| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
* Department of Microbiology and Immunology and
Christchurch School of Medicine, University of Otago, Dunedin, New Zealand; and
Division of Immunobiology, Institute for Genetics, University of Bonn, Bonn, Germany
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
Exosome release has been well documented in a variety of cell types, including B and T cell lines and in vitro-differentiated APCs, such as macrophages and bone marrow or monocyte-derived dendritic cells (DC)4 (1, 2, 3, 4). However, little work has been performed on exosome release from primary APCs directly isolated ex vivo.
Recent data from this laboratory failed to support the notion that APC-derived exosomes are present in the circulation of mice or humans under steady-state conditions (5). Instead, our study identified a pool of circulating MHC-I and MHC-II present in blood that was not exosome associated. However, these findings did not rule out the possibility that cellular activation (e.g., inflammatory signals) might lead to the release of exosomes or other soluble forms of MHC (sMHC).
In this study, we sought to determine whether exosomes, or other forms of sMHC were released from primary leukocytes. We analyzed the release of sMHC from leukocytes subjected to a variety of mitogenic signals, including TLR ligands, cytokines, and CD40 triggering. The highest levels of sMHC triggered in primary splenocytes were released from CD40- and IL-4-stimulated B cells and the sMHC was exclusively associated with exosomes. B cell-derived exosomes were enriched in both MHC-I and MHC-II and expressed Ig, as well as several other components of the BCR complex. IgD and IgM present at the plasma membrane of primary B cells was shuttled into the exosome pathway, demonstrating linkage of the surface BCR to the exosome pathway in B cells that receive T cell help. Together, these results demonstrate that exosome release is not constitutive activity of B cells, but is only induced following the provision of certain cellular activation signals.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
BALB/c mice obtained from The Jackson Laboratory were maintained and bred in specific pathogen-free conditions at the Otago University Hercus/Taieri Unit. Animal studies were approved by the regional Animal Ethics Committees. Peripheral blood was obtained by venesection with the approval of the University of Otago Human Ethics Committee. Mononuclear cells were isolated by Ficoll-Hypaque (Sigma-Aldrich) centrifugation as previously described (6).
Cells, mitogens, and cytokines
Splenocytes were isolated from BALB/c mice and cultured in RPMI 1640 plus 10% FCS (R10) supplemented with streptomycin, penicillin (Invitrogen), and 50 µM 2-ME in 6- or 24-well plates (Nunc) at 2 x 106/ml. Splenocytes were stimulated with the following mitogens: PHA (5 µg/ml), Con A (2.5 µg/ml), PMA (25 ng/ml), ionomycin (750 ng/ml), Leuconostoc spp. dextran sulfate (Mr 500 kDa; 15 µg/ml), Escherichia coli LPS (1 µg/ml), Staphylococcus aureus lipoteichoic acid (5 µg/ml), and staurosporine (250 nM). All reagents were sourced from Sigma-Aldrich. Surface Ig was cross-linked using divalent goat anti-mouse Ig (1 µg/ml; Southern Biotechnology Associates). IL-4 (BD Pharmingen) was used at 50 ng/ml. CD40 (5 µg/ml, clone FGK-45 (7), was provided by A. Rolink, Center for Biomedicine, Basel, Switzerland), CD3 (1 µg/ml, 145-2C11; American Type Culture Collection), CD28 (1 µg/ml, clone 37-51, provided by J. Allison, Memorial Sloan Kettering Cancer Center, New York, NY). Neutralizing mAb to CD 40 ligand (CD40L; clone MR1 (8); BD Biosciences), and anti-IL-4 (clone 11B11 (9), provided by J. Kirman, Malaghan Institute, Wellington, NZ) were included in splenocyte cultures at 10 µg/ml. mAbs were purified from hybridomas grown in a Celline1000 Bioreactor (Integra Biosciences) by ammonium sulfate precipitation or protein G affinity purification. Bone marrow-derived dendritic cells (BMDC) were generated exactly as previously described (5). Human B cells were transformed by EBV using the conditioned culture supernatant of the marmoset B95/8 B cell line. Due to a cross-reaction of the sHLA-DR ELISA with soluble marmoset MHC-II present in B95/8 culture supernatants, we first incubated 1 ml of 107 PBMC with 1 ml of B95/8 supernatant for 4 h in 24-well plates in the presence of 1 µg/ml cyclosporin A (Novartis). Cells were then resuspended and washed twice in warm medium (R10), resuspended in medium plus cyclosporin A, and returned to the original culture wells.
