Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0407249 on July 11, 2007

Published online before print July 11, 2007
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(Journal of Leukocyte Biology. 2007;82:829-838.)
© 2007 by Society for Leukocyte Biology

Nonspecific CD4+ T cells with uptake of antigen-specific dendritic cell-released exosomes stimulate antigen-specific CD8+ CTL responses and long-term T cell memory

Siguo Hao, Jinying Yuan and Jim Xiang1

Research Unit, Division of Health Research, Saskatchewan Cancer Agency, Departments of Oncology, Microbiology and Immunology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

1 Correspondence: Saskatoon Cancer Center, 20 Campus Drive, Saskatoon, SK S7N 4H4, Canada. E-mail: jxiang{at}scf.sk.ca

ABSTRACT

Dendritic cell (DC) and DC-derived exosomes (EXO) have been used extensively for tumor vaccination. However, its therapeutic efficiency is limited to only production of prophylactic immunity against tumors. T cells can uptake DC-released EXO. However, the functional effect of transferred exosomal molecules on T cells is unclear. In this study, we demonstrated that OVA protein-pulsed DC-derived EXO (EXOOVA) can be taken up by Con A-stimulated, nonspecific CD4+ T cells derived from wild-type C57BL/6 mice. The active EXO-uptaken CD4+ T cells (aTEXO), expressing acquired exosomal MHC I/OVA I peptide (pMHC I) complexes and costimulatory CD40 and CD80 molecules, can act as APCs capable of stimulating OVA-specific CD8+ T cell proliferation in vitro and in vivo and inducing efficient CD4+ Th cell-independent CD8+ CTL responses in vivo. The EXOOVA-uptaken CD4+ aTEXO cell vaccine induces much more efficient CD8+ T cell responses and immunity against challenge of OVA-transfected BL6-10 melanoma cells expressing OVA in wild-type C57BL/6 mice than EXOOVA. The in vivo stimulatory effect of the CD4+ aTEXO cell to CD8+ T cell responses is mediated and targeted by its CD40 ligand signaling/acquired exosomal CD80 and pMHC I complexes, respectively. In addition, CD4+ aTEXO vaccine stimulates a long-term, OVA-specific CD8+ T cell memory. Therefore, the EXOOVA-uptaken CD4+ T cells may represent a new, effective, EXO-based vaccine strategy in induction of immune responses against tumors and other infectious diseases.

Key Words: aTEXO • pMHC I • T cell vaccine • antitumor immunity

INTRODUCTION

Stimulation of T cells by APCs involves at least two signaling events: one elicited by TCR recognition of peptide-MHC (pMHC) and the other by costimulatory molecule signaling (e.g., T cell CD28/APC CD80) [1 ]. Dendritic cells (DCs) are professional APCs and are the most powerful stimulators of naive T cells [2 3 4 5 ]. DCs process exogenous antigens in endosomal compartments such as multivesicular endosomes [6 ], which can fuse with a plasma membrane, thereby releasing antigen-presenting vesicles called "exosomes" (EXO) [7 8 9 ], 50–90 nm diameter vesicles containing antigen-presenting (MHC class I, class II, CD1, heat shock protein 70–90), tetraspan (CD9, CD63, CD81), adhesion (CD11b, CD54), and costimulatory (CD86) molecules [10 11 12 ], i.e., the necessary machinery required for generating potent immune responses.

The biology of small vesicles secreted from APCs has attracted a great deal of attention with the demonstration of their potent immunostimulatory functions in tumor models [10 , 13 14 15 ]. The origin of vesicle secretion was first described in differentiating RBCs, where multivesicle bodies fused with the plasma membrane in an exocytic manner. EXO have been described further in several cell types, including B lymphocytes, DCs, reticulocytes, platelets [8 , 9 ], and more recently, epithelial cells [16 ]. Formation of EXO occurs in MHC class II-enriched compartments (MIIC) by macroautophagy of the internal membrane, and then EXO are exocytosed by direct fusion of MIIC with the plasma membrane. However, for each cell type, EXO were described to have distinct and yet not fully understood properties [10 ]. Zitvogel et al. [9 ] first demonstrated vaccination of DC-derived EXO in eradication of tumors in animal models. Subsequently, EXO-based vaccines have been confirmed to stimulate strong CTL responses and induce antitumor immunity. However, its efficiency was less effective, as it only induced prophylatic immunity in animal models [13 , 17 18 19 20 ] or limited immune responses in clinical trials [21 ]. Exosomal MHC I and II complexes are functional but require to be transferred to DCs to promote T cell activation leading to tumor eradication [18 , 22 , 23 ]. Therefore, the potential pathway of in vivo EXO-mediated antitumor immunity may be through up-take of EXO by DCs, which in turn, stimulate antigen-specific T lymphocytes via the MHC complexes and costimulatory molecules on EXO-absorbed DCs. We have demonstrated recently that OVA protein-pulsed DCs (DCOVA)-derived EXO (EXOOVA) displayed MHC class I/OVA I peptide (pMHC I) complexes, CD11c, CD40, CD54, and CD80 molecules. EXOOVA can be uptaken by DCs. EXO-uptaken DC expressing high levels of pMHC I and costimulatory CD40, CD54, and CD80 molecules can stimulate naïve, OVA-specific CD8+ T cell proliferation strongly in vitro and in vivo and induce OVA-specific CTL responses, antitumor immunity, and CD8+ T cell memory (Tm) efficiently [24 ]. We have also demonstrated that CD4+ and CD8+ T cells derived from OVA-specific, TCR-transgenic OT II and OT I mice can acquire DC molecules such as pMHC I and costimulatory molecules by OVA-pulsed DC activation and become Th-APCs and cytotoxic T-antigen presenting cells (Tc-APCs), capable of stimulating CD8+ CTL responses [25 , 26 ]. Recently, it has been reported that T cells can also uptake DC-released EXO but at a much lower efficiency as compared with the amount of exosomal MHC II transferred in conditions where direct cell-to-cell contact takes place [27 ]. This prompted us to make an assumption that T cells with uptake of immunogenic, exosomal molecules derived from antigen-specific DCs may also gain capability in stimulation of antigen-specific CD8+ CTL responses.

