrophoresis (DGE). This technique, which is particularly well established for Mel JuSo cells, separates membrane vesicles by their surface charge. These are then identified by certain markers (17, 18). Late endosomes/lysosomes, early endosomes, Golgi, endoplasmic reticulum, and plasma membrane are positioned in distinct fractions, as indicated in Fig. 3 A and B. Proteins were precipitated from the DGE fractions and analyzed by Western blotting (Fig. 3 B). The majority of MHC class I was detected in the plasma membrane and endoplasmic reticulum fractions. A small pool was also present in the late endosomal/lysosomal fractions because MHC class I complexes undergo endocytosis and are directed to lysosomes for degradation or loading for cross-presentation (19). MHC class II β-chain was detected in both plasma membrane and late endosomal/lysosomal fractions, consistent with its known subcellular distribution (17). CD70 closely followed the distribution of MHC class II throughout fractions representing plasma membrane and MIIC, consistent with the CLSM data. CD70 protein was also detected in fractions 28–32, which include Golgi and early endosomes (Fig. 3 B). Because early endosomes hardly labeled for CD70 (Fig. 2 C), CD70 is probably also present in the (trans-) Golgi.
Localization of CD70 in Mel JuSo cells by subcellular fractionation and EM. (A) CD70-transduced cells were fractionated by DGE. Protein concentration and β-hexosaminidase activity were detected in the odd-numbered fractions. (B) Total protein precipitated from the even-numbered fractions was analyzed by Western blotting with antibodies to CD70 and MHC class I and II. Localization of plasma membrane (PM), endoplasmic reticulum (ER), early endosomes (EE), Golgi (G), and late endosomes (LE)/lysosomes (L) are indicated. (C) EM of CD70-transduced cells. (Left) A cell fragment with organelles indicated (M, mitochondrion). Frame indicates the area with two MIIC shown at higher magnification (Right). CD70 is decorated with small (10 nm) gold particles and MHC class II with large (15 nm) gold particles. The small arrow indicates an internal vesicle and the large arrow indicates the limiting membrane of MIIC. For quantification of the relative distribution of CD70 within MIIC, a total of 400 small gold particles were counted and their distribution was determined: 87% localized to internal vesicles; 13% localized to the limiting membrane. (Scale bar: 100 nm.)
To study the distribution of CD70 within MIIC, we performed immuno-EM. Ultrathin sections of CD70-expressing Mel JuSo cells were labeled with antibodies to CD70 and MHC class II and decorated with small and large gold particles, respectively (Fig. 3 C). CD70 was clearly enriched in MIIC but also was visible at the plasma membrane and in vesicles proximal to Golgi stacks, as shown in Fig. 3 C. Because CD70 was also seen in Golgi stacks (not shown), these vesicles probably represent the trans-Golgi network. Examination of MIIC with its characteristic multivesicular morphology (13) confirmed that CD70 and MHC class II molecules colocalized in this compartment. Quantification indicated that within MIIC, 87% of CD70 molecules localized to the internal vesicles whereas 13% resided at the limiting membrane, indicating that CD70 has a distribution in these compartments similar to that of MHC class II (18).
The Class II Transactivator (CIITA) Master Regulator of Transcription Controls Transport of CD70 to the MIIC.
MIIC is a compartment with features of late endosomes and lysosomes, where antigenic peptides are loaded onto MHC class II molecules for presentation (12, 13). To study which route CD70 travels in cells that lack the attributes for antigen presentation by MHC class II, HeLa cervix carcinoma cells were transfected with CD70 and its intracellular localization was examined by CLSM. Apart from occasional weak perinuclear staining, CD70 was not found within these cells and colocalization with the late endosomal/lysosomal marker CD63 was absent. Rather, CD70 was efficiently transported by default to the plasma membrane (Fig. 4 A). We next tested whether introduction of the MHC class II antigen-presentation pathway in these cells could influence CD70 trafficking. Because CIITA functions as a transcriptional master regulator of genes involved in antigen presentation (20), HeLa cells stably transduced with CIITA were used for this purpose. HeLa cells acquired MHC class II protein on CIITA expression (compare Fig. 4 A and B), confirming the functional activity of CIITA. Moreover, in these cells, CD70 was routed toward compartments that contained CD63 as well as MHC class II, classifying them as MIIC (Fig. 4 B). These results indicate that trafficking of CD70 toward CD63-marked late endocytic structures is actively regulated and occurs only in cells equipped with the machinery for MHC-class-II-mediated antigen presentation.