Ultracentrifugation and exosome isolation
Splenocytes, BMDC, or the A20 cell line were cultured for 3 days in RPMI 1640 plus particulate-depleted 10% FCS. We were unable to confirm the presence of exosomes in FCS (data not shown). However, because numerous protein aggregates were visualized in 100,000 x g FCS pellets by transmission electron microscopy, FCS was subjected to a 16 h, 100,000 x g step to clear debris. Culture supernatants were first spun at 400 x g for 5 min to deplete cells and then at 10,000 x g for 20 min to deplete residual cellular debris. Samples were then 0.2-µm filtered and exosomes were isolated by 100,000 x g ultracentrifugation for 1 h at 4°C. In some experiments, 0.1% sodium deoxycholate (DOC) was added at room temperature 1 h before gel filtration or ultracentrifugation to release lipid-bound MHC Ags from exosomes. Continuous sucrose gradient separation of exosomes was carried as previously described (1, 10).
Abs, CFSE staining, flow cytometric analysis, and sorting
The following Abs were used: TCRβ-PE (PE; clone H57-597), anti-I-Ad-bio (AMS-32.1), anti-I-Ed-bio (14-4-4), anti-I-A/E (2G9), CD5-PE (4H8E6), CD9-bio (KMC8), CD11c-bio (HL3), CD11b-bio (M1/70), CD19-PE (1D3), CD21-bio (7G6), CD23-bio (B3B4), CD27-bio (LG.3A10), CD29-bio (Ha2/5), CD40-PE (3/23), CD44-PE (IM7), CD45R/B220-bio (RA3-6B2), CD62L (MEL-14), CD80-PE (16-10A1), CD81-bio (Eat2), CD86-PE (GL1), 
4-PE (R1-2), β7-PE (M293), and 4β7-bio (DATK32), all from BD Pharmingen, except H2-Da,b,d,j,k,s,u-bio (M1/42; American Type Culture Collection; biotin-conjugated in house). Allophycocyanin-conjugated to streptavidin (BD Biosciences) was used to detect biotinylated Abs. To detect membrane-bound Ig, highly cross-adsorbed donkey anti-murine Ig conjugated to PE (Jackson ImmunoResearch Laboratories; Australian Laboratory Services) was resuspended at 10 µg/ml in PBS plus 50 µg/ml rat Ig and applied to cell or exosome suspensions. For flow cytometric analysis of exosomes, 10 µg of exosomes (isolated by 100,000 x g ultracentrifugation) in 100 µl of PBS was added to 25 µl of aldehyde sulfate microspheres (1.4 x 109/ml) and incubated overnight at 4°C. Microspheres were blocked by the addition of 0.5 ml of 0.5 mg/ml BSA/PBS for 15 min on ice and pelleted by centrifugation at 3185 x g at 4°C. Microspheres were quenched with 0.5 ml of 100 mM glycine (pH 7.4) for 30 min on ice, pelleted, and washed in 0.5 mg/ml BSA/PBS. Beads were finally resuspended at 3.5 x 108/ml in 0.5 mg/ml BSA/PBS. For labeling, 106 exosome-conjugated beads were incubated with 2.5 µg/ml biotinylated Abs or PE Abs for 15 min on ice. In some experiments, exosomes (75 µg in 100 µl) were captured directly onto 25 µl of beads that had been previously coated with anti-I-A/E (2G9) Ab (100 µl of 2G9 at 50 µg/ml coupled to 25 µl of beads overnight). To monitor cell division (see Fig. 1C), splenocytes (5 x 106/ml in PBS) were labeled with 1 µM CFSE (Molecular Probes and Invitrogen) for 6 min at room temperature, quenched in 5 ml of FCS, and washed twice in 10% FCS/PBS. Cells and exosomes were analyzed using a FACSCalibur (BD Biosciences). To isolate newly formed, marginal zone and follicular B cell subsets (11, 12), splenocytes were labeled with CD19-FITC, CD21-PE, and CD23-bio (detected with streptavidin-allophycocyanin) and subsets were sorted to >98% purity using a FACSAria (BD Biosciences).