In this study, we tested the above assumption. We investigated the efficiency of EXOOVA uptaken by Con A-stimulated, nonspecific CD4+ T cells of C57BL/6 mice. We assessed the OVA-specific CD8+ CTL responses and antitumor immunity derived from the vaccine of active EXOOVA-uptaken CD4+ T (aTEXO) cells compared with those derived from EXOOVA vaccination. We further elucidated the important role of acquired exosomal molecules on CD4+ aTEXO cells in a CD4+ aTEXO cell-based vaccine. We demonstrated that CD4+ aTEXO cells are capable of stimulating CD4+ Th cell-independent, OVA-specific CD8+ T cell responses and inducing much more efficient CTL responses and immunity against challenge of OVA-transfected BL6-10 (BL6-10OVA) melanoma cells expressing OVA in wild-type C57BL/6 mice than EXOOVA. In addition, the CD4+ aTEXO vaccine stimulates a long-term, OVA-specific CD8+ Tm.

MATERIALS AND METHODS

Reagents, cell lines, and animals
OVA was obtained from Sigma Chemical Co. (St. Louis, MO, USA). OVA I (SIINFEKL), specific for H-2Kb [28 , 29 ], and Mut1 (FEQNTAQP) peptide, specific for H-2Kb of an irrelevant 3LL lung carcinoma [23 ], were synthesized by Multiple Peptide Systems (San Diego, CA, USA). Biotin-labeled or FITC-labeled antibodies specific for H-2Kb (AF6-88.5), Iab (AF6-120.1), CD8 (53-6.7), CD11c (HL3), CD40 (IC10), CD40 ligand (CD40L; MR1), CD54 (3E2), and CD80 (16-10A1) as well as FITC-conjugated avidin were obtained from PharMingen Inc. (Mississauga, Ontario, Canada). The anti-H-2Kb/OVA I complex (pMHC I) antibody was obtained from Dr. Ronald Germain (National Institutes of Health, Bethesda, MD, USA) [23 ]. The recombinant mouse IL-4 and GM-CSF were purchased from R&D Systems Inc. (Minneapolis, MN, USA). The CFSE was obtained from Molecular Probes (Eugene, OR, USA). The highly lung metastatic BL6-10 and BL6-10OVA melanoma cell lines were generated in our own laboratory [25 ]. Female C57BL/6 and OVA-specific, TCR-transgenic OT II mice and H-2Kb–/–, CD40–/–, CD40L–/–, and CD80–/– gene knockout (KO) mice on a C57BL/6 background were obtained from the Jackson Laboratory (Bar Harbor, MA, USA). All mice were treated according to Animal Care Committee guidelines of the University of Saskatchewan (Canada).

DC generation
The generation of bone marrow (BM)-derived, mature DCs from wild-type C57BL/6 mice in the presence of GM-CSF/IL-4 (20 ng/mL) has been described previously [30 ]. DCs, at Day 6 in culture, were pulsed further with OVA protein (0.3 mg/mL) in AIM-V medium (Gibco, Burlington, Ontario, Canada) for overnight culture and termed DCOVA [24 ]. The DCOVA from H-2Kb–/–, CD40–/–, CD40L–/–, and CD80–/– KO mice were termed (Kb–/–)DCOVA, (CD40–/–)DCOVA, (CD40L–/–)DCOVA, and (CD80–/–)DCOVA, respectively.

EXO preparation
EXO preparation and purification were described previously [8 , 9 ]. Briefly, culture supernatants of OVA-pulsed, BM-derived DCs were subjected to four successive centrifugations at 300 g for 5 min to remove cells, 1200 g for 20 min and 10,000 g for 30 min to remove cellular debris, and 100,000 g for 1 h to pellet EXO [24 ]. The EXO pellets were washed twice in a large volume of PBS and recovered by centrifugation at 100,000 g for 1 h. The amount of exosomal proteins recovered was measured by the Bradford assay (Bio-Rad, Richmond, CA, USA). EXO derived from DCOVA, (Kb–/–)DCOVA, (CD40–/–)DCOVA, (CD40L–/–)DCOVA, and (CD80–/–)DCOVA were referred to as EXOOVA, (Kb–/–)EXOOVA, (CD40–/–)EXOOVA, (CD40L–/–)EXOOVA, and (CD80–/–)EXOOVA, respectively. To generate CFSE-labeled EXO (EXOCFSE), DCs were stained with 0.5 µM CFSE at 37°C for 20 min [31 , 32 ] and washed three times with PBS and then pulsed with OVA protein in AIM-V serum-free medium overnight. The EXOCFSE were harvested and purified from the culture supernatants as described above. An average of 5 µg EXOOVA with 50–90 nm in diameter, as determined by electron microscopy (data not shown), was recovered from an overnight culture of 1 x 106 DCOVA, which is consistent with a previous report [9 ].