Localization of exogenous CD70 in HeLa cells with and without MHC class II antigen-presenting system, imposed by CIITA expression. Cells were transfected with CD70 cDNA, labeled to detect CD70 and CD63 or MHC class II as outlined for Fig. 2, and examined by CLSM. (A) Control cells. (B) Cells stably expressing CIITA. (Scale bars: 10 μm.)
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 same mechanism that regulates intracellular transport of MHC class II. Late endosomal/lysosomal compartments (including MIIC) move along microtubules in a bidirectional manner because of the alternating activities of a plus-end-directed kinesin motor and a minus-end-directed dynein–dynactin motor (21). The active Rab7 GTPase associates with late endosomes/lysosomes and recruits the dynein–dynactin motor via the Rab-interacting lysosomal protein (RILP) effector (22). RILP therefore directs movement of Rab7-containing vesicles toward the minus-end of microtubules. Its overexpression leads to clustering of these vesicles around the microtubule-organizing center. The ΔN mutant of RILP (C-terminal half of RILP) also binds to active Rab7, but fails to recruit the motor protein complex. As a result, expression of ΔN RILP disperses Rab7-associated vesicles throughout the cytoplasm due to the remaining kinesin motor activity (22). Mel JuSo cells stably expressing a GFP-Rab7 chimera and CD70 were used to monitor trafficking of CD70-containing vesicles. When these cells were transfected to overexpress wild-type RILP, the majority of intracellular CD70 colocalized with Rab7 and RILP in the perinuclear region (Fig. 5 Upper). This localization is consistent with RILP enforcing unidirectional dynein motor-driven transport of CD70-containing vesicles, resulting in their clustering around the microtubule-organizing center, as defined by immuno-EM for tubulin in a previous study (22). Conversely, CD70-containing vesicles were dispersed by expression of the ΔN RILP mutant (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 previously for MHC class II (21).
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) or the ΔN 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 by CLSM. Cells transfected with RILP are outlined with solid white lines, untransfected cells with dashed white lines. (Scale bars: 15 μm.) The zooms are ×5 enlargements of the framed areas in the merged images.
CD70 Is Specifically Recruited Toward the Immunological Synapse.
Because CD70 followed the same intracellular route as MHC class II, we surmised that both molecules might have the same destination when an APC contacts a T cell. During antigen-specific interaction of a mature DC with a naïve T cell, MHC class II molecules are directed toward the contact region between these two cells, the so-called immunological synapse (23). To test whether CD70 follows the same route, we studied CD70 distribution in DC–T cell conjugates. To this end, primary DC were allowed to endocytose intact ovalbumin (OVA) protein for 4 h, which suffices for display of OVA peptide-loaded MHC class II molecules on the cell surface (24). LPS was added simultaneously, to induce DC maturation and expression of CD70. Next, DC were allowed to interact with naïve OVA-specific CD4+ T cells (OT-II) and after 30, 60, and 120 min, subcellular distribution of CD70 and MHC class II was analyzed by CLSM. At 30 min, polarization of DC toward the T cell interaction site was apparent and CD70 colocalized with MHC class II in internal vesicles (Fig. 6). In time, both CD70 and MHC class II were progressively recruited toward the contact site (60 min) which was followed by dispersion of both molecules over the plasma membrane of DC at the synapse area (120 min) (Fig. 6).
Recruitment of CD70 toward the T cell contact site. Splenic DC incubated with OVA and LPS for 4 h were allowed to form conjugates with 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 class II followed by Alexa Fluor 568 goat anti-rabbit Ig (red). Images are representative of 60–70% of conjugates (n = 30) analyzed at the indicated time points. T, transmission image. Data are representative of three independent experiments. (Scale bars: 5 μm.)
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. 7 A). 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. 7 B). 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.
