Activated T cells recruit exosomes secreted by dendritic cells via LFA-1

During cognate T cell–dendritic cell (DC) interactions, several proteins, including major histocompatibility complex (MHC) and costimulatory molecules, are transferred from DCs to T cells (reviewed by Davis¹ ). Transfer of MHC class II/peptide complexes from DCs to CD4⁺ T cells occurs in vivo and can play an important role in down-regulation of immune responses via T cell to T-cell presentation of antigen.^2,–4  How these proteins are transferred and what determines their secretion and targeting are unclear. It has been proposed that DC exosomes, approximately 100-nm vesicles formed within multivesicular bodies,⁵  could be unique vectors for intercellular communication. Depending on the activation status of the DCs, they contain a narrow spectrum of molecules involved in immune responses and signal transduction.^6,–8  Here we determine how T cells bind DC exosomes secreted in the extracellular milieu during cognate interactions.

The splenic C57BL/6 immature DC-line D1⁹  and D1 transduced with I-A^kβ¹⁰  were cultured as described.¹¹  Bovine exosomes were removed from fetal calf serum by ultracentrifugation. The p53-specific CD4⁺ T-cell clone (p53 T cells), generated in a C57BL/6 p53^−/− mouse and provided by Prof C. Melief (Leiden University Medical Center, Leiden, The Netherlands),¹²  recognizes the murine p53 77-96 peptide (p53p). A novel CD4⁺ T-cell line recognizing the ovalbumin 323-339 peptide (OVAp) was generated from draining lymph nodes of OVAp/CFA-immunized C57BL/6 mice. T cells were cyclically restimulated with peptide-pulsed irradiated splenocytes and expanded with IL-2. Experiments were performed at standard tissue culture conditions¹⁰  and were approved by the institutional ethical animal committees at Utrecht University (Utrecht, The Netherlands).

DC–T-cell coculture supernatant was cleared from cells and potentially contaminating membranes by sequential centrifugation steps up to 30 minutes at 10 000g.¹³  T cells (10⁶) were incubated with 3 mL of this exosome-containing supernatant for 5 or 24 hours on plates coated or not with 10 μg/mL anti-CD3 and 5 μg/mL anti-CD28 antibody (provided by Dr L. Boon, Bioceros, Utrecht, The Netherlands). Where indicated, T cells were incubated with exosomes in the presence of anti–leukocyte function-associated antigen-1 (LFA-1; M17/4), provided by Prof C. Figdor (Nijmegen, The Netherlands), and/or manganese chloride (Sigma-Aldrich, St Louis, MO).

Cells were harvested and immunolabeled for 30 minutes on ice in phosphate-buffered saline/1% bovine serum albumin. Phycoerythrin and allophycocyanin-conjugated anti–I-A^b (M5.114), anti-CD9 (KMC8), anti-CD3 (145-2C11), anti-CD69 (H1.2F3), anti–interferon-γ (IFN-γ; XMG1.2), anti–I-A^k (Ox6), and isotype control antibodies were from BD PharMingen (San Diego, CA). Where indicated, T cells were labeled with 0.5 μM carboxyfluorescein succinimidyl ester (CFSE; Invitrogen, Carlsbad, CA) for 15 minutes at 37°C or with 2 μM PKH26-GL (Sigma-Aldrich) for 5 minutes at room temperature. Paramagnetic epoxy beads (Dynal Biotech, Lake Success, NY) were coupled to anti-MHC class II (M5/114) or anti–ICAM-1 antibodies (BD PharMingen) according to the manufacturer's instructions. Exosomes from 1.0 mL 10 000g culture supernatant were adsorbed onto 6 × 10⁴ antibody-coated beads during 24 hours at room temperature by end-over-end rotation and immunolabeled for I-A^b or CD9. Beads and cells were analyzed by flow cytometry using a FACSCalibur and CellQuest (BD Biosciences, San Jose, CA) or FCS Express software.

Consistent with reported findings on MHC class II acquisition by murine T cells,^3,14  we found that mouse T cells, which do not synthesize MHC class II,¹⁵  acquired MHC class II during cognate DC–T-cell cocultures (Figure 1A). CD9, a tetraspanin enriched on DC exosomes,⁶  was also acquired by cocultured T cells (Figure 1A), suggesting that acquisition of these molecules was mediated by DC-derived exosomes. Previously, it was shown that DC exosomes associated to bystander DCs.^16,–18  We analyzed whether MHC class II released by DCs during cognate interaction with T cells could be acquired by both T cells and bystander DCs. In a 3-cell culture system, containing nontransduced DC expressing only endogenous I-A^b, DCs also expressing transduced I-A^kβ,¹¹  p53-specific T cells, and p53p, we observed transfer of I-A^kβ to both nontransduced DC and T cells (Figure 1B). These data imply that, besides DC, T cells are targets for DC exosomes. Indeed, our recent electron microscopy data confirm that MHC class II and CD9 containing exosomes are present on the T-cell plasma membrane and that the majority of these exosomes neither fuse with the plasma membrane nor are efficiently internalized (S.I.B., E.N.M.N-‘t.H., Guillaume van Niel, Maaike S. Pols, Toine ten Broeke, Marjolein Lauwen, Ferry Ossendorp, Cornelis J.M. Melief, Graça Raposo, Richard Wubbolts, M.H.M.W., and W.S., manuscript submitted, August 14, 2008).