T cell preparation
Naïve, OVA-specific OT I CD8+ T cells were isolated from OVA-specific, TCR-transgenic OT I mouse spleens, enriched by passage through nylon wool columns (C&A Scientific, Manassas, VA, USA) and then purified by negative selection using anti-mouse CD4 (L3T4) paramagnetic beads (Dynal Inc., Lake Success, NY, USA) to yield populations, which were >98% CD8+/V{alpha}2Vß5+ [25 ]. Naïve CD4+ T cells were isolated from C57BL/6 mouse spleens, enriched by passage through nylon wool columns, and then purified by negative selection using anti-mouse CD8 (L3T8) paramagnetic beads. To generate CD4+ aT cells, the spleen cells from naïve C57BL/6 mice were cultured in RPMI-1640 medium containing IL-2 (20 U/ml) and Con A (1 µg/ml) for 3 days [25 ]. The Con A-activated CD4+ T cells were then purified as described above to yield T cell populations, which were >98% CD4+/V{alpha}2Vß5+ T cells, referred to as CD4+ aT cells. These CD4+ aT cells secreted IL-2 (3.4 ng/ml/106 cells/24 h) and IFN-{gamma} (2.7 ng/ml/106 cells/24 h) but not IL-4 and IL-10 in the culture supernatants. The CD4+ aT cells derived from H-2Kb–/–, CD40–/–, CD40L–/–, and CD80–/– mice were termed aT(Kb–/–), aT(CD40–/–), aT(CD40L–/–), and aT(CD80–/–), respectively.

Uptake of EXO by CD4+ aT cells
For assessment of exosomal uptake, CD4+ aT cells were incubated with EXOCFSE (10 µg/1x106 T cells) at 37°C for 4 h, washed with PBS for two times, and then analyzed for CFSE expression by flow cytometry [27 ]. In another set of experiments, CD4+ aT cells were cocultured with EXO and then analyzed by flow cytometry using a panel of FITC-labeled anti-H-2Kb, Iab, CD54, CD80, and pMHC I antibodies, respectively. CD4+ aT cells cocultured with or without EXOOVA were termed aTEXO and aT cells, respectively. CD4+ aT(Kb–/–), aT(CD40–/–), aT(CD40L–/–), and aT(CD80–/–) cells cocultured with (Kb–/–)EXOOVA, (CD40–/–)EXOOVA, (CD40L–/–)EXOOVA, and (CD80–/–)EXOOVA were termed aTEXO(pMHC I–/–), aTEXO(CD40–/–), aTEXO(CD40L–/–), and aTEXO(CD80–/–), respectively. Except for the respective gene deficiency, these CD4+ aTEXO cells with different gene KO displayed a similar profile of immunologically important cell-surface molecule expression and cytokine secretion as the CD4+ aT cells (data not shown).

In vitro T cell proliferation assay
To assess the functional effect of aTEXO cells, we performed CD8+ T cell proliferation assay. The irradiated (3000 rad) CD4+ aTEXO or aT (0.6x105 cells/well) cells, DCOVA (0.3x105 cells/well), and their twofold dilutions were cultured with a constant number of naïve OT I CD8+ T cells (1x105 cells/well). EXOOVA (10 µg/ml) and its twofold dilutions were cultured with a constant number of OT I CD8+ T cells (1x105 cells/well). To examine the molecular mechanism, a panel of reagents including anti-H-2Kb, I-Ab, and LFA-1 antibodies and CTLA-4/Ig fusion protein (each 10 µg/ml), a mixture of the above reagents (as mixed reagents), and a mixture of isotype-matched, irrelevant antibodies (as control reagents) were added to the cell cultures, respectively. After culturing for 3 days, thymidine incorporation was determined by liquid scintillation counting [31 ].

ELISPOT assay
In ELISPOT assay [33 ], the splenocytes were harvested from immunized mice 6 days after the primary i.v. immunization with irradiated (4000 rad) DCOVA (1x106 cells/mouse) and CD4+ aT and aTEXO (3x106 cells/mouse) cells, respectively, or EXOOVA (10 µg/mouse) [24 ]. These splenocytes (1x106 cells) were then seeded into each well of filtration plates (96 wells; Millipore, Bedford, MA, USA) in the absence (as control) or presence of OVA I (2 µM), which was coated previously with purified anti-IFN-{gamma} antibody for 24 h and blocked with 10% FCS. The plates were then incubated at 37°C for 24 h. After washing, biotin-conjugated, anti-IFN-{gamma} mAb were added and incubated for 2 h at room temperature. The plates were then washed three times with distilled water. The streptavidin-alkaline phosphatase (Invitrogen, Carlsbad, CA, USA) was added, and the plates were incubated for 1–2 h at room temperature. After three washes with distilled water, the alkaline phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate/NBT (Sigma Chemical Co.) was added, and the color was developed, according to the manufacturer's instructions. Spots were counted under a microscope.

Tetramer staining assay
Six days after the immunization, 100 µL blood was taken from the tail of the mice with i.v. immunization with irradiated (4000 rad) DCOVA (1x106 cells/mouse), CD4+ aT, aTEXO, aTEXO(pMHC I–/–), aTEXO(CD40–/–), aTEXO(CD40L–/–), and aTEXO(CD80–/–; 3x106 cells/mouse) cells, respectively, or EXOOVA (10 µg/mouse) [24 ]. The blood samples were incubated with PE-conjugated H-2Kb/OVA257–264 tetramer (Beckman Coulter, Mississauga, Ontario, Canada) and FITC-conjugated anti-CD8 antibody for 30 min at room temperature. The erythrocytes were then lysed using lysis/fixed buffer (Beckman Coulter). The cells were washed and analyzed by flow cytometry.