|
To investigate the entry of surface Ig into the exosomal pathway, RBC lysed splenocytes (2.5 x 107/ml) were surface biotinylated with 1 mg/ml NHS-LC-Biotin (catalog no. 21335; Pierce and Global Science) for 10 min at 4°C. B cells were then washed twice in 100 mM glycine/PBS (pH 7.4) and once in PBS. Cells were then either lysed immediately ("fresh B cells") or resuspended in medium and stimulated with CD40/IL-4 for 3 days. A20 lymphoma cells were cultured in R10 medium alone. Exosomes (
100 µg from 108 splenocytes or A20 cells) were then isolated by ultracentrifugation, lysed in lysis buffer (0.5% Triton X-100, 0.25% CHAPS, 150 mM NaCl, 100 mM Tris-HCl (pH 8.0) with Complete protease inhibitor mixture; Roche) and Ig was immunoprecipitated from lysed exosomes (100 µg of protein in 100 µl lysis buffer) using 15 µl of 10x concentrated BioMag goat anti-mouse Ig beads (Polysciences). Beads were rotated overnight at 4°C and washed twice in lysis buffer, once in 0.25% Nonidet P-40/PBS, and once in PBS. Reduced and boiled precipitates (note, samples used for CD9, CD63, and CD81 analysis were not reduced or boiled) were then separated by 12% Bis-Tris-PAGE and transferred to nitrocellulose and membranes were blocked with 1% BSA. Biotinylated H and L chains of Ig were detected by streptavidin-HRP (1/1000 in 0.1% caseinate/PBS). For immunoblotting, we blocked membranes with 0.1% caseinate and used rabbit antisera recognizing the cytoplasmic domains of I-A and I-Aβ at 1/500 dilution, a gift from Dr. J. Villadangos, Walter and Eliza Hall Institute, Melbourne, Australia). The rabbit anti-calnexin, CD63 (LAMP3), and Tsg101 were all from Santa Cruz Biotechnology (Global Science). HRP-conjugated goat anti-mouse Ig whole molecule (A-4416) was from Sigma-Aldrich.
Soluble MHC and Ig ELISA
Soluble murine or human MHC-II was detected by double determinant ELISA, exactly as previously described (5). OD readings were quantified into unit values against a standard curve prepared from LPS-treated BMDC or from Raji human B cell lymphoma; 106 U/ml is equivalent to the signal obtained from a lysate of 106 cells/ml (5). The isotype of secreted soluble or exosomal Ig was determined using the mouse monoclonal isotyping reagents (ISO-2; Sigma-Aldrich).
Gel permeation
Gel permeation fast protein liquid chromatography (FPLC) was conducted using a Superose 12 HR 10/30 column (GE Healthcare Bio-Sciences) at a constant flow rate of 0.5 ml/min using PBS as the eluant. Culture supernatants were clarified by differential centrifugation, filtered (0.22 µm), and 0.5 ml injected onto the column. Fractions (0.7 ml) were collected and analyzed by soluble I-Ad ELISA (see above). The molecular mass of sMHC was estimated by comparison of the elution volumes with those of standard compounds of known mass (Blue Dextran, 2000 kDa; catalase, 240 kDa; human albumin, 65 kDa; deoxyribonuclease, 37 kDa; cytochrome c, 13 kDa; vitamin B12, 1.35 kDa) monitored by absorbance at 280 nm.
Transmission electron microscopy and immunolabeling
Exosomes isolated by 100,000 x g ultracentrifugation were applied to formvar-coated copper grids. The formvar surface was rendered hydrophilic before use by exposure to plasma discharge in an Edwards E306A vacuum coating system (Edwards High Vacuum). The exosomes were negatively stained with 1% phosphotungstic acid (pH 7.0) and the grids were visualized using a Philips CM100 BioTWIN transmission electron microscope (Philips/FEI) and images were captured using a MegaView lll digital camera (Olympus Soft Imaging Solutions).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
To determine the possible hematopoietic source of the circulating MHC identified in earlier studies (5, 13, 14, 15), we cultured splenocytes in medium alone or in the presence of stimulatory cytokines, mitogens, TLR ligands, or anti-Ig and measured sMHC release over several days by ELISA. Although several mitogens were able to induce the release of modest amounts of sMHC-II, the highest levels of sMHC were released by B cells stimulated with agonistic CD40 mAb plus IL-4 (Fig. 1A). Soluble MHC-II release displayed similar kinetics to the proliferation of B cells (Fig. 1B) and, in general, soluble MHC release correlated with the number of viable cells present in the mitogen-stimulated splenocyte suspensions (Fig. 1A). An exception was dextran plus LPS which induced extensive B cell proliferation as determined by CFSE analysis of cell division (Fig. 1C). However, compared with CD40/IL-4 stimulation, dextran plus LPS induced 53.9% ± 26.5 SD (n = 5) of B cell proliferation, but only 7.0% ± 7.3 SD (n = 4) of soluble MHC-II release. The release of exosomes was also triggered in splenocytes by CD3 and CD28 stimulation (Fig. 1D). A combination of neutralizing Ab against CD40L and IL-4 reproducibly inhibited of sMHC-II release, demonstrating that CD3/CD28 stimulation of exosome release is at least partly dependent on CD40L and IL-4 signaling (Fig. 1D).