Here we determined the requirements for T-cell recruitment of DC exosomes secreted during cognate T-cell–DC interactions. To uncouple exosome secretion from recruitment, T cells were incubated with exosome containing 10 000g supernatants¹³  prepared from DC–T-cell cocultures. In this supernatant, MHC class II was exclusively associated to exosomes, as confirmed by its characteristic buoyant density (1.14 g/mL) in a sucrose density gradient^19,20  (Figure 1C), and by electron microscopy analysis (S.I.B., E.N.M.N-‘tH., Guillaume van Niel, Maaike S. Pols, Toine ten Broeke, Marjolein Lauwen, Ferry Ossendorp, Cornelis J.M. Melief, Graça Raposo, Richard Wubbolts, M.H.M.W., and W.S., manuscript submitted, August 14, 2008). We found that only during activation, and not in the resting state, T cells efficiently recruited these exosomes (Figure 2A). This suggests that DC exosomes secreted in the extracellular milieu during cognate T-cell–DC interactions can be efficiently targeted to activated T cells present in that microenvironment.

Next, we asked which T-cell proteins were responsible for recruiting DC exosomes. We first tested whether T-cell receptor (TCR)–mediated recognition of MHC class II/peptide complexes on exosomes was essential for T-cell binding. We found that exosomes produced during cognate cultures of DCs with ovalbumin-specific T cells or p53-specific T cells could be recruited efficiently by both p53-specific and ovalbumin-specific T cells (Figure 2B). Although we do not exclude a role for specific TCR-MHC class II/peptide interactions in intercellular protein transfer during cognate T-cell–DC interactions, we conclude that DC exosomes secreted in the extracellular milieu are efficiently transferred to activated T cells irrespective of their TCR specificity. During T-cell activation, TCR signaling induces a transient conformational change in LFA-1, therewith highly increasing the affinity for ICAM-1 (reviewed by Kinashi²¹ ). Because only activated T cells recruited DC exosomes, binding to activated T cells could involve high-affinity LFA-1/ICAM-1 interactions. Exosomes secreted by lipopolysaccharide-activated DCs were shown to contain ICAM-1, which was required for exosome binding to bystander DCs.¹⁷  We found that MHC class II and CD9 bearing exosomes were efficiently captured from DC–T-cell coculture 10 000g supernatant by both anti–MHC class II– and anti–ICAM-1–coated beads (Figure 2C). Thus, DC exosomes produced during cognate interaction with T cells contained ICAM-1. To test whether LFA-1 on T cells mediated exosome binding, DC exosome recruitment assays were performed in the presence of an LFA-1 blocking antibody. Indeed, anti–LFA-1 antibody interfered dose-dependently with the recruitment of DC exosomes by activated T cells without affecting T-cell activation (Figure 2D), suggesting that LFA-1 on activated T cells is involved in DC exosome binding. Furthermore, these data support the exosomal origin of transferred MHC class II and argue against a major contribution of soluble nonexosomal MHC class II.²²  If active LFA-1 was the only determining factor in DC exosome binding, induction of a high-affinity conformation of LFA-1 on resting T cells should allow these cells to recruit exosomes. Because the divalent cation Mn²⁺ induces and stabilizes the high-affinity state of LFA-1,²³  we measured the exosome binding capacity of resting T cells in the presence of MnCl₂. Indeed, MnCl₂ rendered resting T cells capable of binding DC exosomes in a dose-dependent fashion (Figure 2E). The specificity of this process was indicated by complete blocking of exosome binding to MnCl₂-treated resting T cells with anti–LFA-1 (Figure 2E). Thus, the affinity status of LFA-1 determined the efficacy of exosome acquisition by recipient T cells.

Previously, DC exosomes were shown to associate with bystander DCs, and it was proposed that the exosomal transfer of MHC-peptide complexes to recipient antigen presenting cells could be involved in spreading of antigen and amplification of immune responses.¹⁶  We here show that also T cells recruit MHC class II–bearing DC exosomes. Presentation of acquired MHC class II/peptide complexes by T cells can inhibit other activated/memory T cells.⁴  Our finding that only activated T cells efficiently recruited DC exosomes secreted during cognate T-cell–DC interactions could be a way to tightly regulate T cell–mediated down-regulation of immune responses.

We thank Drs R. Wubbolts, T. ten Broeke, M. Nolte, and L. Taams for helpful discussions and critical reading of the manuscript; Prof C. Melief (Leiden University Medical Center, Leiden, The Netherlands) for the kind gift of the p53 T-cell clone; and Dr L. Boon (Bioceros, Utrecht, The Netherlands) and Prof C. Figdor (Nijmegen, The Netherlands) for the kind gifts of antibodies.

Contribution: E.N.M.N.-‘t.H. designed and performed research and wrote the paper; S.I.B. performed research; S.M.A. contributed a vital new T-cell line; W.S. designed research; and M.H.M.W. designed research and wrote the paper.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Marca H. M. Wauben, Faculty of Veterinary Medicine, Department of Biochemistry & Cell Biology, Utrecht University, Yalelaan 2, 3584CM Utrecht, The Netherlands; e-mail: M.H.M.Wauben@uu.nl.