Cytotoxicity assay
The in vivo cytotoxicity assay was performed as described previously [25 ]. Briefly, splenocytes were harvested from naive mouse spleens and incubated with high (3.0 µM, CFSEhigh) or low (0.6 µM, CFSElow) concentrations of CFSE to generate differentially labeled target cells. The CFSEhigh cells were pulsed with OVA I peptide, whereas the CFSElow cells were pulsed with Mut1 peptide and served as internal controls. These peptide-pulsed target cells were washed extensively to remove free peptides and then i.v.-coinjected at a 1:1 ratio into the above immunized mice 6 days after immunization. Sixteen hours after the target cell delivery, the spleens of immunized mice were removed, and residual CFSEhigh and CFSElow target cells remaining in the recipients' spleens were analyzed by flow cytometry.

Animal studies
To examine the antitumor protective immunity conferred by EXOOVA-targeted CD4+ T cells, wild-type C57BL/6, Iab–/– , or Kb–/– KO mice (n=8) lacking CD4+ or CD8+ T cells were injected i.v. with irradiated (4000 rad) DCOVA (1x106 cells/mouse), CD4+ aT, aTEXO, aTEXO(pMHC I–/–), aTEXO(CD40–/–), aTEXO(CD40L–/–), and aTEXO(CD80–/–; 3x106 cells/mouse) cells, respectively, or EXOOVA (10 µg/mouse) [24 ]. The mice injected with PBS were used as a control. The immunized mice were challenged i.v. with 0.5x106 BL6-10OVA or BL6-10 cells 6 days subsequent to the immunization. The mice were killed 4 weeks after tumor cell injection, and the lung metastatic tumor colonies were counted in a blind manner. Metastases on freshly isolated lungs appeared as discrete, black-pigmented foci, which were easily distinguishable from normal lung tissues and confirmed by histological examination. Metastatic foci too numerous to count were assigned an arbitrary value of >100 [25 ].

RESULTS

Acquisition of exosomal molecules by CD4+ aT cells
DCOVA-derived EXOOVA were systemically characterized by flow cytometry. We demonstrated that similar to OVA-pulsed DCOVA, MHC class I (Kb) and class II (Iab), CD11c, CD40, CD54, CD80, and pMHC I complex were detected on DCOVA-derived EXOOVA but with a lesser amount compared with DCOVA (Fig. 1 ). DCOVA and EXOOVA did not express the T cell surface molecule CD40L (data not shown). To explore the potential uptake of EXO by T cells, Con A-stimulated CD4+ aT cells derived from C57BL/6 mice were incubated with EXOCFSE derived from CFSE-labeled DCOVA and then analyzed by flow cytometry. As shown in Figure 2a , the CFSE dye was detectable on CD4+ aT cells in a moderate amount but not on C57BL/6 CD4+ naïve T cells, indicating that EXO can be uptaken by CD4+ aT cells. The uptake of EXOCFSE by CD4+ aT cells increased with the time of incubation and reached a maximal level after incubation with EXOCFSE for 4 h (Fig. 2b) . CD4+ aT cells displayed some expression of MHC II, CD40L, and CD80 molecules, whereas CD4+ naïve T cells did not express any MHC II, CD40L, and CD80 molecules (Fig. 2c) . All naive and CD4+ aT cells did not express pMHC I. Similar to CFSE dye transfer, other exosomal molecules such as MHC classes I and II, CD54, CD80, and pMHC I, the critical components in stimulation of OVA-specific, CD8+ CTL responses, were also transferred onto CD4+ aT cells (Fig. 2c) after 4 h incubation, indicating an antigen-independent acquisition of exosomal molecules by CD4+ T cells, which is consistent with a previous report by Undale et al. [27 ]. To confirm the acquisition of CD40, CD80, and pMHC I from EXOOVA, we used CD4+ aT cells derived from CD40–/–, CD80–/–, and Kb–/– gene KO mice. As shown in Figure 2d , the original CD4+ aT cells derived from gene KO mice did not express pMHC I, CD40, and CD80, respectively. However, after incubation with EXOOVA, they displayed CD40, CD80, and pMHC I, indicating that the exosomal CD40, CD80, and pMHC I complexes can be transferred onto the CD4+ aT cells. This was confirmed further by confocal fluorescence microscopy (Fig. 2e) .


Figure 1
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  		Figure 1. Phenotypic analysis of DC and DC-drived EXO by flow cytometry. DCOVA and EXOOVA (thick, solid lines) were stained with a panel of antibodies and then analyzed by flow cytometry. They were also stained with irrelevant, isotype-matched antibodies (dotted lines). One representative experiment of two is displayed.

 

Figure 2
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  		Figure 2. EXO uptake by CD4+ T cells. (a) Naïve (nT) and CD4+ aT cells were incubated with EXOCFSE for 4 h (thick, solid lines) and analyzed for CFSE expression by flow cytometry. The original CD4+ T cells (thin, dotted lines) were also analyzed by flow cytometry. (b) The CD4+ aT cells were incubated with EXOCFSE for different times and examined by confocal fluorescence microscopy. (c) Naïve CD4+ T, CD4+ aT, and aTEXO (thick, solid lines) were stained with a panel of antibodies and analyzed by flow cytometry. Irrelevant, isotype-matched antibodies were used as controls (thin, dotted lines). (d) CD4+ aT cells from CD40, CD80, and H-2Kb KO mice [(CD40–/–)aT, (CD80–/–)aT, and (Kb–/–)aT] were also cocultured with (thick, solid lines) and without (thick, dotted lines) EXOOVA, stained with biotin-labeled anti-CD40, -CD80, and -pMHC I antibodies, followed by FITC-avidin, and then anayzed by flow cytometry. Irrelevant, isotype-matched antibodies were used as controls (thin, dotted lines). (e) The above CD4+ aT cells were also examined under differential interference contrast (DIC) and by confocal fluorescence microscopy. One representative experiment of two is displayed.