CD70 localization in the immunological synapse. DC–T cell conjugates were generated as outlined for Fig. 6. (A) Cells were fixed at 120 min, stained with anti-ICAM-1 mAb followed by Alexa Fluor 568 goat anti-rat Ig (red), fixed again, and stained with FITC-conjugated anti-CD70 mAb (green). (Scale bar: 5 μm.) (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 Fig. 6 were processed for three-dimensional imaging. The lines reveal the position of the x–z and y–z stack as represented at the bottom or right side, respectively. All images are representative of 60–70% of conjugates analyzed (n = 30). T, transmission image. Data are representative of three independent experiments.
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 stores as well as at the plasma membrane, which suggested the existence of a mechanism regulating delivery of CD70 to the cell surface. Transport of newly synthesized transmembrane proteins from the endoplasmic reticulum to the cell surface occurs by default. Their delivery from the Golgi to certain intracellular compartments such as endosomes and lysosomes is accomplished by sorting the proteins into membrane microdomains, which pinch off as vesicles (25). Sorting occurs via specific sequences in the protein, such as tyrosine- and leucine-based motifs (26). We have demonstrated here that CD70, which contains no classical sorting motifs, is nevertheless specifically transported to late endocytic structures in cells that have features of professional APC. In cells that lack such characteristics, CD70 follows the default route to the plasma membrane.
MHC class II also does not contain any sorting motifs. Its transport to late endocytic structures is fully dependent on association with the invariant chain (Ii), a chaperone that has two leucine-based motifs (27–29). Transport of CD70 most likely also depends on a chaperone(s) with adequate sorting motifs. Such potential chaperones are apparently targets of CIITA, because its expression in HeLa cells directed CD70 toward MIIC. Cell surface expression of other TNF family members, i.e., CD40 ligand, FasL, and TRAIL, is apparently also regulated posttranslationally (30–33). The case of FasL is reminiscent of what we have found for CD70: it is sorted to secretory lysosomes of immune cells, but travels to the plasma membrane in HeLa cells (31). However, the proline-rich motif that delivers FasL to lysosomes (33) is absent from CD70 and other TNF family members.
DC exist in immature or mature states, providing either tolerogenic or immunogenic contexts for naïve T cells (34). Immature DC are highly endocytic and express relatively few MHC class II molecules at the cell surface, which are primarily loaded with self peptides (35). On DC maturation, cell surface expression of antigenic peptide-loaded MHC class II and of various costimulatory ligands is elevated (8, 14). MHC class II molecules are recruited toward the plasma membrane from MIIC (14, 15, 36–38) and are retained there because of decreased endocytosis (35). On cognate contact between a maturing DC and an antigen-specific T cell, the DC is polarized and positions its nucleus opposite the contact point (immunological synapse) (23). Microtubules are directed toward the synapse with consequent trafficking of MIIC in this direction (37). It has been observed that in maturing DC, multivesicular MIIC dramatically alter their morphology, changing from vacuolar to long tubular organelles that extend along microtubules toward the plasma membrane. MHC class II relocalizes from the internal membranes of multivesicular MIIC to the limiting membrane of the tubular organelles. Vesicles forming at the tip of the tubules are suggested to carry MHC class II to the plasma membrane (36–38). Like MHC class II, CD70 may relocalize from internal vesicles in MIIC to the limiting membrane of the tubules and thus be incorporated in the plasma membrane at the synapse. Another possible mechanism of delivery to the plasma membrane involves transport of intact multivesicular MIIC toward the synapse along the microtubular cytoskeleton. On exocytosis of such MIIC, the internal vesicles containing CD70 and MHC class II may fuse with the DC membrane (39) and thus be in the correct orientation to bind their respective receptors at the T cell membrane. Lack of intrinsic lysosomal targeting sequences in CD70 presumably allows for more stable surface expression as compared with other MIIC-resident proteins. Such proteins are rapidly internalized by their MIIC-targeting signals (40).
