HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Hao, S. Articles by Xiang, J. Search for Related Content PubMed PubMed Citation Articles by Hao, S. Articles by Xiang, J.

Antigen Specificity Acquisition of Adoptive CD4⁺ Regulatory T Cells via Acquired Peptide-MHC Class I Complexes¹

Siguo Hao^*, Jinying Yuan^*, Shulin Xu^*, Manjunatha Ankathatti Munegowda^*, Yulin Deng^*, John Gordon, Zhou Xing and Jim Xiang^2,*

^* Departments of Oncology, Microbiology, and Immunology, Research Unit, Saskatchewan Cancer Agency, College of Medicine, Department of Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, and Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The Ag-specific CD4⁺ regulatory T (Tr) cells play an important role in immune suppression in autoimmune diseases and antitumor immunity. However, the molecular mechanism for Ag-specificity acquisition of adoptive CD4⁺ Tr cells is unclear. In this study, we generated IL-10- and IFN--expressing type 1 CD4⁺ Tr (Tr1) cells by stimulation of transgenic OT II mouse-derived naive CD4⁺ T cells with IL-10-expressing adenovirus (AdV_IL-10)-transfected and OVA-pulsed dendritic cells (DC_OVA/IL-10). We demonstrated that both in vitro and in vivo DC_OVA/IL-10-stimulated CD4⁺ Tr1 cells acquired OVA peptide MHC class (pMHC) I which targets CD4⁺ Tr1 cells suppressive effect via an IL-10-mediated mechanism onto CD8⁺ T cells, leading to an enhanced suppression of DC_OVA-induced CD8⁺ T cell responses and antitumor immunity against OVA-expressing murine B16 melanoma cells by 700% relative to analogous CD4⁺ Tr1 cells without acquired pMHC I. Interestingly, the nonspecific CD4⁺25⁺ Tr cells can also become OVA Ag specific and more immunosuppressive in inhibition of OVA-specific CD8⁺ T cell responses and antitumor immunity after uptake of DC_OVA-released exosomal pMHC I complexes. Taken together, the Ag-specificity acquisition of CD4⁺ Tr cells via acquiring DC’s pMHC I may be an important mean in augmenting CD4⁺ Tr cell suppression.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Natural self-Ag-reactive CD4⁺25⁺ regulatory T (Tr)³ cells expressing Foxp3 (1) play important roles in the maintenance of self-tolerance and control of autoimmunity (2). They develop in the thymus and then enter peripheral tissues, where they suppress the activation of self-reactive T effector cells in a non-Ag-specific manner (3). Adaptive CD4⁺ Tr cells are generated in the periphery through dendritic cell (DC) presentation to naive T cells and can take on varying phenotypes, depending upon the conditions under which they are induced. IL-10 or TGF-β-secreting CD4⁺ Tr cells are generated from Ag presentation by immature DCs or IL-10- or TGF-β-expressing DCs (4, 5). These CD4⁺ Tr cells, which are Ag specific, can suppress CD8⁺ T cell-mediated autoimmune diseases (4, 5), infectious diseases (6, 7), and antitumor immunity (8, 9) in either cell contact-dependent or -independent fashions. Our current knowledge of the Ag specificity of CD4⁺ Tr cells has come largely from studies with Ag-specific TCR-transgenic mice (5, 10, 11). However, the molecular mechanism responsible for the specifically targeted delivery of adoptive CD4⁺ Tr cell suppression to cognate CD8⁺ T cells in vivo is still largely unknown.

One important feature of synapse physiology is that DC surface molecules can be transferred to the CD4⁺ Th cells during the course of their normal TCR recycling (12, 13). We have recently demonstrated that during OVA presentation by DCs, CD4⁺ T cells from OVA-specific TCR-transgenic OT II mice acquired peptide MHC class I (pMHC I) and costimulatory molecules colocalizing in the same synapse comprising pMHC class II (14) from OVA-pulsed DCs and were by themselves able to directly stimulate CD8⁺ CTL responses (15). We also confirmed that the pMHC I acquired by the CD4⁺ T cells was the critical factor that allowed this specific targeting to the CD8⁺ T cells in vivo (15, 16). Based on this principle, we hypothesize that the adoptive CD4⁺ Tr cells may similarly acquire Ag specificity for CD8⁺ T cells following interactions with cognate Ag-presenting DCs (i.e., following transfer of pMHC I complexes onto CD4⁺ Tr cells from the DCs) via acquired pMHC I.