 
Stimulation of in vitro CD8+ T cell proliferation by CD4+ aTEXO
As EXO harbor many immune molecules, including pMHC I and costimulatory molecules, they may have a potent effect in stimulation of CD8+ T cells. As shown in Figure 3a , EXOOVA was able to stimulate CD8+ T cell proliferation but to a lesser extent, which is consistent with previous reports [34 , 35 ]. The aTEXO stimulated CD8+ T cell proliferation efficiently, as did DCOVA. The stimulation of CD8+ T cell proliferation by aTEXO cells is critically dependent on antigen specificity, as no proliferative response was observed in the aT group with no expression of acquired exosomal pMHC I. To investigate the molecular mechanism involved in CD8+ T cell proliferation, a panel of reagents was added to the cell cultures. As shown in Figure 3b , anti-H-2Kb, anti-LFA-1, and CTLA-4/Ig inhibited CD8+ T cell-proliferative responses significantly in the cocultures by ~30%, ~32%, and ~62% (P<0.05), respectively. A more effective inhibition in proliferation of CD8+ T cell by ~90% was observed in the mixed reagents group, indicating that the CD8+ T cell proliferation is critically dependent on the OVA-specific pMHC I/TCR interaction and greatly affected by costimulations (CD80/CD28 and CD54/LFA-1).


Figure 3
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  		Figure 3. Stimulation of CD8+ T cell proliferation in vitro. (a) In an in vitro CD8+ T cell proliferation assay, The irradiated (3000 rad) DCOVA, CD4+ aTEXO, and aT cells (0.3x105 cells/well) and their twofold dilutions were cocultured with a constant number of OT I CD8+ T cells (1x105 cells/well). EXOOVA (10 µg/ml) and its twofold dilutions were cultured with a constant number of OT I CD8+ T cells (1x105 cells/well). After 3 days, the proliferation response of CD8+ T cells was determined by 3H-thymidine uptake assay. (b) The impact of aTEXO cell stimulation of OT I CD8+ T cell proliferation by adding each of the neutralizing reagents, a mixture of neutralizing reagents together (mixed reagents), and a mixture of control antibodies and fusion proteins (control reagents) was assessed. *, P < 0.05, versus cohorts without adding any neutralizing reagent (Student's t-test). One representative experiment of three is displayed.

 
Stimulation of in vivo CD8+ T cell proliferation by CD4+ aTEXO
To assess whether EXOOVA-uptaken CD4+ T cells can also stimulate CD8+ T cell proliferation in vivo, we performed ELISPOT and tetramer staining assays to detect OVA-specific and IFN-{gamma}-secreting CD8+ T cell proliferation in mice 6 days after immunization with DCOVA and aTEXO cells, respectively. As shown in Figure 4a , we detected an average of 612 IFN-{gamma}-secreting cells/106 splenocytes in mice immunized with DCOVA. We also detected an average of 250 IFN-{gamma}-secreting cells/106 splenocytes in mice immunized with EXOOVA, which is consistent with our previous report [24 ]. It is interesting that we further detected an average of 560 IFN-{gamma}-secreting cells/106 splenocytes in mice immunized with aTEXO cells, indicating that EXOOVA-uptaken CD4+ TEXO cells can induce significantly stronger activation of naïve, antigen-specific CD8+ T cell responses in vivo than EXOOVA (P<0.05). No IFN-{gamma}-secreting cells were detectable in the control group and aT group. To confirm the above observations further, we performed the tetramer staining assay to detect OVA-specific CD8+ T cells in wild-type or Iab gene KO mice 6 days after immunizations with DCOVA and aTEXO cells, respectively. As shown in Figure 4b , EXOOVA stimulated H-2Kb/OVA257–264 tetramer-positive CD8+ T cell responses, accounting for 0.56% of the total spleen CD8+ T cells of the wild-type C57BL/6 mice, whereas DCOVA and aTEXO cells stimulated H-2Kb/OVA257–264 tetramer-positive CD8+ T cell responses, accounting for 2.41% and 1.89% of the total spleen CD8+ T cells, respectively, indicating that the aTEXO cell is a much stronger stimulator in activation of antigen-specific CD8+ T cell responses in vivo than EXOOVA (P<0.05). In Iab gene KO mice lacking CD4+ T cells, however, only aTEXO, but not DCOVA, could still stimulate OVA-specific CD8+ T cell responses; i.e., 1.67% of tetramer-positive CD8+ T cells were detectable, indicating that the OVA-specific CD8+ T cell response stimulated by aTEXO is CD4+ T cell-independent, whereas the OVA-specific CD8+ T cell response stimulated by DCOVA is mainly CD4+ T cell-dependent.