CD27–CD70 interactions are essential for efficient T cell priming (2–4). During an in vivo response, communication between recently activated DC and naïve T cells takes place in lymphoid organs. We have demonstrated here that newly synthesized CD70 in such DC travels to the immunological synapse, where it can contribute to T cell priming. In mature DC, MIIC can travel to the plasma membrane also in absence of T cell contact (14) and deposit CD70 and MHC class II there. Indeed, mature DC contain a cell surface resident as well as an intracellular pool of both molecules (Fig. 1). Whether these two pools of CD70 have distinct roles in evoking or sustaining T cell responses remains to be established. In summary, we have uncovered that in professional APC, CD70 deviates from the default pathway and is specifically delivered to MIIC. The resulting regulated trafficking of CD70 to the cell surface via the MHC class II pathway is expected to have important consequences for T cell priming.
Materials and Methods
Cell Culture.
Human melanoma cell line Mel JuSo, human cervix carcinoma cell line HeLa, and their stably transduced or transfected derivatives were grown in Iscove's modified Dulbecco's medium (GIBCO/BRL, Paisley, U.K.) supplemented with 8% FCS, penicillin, and streptomycin. Mice were bred and used for experiments in accordance with institutional and national ethical guidelines. Bone marrow DC of C57BL/6 WT mice were obtained as described in ref. 41. In short, bone marrow was flushed out of femurs and tibiae from 6- to 7-week-old mice and cultured for 6 days. Cells were grown in Iscove's modified Dulbecco's medium supplemented with 10% FCS, penicillin, streptomycin, 50 μM 2-mercaptoethanol, and 5% X63-GM-CSF supernatant (42). DC were matured by 16-h incubation with 1 μg/ml LPS of Escherichia coli serotype O55:B5 (Sigma–Aldrich, St. Louis, MO).
Constructs and Gene Transfer.
The retroviral LZRS-mCD70 vector was generated by ligation of the mCD70 cDNA (6) into LZRS-MS-IRES-Zeo/pBR, a derivative of LZRS-pBMN-lacZ (43) by using BamHI and NotI sites. Other constructs were described previously: pcDNA3-RILP and pcDNA3-ΔN RILP (C-terminal amino acids 199–401 of RILP) (22) and pREP4-CIITA (44). Mel JuSo cell lines stably expressing mCD70 were generated by retroviral transduction. Retroviral supernatants were obtained by transfection of LZRS-mCD70 into φNX-Ampho packaging cells (43). Mel JuSo stably expressing a GFP-Rab7 chimera (22) were transduced with LZRS-mCD70 and subsequently transiently transfected to express WT- or ΔN-RILP, by using linear polyethylenimine (Polysciences, Warrington, PA).
Antibodies.
The following antibodies were used: (i) affinity-purified, FITC- or Alexa Fluor 647-conjugated rat anti-mCD70 clone FR70 (eBioscience, San Diego, CA) and rat anti-mouse ICAM-1 clone YN-1; (ii) mouse mAb: anti-human EEA-1 (Transduction Laboratories, Lexington, KY), anti-human CD63 (NKI-C3) (45), anti-human MHC class I heavy chain (HC-10) (13), and anti-HLA-DR (1B5) (12); and (iii) polyclonal rabbit sera: anti-DR (12), anti-I-Abβ (JV2) (46), anti-human RILP (22). Fluorescent secondary antibodies, conjugated to Alexa Fluor 488 (green), Alexa Fluor 568 (red), and Alexa Fluor 633 (far red, shown as blue in Fig. 5), were from Molecular Probes (Leiden, The Netherlands). Antibody cross-reactivity was excluded because nonspecific combinations of primary and secondary antibodies did not show any staining (data not shown). Secondary antibodies conjugated to horseradish peroxidase were from DakoCytomation (Carpinteria, CA). For all stainings of DC, cells were preincubated with Fc Block (mAb to CD16/CD32, 2.4G2; BD Biosciences, Mountain View, CA).
CLSM.