We next confirmed the results for humans using PBMC. PBMC showed a similar release of sMHC-II in response to several mitogens, CD40 plus IL-4, or following infection of B cells with EBV (Fig. 1, E and F). A variety of TLR ligands, namely, LPS, lipoteichoic acid, N-palmitoyl-S-[2,3-bis(palmitoyloxy)propyl]cysteine, poly(I:C), peptidoglycan, flagellin, and CpG, failed to induce sMHC-II release in human or murine B cells (data not shown).
As a control for nonspecific release of sMHC by dying cells, splenocytes were rendered apoptotic by inclusion of staurosporine (Fig. 1, A and D). Despite the induction of extensive death in splenocytes, sMHC-II was not released from apoptotic cells.
Identification of APCs responsible for sMHC release
To more precisely determine the source of released sMHC-II, we sorted splenocytes into CD19+ B cells and TCRβ+ T cells (Fig. 2A). Although CD19+ B cells alone were capable of secreting sMHC-II in response to CD40/IL-4, admixing of CD19+ B cells with TCRβ+ T cells was required for release of sMHC-II during CD3/CD28 stimulation (Fig. 2B). To determine whether there existed a heterogeneity in the sMHC-II release by different B cell subsets present in spleen in response to CD40/IL-4 stimulation, we isolated newly formed, follicular and marginal zone B cells by FACS, as previously described (11, 12) (Fig. 2C). All B cell subsets were capable of releasing sMHC-II in response to CD40/IL-4 triggering. However in all four experiments, follicular B cells released 30–70% more sMHC-II than the other subsets (Fig. 2D).
|
Having established that B cells release sMHC in response to T cell-derived signals, we next sought to establish whether the sMHC was bound to membranes or vesicles, or was present as an oligomeric form, not associated with lipid-rich structures (5). Almost all sMHC pelleted at 100,000 x g and the observed sMHC fractionation into the pellet fraction was detergent sensitive (Fig. 3A). Gel filtration showed that almost all sMHC was present in the void volume of FPLC-fractionated, conditioned cell culture medium (Fig. 3B). DOC caused a shift in the molecular mass of sMHC-II from the void volume into fractions that corresponded to the expected molecular mass of dimeric MHC-II (100 kDa). To further confirm that the released sMHC was exosome associated, we subjected 100,000 x g isolated exosomes to sucrose gradient ultracentrifugation and detected the MHC-II β-chain in the fractions by Western blotting. As expected, MHC-II-positive vesicles migrated to densities between 1.13 and 1.15 (Fig. 3C), consistent with densities previously reported for exosomes released by immortalized B cell lines (1, 16). We also examined 100,000 x g pellets by transmission electron microscopy and confirmed the presence of typical features of exosomes such as cup-shaped morphology and 50- to120-nm diameter (Fig. 3D).