Figure 4
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  		Figure 4. Stimulation of CD8+ T cell proliferation and differentiation in vivo. (a) Wild-type C57BL/6 mice were i.v.-immunized with irradiated DCOVA and CD4+ aT and aTEXO cells or EXOOVA. Six days after immunization, the splenocytes were harvested, and IFN-{gamma}-secreting cells were analyzed by ELISPOT as described in Materials and Methods. (b) In tetramer staining, C57BL/6 mice and Iab gene KO mice were i.v.-immunized with irradiated DCOVA and CD4+ aT, aTEXO, and aTEXO cells with different gene deficiency or EXOOVA. The tail blood samples of immunized mice were incubated with PE-H-2Kb/OVA I tetramer and FITC-anti-CD8 antibodies and analyzed by flow cytometry. The value in each panel represents the percentage of tetramer-positive CD8+ T cells versus the total CD8+ T cell population. The value in parentheses represents the SD. *, P < 0.05, versus cohorts of aTEXO cells (Student's t-test). (c) In in vivo cytotoxicity assay, the above-immunized mice were i.v.-coinjected at a 1:1 ratio of splenocytes labeled with high (3.0 µM, CFSEhigh) and low (0.6 µM, CFSElow) concentrations of CFSE and pulsed with OVA I and Mut1 peptide, respectively, 6 days after immunization with aTEXO and aTEXO with various gene KO, respectively. Sixteen hours after target cell delivery, the residual CFSEhigh and CFSElow target cells remaining in the spleens of the recipients were sorted and analyzed by flow cytometry. The value in each panel represents the percentage of CFSEhigh cells versus CFSElow cells remaining in the spleens. The value in parentheses represents the SD. *, P < 0.05, versus cohorts of aTEXO cells (Student's t-test). One representative experiment of three in the above different experiments is shown. L, Low; H, high.

 
Stimulation of in vivo CD8+ T cell differentiation into CTL effectors by CD4+ aTEXO
Next, we assessed the ability of aTEXO to induce the differentiation of naïve CD8+ T cells into CTL effectors in vivo. We adoptively transferred OVA I peptide-pulsed splenocytes, strongly labeled with CFSE (CFSEhigh), as well as the control peptide Mut1-pulsed splenocytes, weakly labeled with CFSE (CFSElow), into recipient mice, which had been vaccinated with DCOVA and aTEXO cells, respectively. As expected, there was substantial loss of the CFSEhigh (OVA I peptide-pulsed) cells in the immunized mice. Among them, the mice immunized with EXOOVA had lost 23% CFSEhigh (OVA I peptide-pulsed) cells (Fig. 4c) , whereas the mice immunized with DCOVA and aTEXO cells had 92% and 82% losses, respectively, indicating that aTEXO cells stimulate CD8+ T cell differentiation more efficiently into CTL effectors than EXOOVA (P<0.05). However, no cytotoxicity was induced in mice immunized with aT cells without acquired exosomal molecules, including pMHC I, thus confirming that the stimulatory effect of aTEXO in vivo is antigen-specific.

Induction of efficient antitumor immunity by CD4+ aTEXO
Next, we assessed the potential antitumor immunity derived from the EXOOVA-uptaken CD4+ aTEXO cell vaccine. As shown in Experiment I of Table 1 , all of the mice injected with PBS had large numbers (>100) of lung metastatic BL6-10 tumor colonies. We demonstrated that the DCOVA and aTEXO cell vaccines induced a complete immune protection against BL6-10OVA tumor cell challenge in eight of eight (100%) mice, whereas EXOOVA only protected three of eight (37%) mice, indicating that CD4+ aTEXO cells induce much stronger antitumor immunity than EXOOVA. The specificity of the protection was confirmed with the observation that aTEXO cells did not protect against BL6-10 tumors, which did not express OVA, and all mice had large numbers (>100) of lung metastatic tumor colonies after tumor cell challenge. To study the immune mechanism, Iab and H-2Kb gene KO mice were used for immunization. As shown in Experiment II of Table 1 , most of Iab gene KO (88%) mice lacking CD4+ T cells were still tumor-free. However, all H-2Kb gene KO mice (eight of eight) lacking CD8+ T cells had numerous lung tumor metastases, indicating that aTEXO-induced antitumor immunity is CD4+ Th cell-independent but mediated by CD8+ CTLs.


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  		Table 1. EXO-Targeted CD4+ T Cell Vaccine Protects against Lung Tumor Metastases

 
The stimulatory effect of the CD4+ aTEXO cell is mediated and targeted to in vivo CD8+ CTL responses and antitumor immunity by its CD40L signaling/acquired exosomal CD80 costimulation and pMHC I, respectively
To elucidate the molecular mechanism, CD4+ aTEXO cells with respective gene deficiency were used for immunizations. In an in vivo CD8+ T cell proliferation assay, we found that aTEXO(CD40–/–) cells still stimulated H-2Kb/OVA257–264 tetramer-positive CD8+ T cell responses, accounting for 1.57% of the total spleen CD8+ T cells, whereas aTEXO(CD40L–/–) and aTEXO(CD80–/–) significantly lost more stimulatory effect for only stimulation of 0.02% and 0.45% CD8+ T cell responses, respectively (P<0.05; Fig. 4b ). It is interesting that we also found that aTEXO(pMHC I–/–) lost their stimulatory effect completely, indicating OVA-specific, CFSEhigh-labeled target cells. In an in vivo cytotoxicity assay, we demonstrated that there was substantial loss (73%) of the OVA-specific, CFSEhigh-labeled target cells in aTEXO(CD40–/–)-immunized mice similarly as in aTEXO-immunized mice (82%; Fig. 4c ), whereas aTEXO(CD40L–/–)-, aTEXO(CD80–/–)-, and aTEXO(pMHC I–/–)-immunized mice mostly and completely maintained their OVA-specific, CFSEhigh-labeled target cells (100%, 64%, and 97%), respectively, indicating that aTEXO(CD40L–/–), aTEXO(CD80–/–)-, and aTEXO(pMHC I–/–) significantly lost their stimulatory effects on CTL responses compared with aTEXO (P<0.05). In animal studies, 100 (eight of eight) of aTEXO(CD40L–/–)- and 62% (five of eight) of aTEXO(CD80–/–)-immunized mice but none (zero of eight) of aTEXO(CD40–/–)-immunized mice lost their antitumor immunity (Experiment III of Table 1 ). It is interesting that 88% (seven of eight) of mice immunized with aTEXO(pMHC I–/–) had large numbers (>100) of lung tumor colonies. These data clearly indicate that the stimulatory effect of the CD4+ aTEXO cell is mediated by its CD40L signaling and its acquired exosomal CD80 costimulation, and the acquired exosoaml pMHC I complexes play a critical role in targeting its stimulatory effect to OVA-specific CD8+ T cell proliferation and cytotoxicity and antitumor immunity in vivo.