Mel JuSo cells were allowed to attach to glass coverslips for 24 h. DC were allowed to attach for 1 h to coverslips coated with poly(l-lysine) (Sigma–Aldrich). Cells were washed in PBS containing 1 mM MgCl2 and 1 mM CaCl2, fixed for 10 min with 3.7% paraformaldehyde in PBS, and permeabilized with 0.1% Triton X-100 in PBS for 3 min. Nonspecific binding sites were blocked for 30 min by using 1% BSA in PBS. Incubations were performed with antibodies diluted in blocking buffer for 45 min, after which coverslips were washed and incubated for 30 min with the appropriate secondary antibodies diluted in blocking buffer. Coverslips were washed and mounted in Vectashield (Vector Laboratories, Burlingame, CA) and viewed under a Leica TCS NT confocal laser-scanning microscope (Leica Microsystems, Heidelberg, Germany). Images with two fluorochromes were taken by simultaneous scanning, and images with three fluorochromes by sequential scanning. Degree of colocalization was tested by using Manders' coefficients plugin (by T. Collins and W. Rasband) of Wright Cell Imaging Facility ImageJ software. Pearson's coefficient was used to express degree of colocalization between the two channels. Serial CLSM images of DC–T cell conjugates as shown in SI Movie 1 were processed for z-stack animation with ImageJ software.
Immuno-EM.
Mel JuSo cells were processed for cryosectioning as described in ref. 47. For further procedures, see SI Materials and Methods.
Flow Cytometry.
Mel JuSo cells or DC were washed in PBS and fixed for 10 min with 3.7% paraformaldehyde in PBS. Nonspecific binding sites were blocked for 30 min by using 1% BSA in PBS. Incubations were performed with Alexa 647-conjugated anti-CD70 mAb diluted in staining buffer (PBS/1% BSA/0.01% sodium azide) for 30 min on ice. For intracellular staining, cells were permeabilized with 0.1% Triton X-100 for 3 min at room temperature and incubated with antibody diluted in staining buffer for 30 min on ice. Cells were analyzed by using FACSCalibur (Becton Dickinson, Mountain View, CA) and FlowJo software (Tree Star, Ashland, OR).
Western Blotting.
Mel JuSo cell lysates and DGE fractions were immunoblotted according to standard procedures. For details, see the SI Materials and Methods.
Subcellular Fractionation by DGE.
Samples were prepared and applied to the DGE device as described in ref. 18. For details, see the SI Materials and Methods.
Formation of the Immunological Synapse.
DC were isolated from the spleen by collagenase D (Roche, Mannheim, Germany) and digestion (2.5 mg/ml in Hanks's balanced salt solution) was 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 (Miltenyi Biotec, Bergisch Gladbach, Germany) and suspended in RPMI medium 1640 supplemented with 10% FCS, penicillin, streptomycin, and 50 μM 2-mercaptoethanol. DC were incubated for 4 h at 37°C with 40 μM OVA (Sigma–Aldrich) and 1 μg/ml LPS and washed in medium. T cells from OT-II transgenic mice that express a T cell antigen receptor specific for an OVA/MHC class II complex (48) were isolated from spleens by using the Mouse T Lymphocyte Enrichment Set (BD Biosciences, San Jose, CA). OT-II T cells were added to DC at a 4:1 ratio in medium in a 96-well plate and centrifuged for 1 min at 200 × g. After incubation at 37°C, cells were allowed to adhere briefly to poly(l-lysine)-coated (Sigma–Aldrich) coverslips, fixed, and processed for CLSM analysis.
Acknowledgments
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 discussions; and T. N. M. Schumacher, R. Arens, and K. Schepers for critical comments on the manuscript. This work was supported by grants from the Dutch Cancer Society (to J.B. and J.N.).
Footnotes
- ‡To whom correspondence should be addressed at: Division of Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands. E-mail: j.borst{at}nki.nl
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Author contributions: A.M.K., J.N., and J.B. designed research; A.M.K., T.A.G., E.A.M.V., 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; and A.M.K., J.N., and J.B. wrote the paper.
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The authors declare no conflict of interest.
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This article contains supporting information online at www.pnas.org/cgi/content/full/0700946104/DC1.
- Abbreviations:
- APC,
- antigen-presenting cells;
- CLSM,
- confocal laser scanning microscopy;
- CIITA,
- class II transactivator;
- DC,
- dendritic cells;
- DGE,
- density gradient electrophoresis;
- ICAM-1,
- intracellular adhesion molecule 1;
- mCD70,
- murine CD70;
- MIIC,
- MHC class II compartment;
- OVA,
- ovalbumin;
- RILP,
- Rab-interacting lysosomal protein.
- © 2007 by The National Academy of Sciences of the USA