|
Exosomes released from primary B cells were analyzed by flow cytometry following coupling to aldehyde sulfate beads or by Western blotting. CD40/IL-4-elicited exosomes contained several surface markers in common with both freshly isolated (fresh) and CD40/IL-4-cultured B cells, including B220 (CD45R) and surface markers of the BCR complex (Fig. 4A and Table I). The absence of the endoplasmic reticulum molecule calnexin in exosome preparations demonstrated a lack of contamination of exosome preparations by cell debris containing endoplasmic reticulum membranes (Fig. 4B). Both the CD9 and CD81 tetraspanin markers associated with the BCR were found to be enriched on B cell-derived exosomes (Fig. 4); however, the tetraspanin CD63 (LAMP-3) was weakly detectable in B cell lysates, but absent from exosomes. Tsg101 has been reported to be expressed by exosomes released from the DC-like cell line D1 (17), but was only weakly expressed by either CD40/IL-4-stimulated B cells or exosomes. The CD21 BCR-associated protein was absent or only very weakly expressed, on exosomes, but was expressed by both fresh and CD40/IL-4- stimulated B cells (Fig. 4A). High levels of MHC-I and MHC-II were present on the surface of B cell-derived exosomes (Fig. 4). Analysis of costimulator molecule expression showed that CD80 was absent or only weakly expressed by B cell exosomes, but moderate levels of CD40 and CD86 were detected (Table I). CD3/CD28-elicited exosomes contained a similar composition of surface markers as compared with CD40/IL-4 exosomes; however, TCRβ was found to be expressed by CD3/CD28 exosomes, suggesting contamination with T cell-derived exosomes. However, when only MHC-II+ exosomes were analyzed from CD3/CD28-stimulated splenocytes using anti-MHC-II mAb-coated beads to bind exosomes, CD3/CD28 elicited exosomes were found to be devoid of TCRβ and CD5, markers expressed by the majority of splenic T cells (Table I). These results suggest that both T cell- and B cell-derived exosomes are secreted from CD3/CD28- stimulated splenocytes. Interestingly, Ig was detected on exosomes by flow cytometry and its expression was confirmed by Western blotting (Fig. 4B and Fig. 5B) and isotype-specific ELISA (Fig. 5A). Although CD40/IL-4-stimulated B cells secreted a range of soluble, Ig isotypes (IgA, IgG1, IgG2a/2b, and IgG3) as nonmembrane-bound forms into the supernatant, IgM and IgD were found to be the predominant isotypes enriched on the surface of exosomes (Fig. 5).
|
|
|
To determine whether surface Ig was capable of entering the exosome pathway, we surface biotinylated B cells before stimulation with CD40/IL-4. After 3 days, exosomes were isolated and Western blotting was used to confirm that biotinylated, plasma membrane-derived IgM and IgD H chains, as well as Ig L chains, were present in the exosome preparations (Fig. 5B). In contrast, the IgG H chain band was present at only low intensity. We next tested whether the trafficking of Ig into the exosome pathway was specific to the IgM and IgD isotypes or was determined by the density of surface expression of Ig isotypes on B cells. To distinguish between these two possibilities, we biotinylated the cell surface of the IgG2a-positive A20 B cell lymphoma line and precipitated Ig from released exosomes. As shown in Fig. 5B, A20 exosomes contained biotinylated IgG2a derived from pools at the cell surface. This suggests that the entry of Ig into the exosome pathway is primarily determined by the density of Ig present on the cell surface, rather than isotype-specific factors.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
The combination of CD40 and IL-4 has previously been shown to induce B cell proliferation (13, 14, 15). Continuous stimulation of B cells with CD40L and IL-4 leads to long-term proliferation of B cells, but CD40L/IL-4 B cell lines are not immortal and remain factor dependent (18). IL-4- and CD40-stimulated human B cells are potent APCs, with similar potential for immunotherapy as DC (19, 20). Our results now show that CD40/IL-4-treated primary B cells possess a similar exosome secretory mechanism to DC (21, 22).
Our results using neutralizing Abs clearly demonstrate that IL-4 and CD40L signaling are required for release of exosomes from B cells incubated with CD3/CD28-stimulated T cells. NKT cells express CD40L and produce large amounts of IL-4 when triggered via the TCR and thus were also candidates for stimulating exosome release in B cells. However, similar levels of exosomes were released from B cells within cd1d–/– splenocyte suspensions devoid of NKT cells (Ref. 23 and data not shown). This is consistent with our sorting experiments (Fig. 2A) demonstrating that conventional TCRβ+ T cells provide the main source of CD40L and IL-4 required for triggering exosome release in B cells. Interestingly, IL-4 triggers exosome release in mast cells, suggesting that IL-4 may be a common trigger for exosome release in distinct cell types (24).
Surface Ig (mostly IgD and IgM) on primary B cells and IgG2a present on a class-switched B cell line was targeted to exosomes. These findings are consistent with the recent data of Rialland et al. (25) showing that IgG was targeted to exosomes in a human lymphoma cell line. However, in their system, cross-linking of the BCR was required, a stimulus previously shown to induce endocytosis of surface Ig and redistribution into MHC-II-rich endosomal vesicles (26, 27). Similarly, naive B cells triggered with anti-Ig plus CD40 mAb were recently reported to traffic surface-derived MHC-II into the exosome pathway, although the transfer of Ig into the exosome pathway was not investigated in this report (16).