Induction of efficient tumor-specific CD8+ Tm by CD4+ aTEXO
CD8+ aT cells can become long-lived Tm cells after adoptive transfer in vivo [36 ]. As aTEXO cells stimulated CD8+ T cell differentiation into CTL effectors in vitro and in vivo, we then assessed whether these activated CD8+ T cells can become long-lived Tm cells. As shown in Figure 5a , 3 months after the immunization, we still detected 0.07%, 0.16%, and 0.25% CD8+ T cells expressing H-2Kb/OVA257–264 tetramer-specific TCR in peripheral blood of mice immunized with EXOOVA, DCOVA, and aTEXO cells, respectively, indicating that these T cells are Tm cells, and aTEXO immunization induces more efficient CD8+ Tm cell development than DCOVA and EXOOVA (P<0.05). To investigate the functionality of these CD8+ Tm cells, the immunized mice were boosted with DCOVA. The recall responses were examined using H-2Kb/OVA257–264 tetramer staining on Day 4 after the boost. As shown in Figure 5b , there were few OVA-specific CD8+ T cells detected in the peripheral blood of the control mice, which were injected with PBS 3 months ago and boosted with DCOVA 4 days ago, indicating that the primary proliferation of OVA-specific CD8+ T cells by the DCOVA boost is almost undetectable at that time-point. As expected, the number of CD8+ Tm cells was expanded by approximately eightfold in the EXOOVA-, aTEXO-, and DCOVA-immunized mice after the boost, indicating that these CD8+ Tm cells are functional.


Figure 5
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  		Figure 5. Development of antigen-specific CD8+ Tm cells. C57BL/6 mice were i.v.-immunized with irradiated DCOVA and CD4+ aT and aTEXO cells or EXOOVA, respectively. (a) Three months later, the tail blood was taken from these immunized mice and stained with PE-H-2Kb/OVA tetramer (PE tetramer) and FITC-anti-CD8 (FITC-CD8) antibodies and analyzed by flow cytomery. (b) The above-immunized mice were also boosted with DCOVA. Four days after the boost, the recall responses were examined using staining with PE tetramer and FITC-CD8 antibodies and analyzed by flow cytometry. The results presented are representative of five separate mice per group. The values in parentheses represent the SD. *, P < 0.05, versus cohorts of aTEXO cells (Student's t-test). One representative experiment of three is shown.

 
Taken together, our data showed that DCOVA-released EXO can be uptaken by nonspecific CD4+ aT cells via CD54/LFA-1 interactions. EXO-uptaken CD4+ T cells acquired exosomal pMHC I and costimulatory CD80 molecules, which express CD40L and induce OVA-specific CD8+ T cell responses via pMHC I/TCR (Signal I), CD40L and CD80 costimulations (Signal II), and IL-2 secretion (Signal III) [25 ] (Fig. 6 ).


Figure 6
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  		Figure 6. Nonspecific CD4+ T cells with uptake of EXO derived from antigen-specific DC stimulate antigen-specific CD8+ T cell responses. EXO carrying pMHC I and CD80 are fused with nonspecific aT cells via CD54/LFA-1 interactions and become CD4+ aTEXO, and CD4+aTEXO expressing CD40L and acquired exosomal pMHC I and CD80 stimulate antigen-specific, naive CD8+ T cell responses via IL-2 secretion and CD40L and CD80 costimulations.

 
DISCUSSION

Intercellular transfer of cell-surface molecules has increasingly attracted attention for study [37 ]. The phenomenon of intercellular membrane/protein transfer between cells has been observed, including immune cells such as DCs [38 ], T cells [39 , 40 ], leukocytes [41 ], B cells [42 , 43 ], NK cells [44 , 45 ], macrophages [46 ], and basophils [38 ]. This membrane transfer has been reported abundantly in systems requiring or not requiring cell-to-cell contacts [47 ]. Several mechanisms have been proposed by which membrane molecules may be transferred between immune cells. The formation of a CD8+ T cell/target cell immunological synapse resulted in the formation of a physical bridge between cells, which facilitated the lateral diffusion of proteins and lipids between fused membranes [48 ]. Transfer can also involve an intimate interaction between cells at an immunological synapse. DC-derived membrane molecules can be transferred to the T cells during the course of their TCR-mediated internalization followed by recycling [49 50 51 ]. Recently, Wetzel et al. [52 ] demonstrated that upon dissociation from DCs, T cells captured GFP-tagged MHC:peptide complexes and CD80 directly from the engineered DCs, and DC membrane molecules may thus be transferred onto CD4+ T cells by this pathway. In addition, membrane nanotubes, long membrane techers between cells, provide another possible mechanism for intercellular transfer of cell-surface proteins [53 ]. It has been demonstrated that nanotubes may allow the intercellular exchanges of proteins and lipids [54 , 55 ]. Recently, it has been reported that T cells can uptake DC-released EXO but at a much lower efficiency as compared with the amount of exosomal MHC II transferred in conditions where direct cell-to-cell contact takes place [27 ]. DC-released EXO expressing MHC classes I and II costimulatory molecules [56 ] can dock and fuse with the T cell membrane [56 ] through its binding to TCR or CD28 on T cells [50 ]. It has been demonstrated that the uptake of EXO by T cells was a saturable process, which required close cell proximity, actin polymerization, and a permissive temperature [57 ]. Therefore, the fusion of DC-released EXO onto CD4+ T cells may be another alternative pathway of DC membrane molecule acquisition. However, the functional effect of transferred exosomal molecules on T cells is unclear.

EXO-based vaccines have been shown to induce antitumor immunity [13 , 17 18 19 20 ]. It has been demonstrated that DC-derived EXO can stimulate naïve T cell proliferation directly in vitro [34 ]. The potential pathway of in vivo EXO-mediated T cell immune responses may be through uptake of EXO by DCs, which in turn, stimulate antigen-specific T lymphocytes via the MHC complexes and costimulatory molecules on EXO-absorbed DCs [18 , 22 , 23 ]. However, its efficiency of stimulation of T cell immune responses was less effective, as it only induced prophylatic immunity in animal models [13 , 17 18 19 20 ]. In clinical trials, the ascites of patients became a good source for purification of tumor antigen-specific EXO for use in an EXO-based vaccine [13 , 58 ]. However, vaccination of ascites-derived TEXO only induced limited tumor antigen-specific CTL responses in clinical trials [59 ]. Administration of attenuated T cells to animals has been shown to stimulate immune suppression and to prevent the development of experimental autoimmune diseases [60 , 61 ]. Vaccination using myelin basic protein autologous T cells has also been applied to a clinical trial in multiple sclerosis [62 ]. However, application of T cell vaccination capable of stimulation of CD8+ CTL responses and induction of protective immunity have not been reported.

In this study, we assessed the potential stimulation of CD8+ CTL responses and antitumor immunity derived from an EXOOVA-uptaken CD4+ aTEXO cell vaccine. We demonstrated that CD4+ aTEXO cells induced much stronger OVA-specific CD8+ CTL responses and antitumor immunity than EXOOVA, possibly as a result of lacking CD40L signaling and IL-2 secretion in EXOOVA. It has been demonstrated recently that CD40L signaling of CD4+ T cells plays an important role in induction of CD8+ CTL responses [63 , 64 ], and the acquisition of CD80 from APCs by CD4+ T cells plays an important role in retaining CD4+ T cell activation in the absence of APCs via up-regulation of NF-{kappa}B and Stat5 [65 ]. In this study, for the first time, we elucidated the molecular mechanism of the stimulatory effect of the CD4+ aTEXO cell. We demonstrated that the stimulatory effect of the CD4+ aTEXO cell is mediated by its CD40L signaling and its acquired exosomal CD80 costimulation, and the acquired exosomal pMHC I complexes play a critical role in targeting its stimulatory effect to OVA-specific CD8+ T cell proliferation and cytotoxicity in vivo. In addition, we demonstrated that the OVA-specific CD8+ T cell response stimulated by aTEXO is CD4+ T cell-independent, whereas the OVA-specific CD8+ T cell response stimulated by DCOVA is mainly CD4+ T cell-dependent. Therefore, the EXO-targeted T cell vaccine, as described in this study, may be useful in induction of anti-HIV immunity in HIV patients with CD4+ T cell deficiency [66 ]. It may also provide an alternative EXO-based strategy for cancer treatment, in which CD4+ T cells can easily be harvested from the peripheral blood of a patient and activated in vitro. These activated, nonspecific CD4+ T cells can acquire the tumor antigen specificity and the stimulatory effect for tumor antigen-specific CD8+ CTL responses after incubation with EXO derived from the ascites of a patient and thus be used as an alternative EXO-based vaccine.

Taken together, our data showed that DCOVA-released EXOOVA can be uptaken by nonspecific CD4+ aT cells. EXO-uptaken CD4+ T cells with acquired exosomal pMHC I and costimulatory CD80 molecules can induce more efficient OVA-specific CD8+ CTL responses, antitumor immunity, and CD8+ Tm in vivo than EXOOVA. The stimulatory effect of the in vivo CD4+ aTEXO cell to CD8+ T cell responses is mediated and targeted by its CD40L signaling/acquired exosomal CD80 and pMHC I complexes, respectively. Therefore, the EXO-uptaken CD4+ T cells may represent a new, effective EXO-based vaccine strategy in induction of immune responses against tumors and other infectious diseases.

ACKNOWLEDGEMENTS

This research work was supported by research grants (MOP 79415 and 81228) from Canadian Institutes of Health Research. S. H. was supported by the Postdoctoral Fellowship from Saskatchewan Health Research Foundation (SHRF). We appreciate Mark Boyd for help in flow cytometry.

Received April 26, 2007; revised May 29, 2007; accepted June 12, 2007.

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