Our findings that CD40 and IL-4 signaling switches on a program of exosome synthesis in primary B cells suggest that exosome synthesis is a process tightly regulated by T cell-derived stimuli. Thus, exosomes would only be released once B cells have received help from CD4+ T cells. Due to a low precursor frequency of Ag-specific B cells early in the immune response, it is likely that only low numbers of exosomes could be released at early time points. However, T cell help induces extensive B cell proliferation: at day 3 in our experiments, CD40 and IL-4 had induced up to five cell divisions in the first 3 days, representing a 32-fold expansion in some subsets (Fig. 1C). Thus, amplification of exosome release by Ag-specific B cells might occur early in the humoral immune response. Likely targets of B cell-derived exosomes identified in our study are follicular DC. Exosomes were reported to decorate the surface of follicular DC in human tonsils in vivo and B cell line-derived exosomes were shown to bind to isolated follicular DC (28). Due to the inflamed nature of the tissue obtained from routine tonsillectomies, it seems likely that follicular DC acquired the exosomes from in vivo-activated B cells present in the tissue used as the source of follicular DC.
We have previously demonstrated that circulating MHC-I and -II in normal individuals is present as a simple dimeric form not associated with exosomes (5). Our present results do not clarify the source of circulating sMHC in serum of normal animals, but suggest that exosomal Ag may be released only during an immune response. Given the rapid removal of vesicles by the liver (29), we predict that released exosomes would display a very short half-life in the circulation, but may be of importance during cellular communication between closely apposed cells, such as T cells, B cells, and follicular DC or conventional DC in lymphatic organs. Importantly, our results show that predictions of exosome release from resting, primary cell types cannot always be determined from studies of their malignant or immortal counterparts, such as B cell lines. Furthermore, this work is in agreement with a recent report showing that resting B cells require activation for exosome release (16). Together, these findings emphasize that cell: cell signaling is an essential mechanism of exosome synthesis in primary cells.
|
Acknowledgments |
|---|
|
Disclosures |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
|
Footnotes |
|---|
1 This work was supported by the Marsden Research Fund (Fast Start to A.D.M.), the Otago Research Committee, Lotteries Health, the Bendigo Trust, the H.S. & J.C. Anderson Trust, and a Senior Smeaton Scholarship for Experimental Science (to P.A.M.). 
2 Current address: School of Chemistry, University of Sydney, Sydney, New South Wales, Australia.
3 Address correspondence and reprint requests to Dr. Alexander D. McLellan, Department of Microbiology and Immunology, University of Otago, Dunedin, 9001, New Zealand. E-mail address: alex.mclellan{at}stonebow.otago.ac.nz
4 Abbreviations used in this paper: DC, dendritic cell; MHC-I/II, MHC class I/II; sMHC, soluble MHC; DOC, deoxycholate; FPLC, fast protein liquid chromatography; CD40L, CD40 ligand; BMDC, bone marrow-derived DC.
Received for publication August 27, 2007. Accepted for publication April 15, 2008.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
heavy chain class switching. Immunity 5: 319-330. [Medline]This article has been cited by other articles:
|
|
 |
|
  Y. Qu, L. Ramachandra, S. Mohr, L. Franchi, C. V. Harding, G. Nunez, and G. R. Dubyak P2X7 Receptor-Stimulated Secretion of MHC Class II-Containing Exosomes Requires the ASC/NLRP3 Inflammasome but Is Independent of Caspase-1 J. Immunol., April 15, 2009; 182(8): 5052 - 5062. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and the number of viable B cells by trypan blue counting along with flow cytometric analysis of CD19+ B cells (
). B, Splenocytes were stimulated with CD40 and IL-4 and the number of viable B cells and soluble I-Ad release was determined by ELISA at the indicated times. C, Splenocytes were labeled with CFSE and cultured with the indicated reagents. After 3 days, proliferation of CD19+ B cells was determined by flow cytometry. Dot plots show total splenocytes and histogram shows CD19+ gated B cells. Thin line, Unstimulated cells; thick line, stimulated cells. D, sMHC-II secretion in CD3/CD28-stimulated cultures is dependent on CD40L and IL-4 signaling: splenocytes were stimulated with CD3 and CD28 mAb in the presence of the 5 µg/ml of the indicated neutralizing mAb for 3 days and soluble I-Ad levels were determined by ELISA. E, Human PBMC were stimulated with the indicated reagents for 5 days before determining the levels of soluble HLA-DR in the culture supernatants. F, Human PBMC were infected with EBV and soluble HLA-DR was determined by ELISA at the indicated times. Controls were PBMC cultured in medium alone (Nil) or in medium plus cyclosporin A (Cyc A). Representative of four experiments.
, and 
H chains (HC) and the L chains (LC) are shown. Representative of three experiments.