Ag presentation by DCs in the presence of immunosuppressive cytokine IL-10 induces in vitro CD4⁺ Tr cell responses (17, 18). DCs transfected with adenoviral vector (AdV_IL-10) expressing IL-10 (19) induced both in vitro CD4⁺ Tr cell responses and in vivo immune tolerance. In this study, we transfected the OVA-pulsed DCs (DC_OVA) with a recombinant adenoviral vector AdV_IL-10 expressing IL-10 for in vitro and in vivo generation of CD4⁺ Tr cells derived from OVA-specific TCR- transgenic OT II mice. We phenotypically characterized these CD4⁺ Tr cells and investigated 1) the pMHC I acquisition of CD4⁺ Tr cells and 2) the targeting role of acquired pMHC I in delivery of CD4⁺ Tr suppressive effect onto in vivo OVA-specific CD8⁺ T cell responses and antitumor immunity.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Tumor cells, reagents, and animals

The OVA-transfected BL6-10 (BL6–10_OVA) melanoma cell line was generated in our laboratory (15). The mouse B cell hybridoma cell line LB27 expressing Ia^b and Ia^d was obtained from American Type Culture Collection. The RF3370 T cell hybridoma cell line bearing TCR specific for OVA pMHC I was obtained from Dr. K. Rock (University of Massachusetts Medical School, Worcester, MA) (20). The biotin- or fluorescent dye (FITC, PE-Texas Red-X, and energy-coupled dye)-labeled Abs specific for H-2K^b (AF6-88.5), Ia^b (AF6-120.1), CD4 (GK1.5), CD8 (53-6.7), CD11c (HL3), CD25 (7D4), CD40 (K19), CD45.1 (A20), CD54 (3E2), CD69 (H1.2F3), CD80 (16-10A), glucocorticoid-induced TNFR (GITR; DTA-1), and V2Vβ5 TCR (MR9-4) were obtained from BD Pharmingen. The anti-H-2K^b/OVAI peptide (pMHC I) Ab was obtained from Dr. J. Germain (National Institutes of Health, Bethesda, MD) (21). The anti-CTLA-4 and Foxp3 Abs were obtained from eBioscience. The anti-IFN-, -IL-4, -IL-10, and -TGF-β Abs as well as the rGM-CSF, IL-2, IL-4, and IL-10 were obtained from R&D Systems. The PE-H-2K^b/OVAI peptide tetramer and FITC-anti-CD8 Ab (PK135) were obtained from Beckman Coulter. The OVAI (SIINFEKL) and OVAII (ISQAVHAAHAEINEAGR) peptides specific for H-2K^b and Ia^b, respectively, and the H-2K^b-specific irrelevant 3LL lung carcinoma peptide Mut1 (FEQNTAQP) (15) were synthesized by Multiple Peptide Systems. The C57BL/6 (B6, CD45.2⁺), B6.SJL-Ptpcra (B6.1, CD45.1⁺), OVA-specific TCR-transgenic OT I and OT II mice and H-2K^b, Ia^b, IFN-, and IL-10 gene knockout (KO) mice on a C57BL/6 background were obtained from The Jackson Laboratory. Homozygous OT II/H-2K^b–/–, OT II/IFN-^–/–, and OT II/IL-10^–/– mice were generated by backcrossing the designated gene KO mice onto the OT II background for three generations; homozygosity was confirmed by PCR according to The Jackson Laboratory’s protocols. OT II/B6.1 mice were generated by backcrossing B6.1 mice onto the OT II background. All mice were treated according to animal care committee guidelines of the University of Saskatchewan.

Preparation of DCs

Bone marrow (BM)-derived DCs were generated as described previously (15). Briefly, BM cells were collected from the femora and tibiae of normal or designated gene-deleted C57BL/6 mice depleted of RBC with 0.84% ammonium chloride, and plated in DC culture medium (DMEM plus 10% FCS, 20 ng/ml GM-CSF, and 20 ng/ml IL-4). On day 3, the nonadherent granulocytes and T and B cells were gently removed and fresh medium was added, and 2 days later, the loosely adherent proliferating DC aggregates were dislodged and replated until day 6, when the nonadherent DCs were harvested. DCs generated in this manner were pulsed with 0.5 mg/ml OVA (Sigma-Aldrich) overnight at 37°C in the absence or presence of a recombinant AdV (AdV_IL-10) expressing IL-10 (22) at a multiplicity of infection of 100, as previously described (23), and referred to as DC_OVA or DC_OVA/IL-10. DCs generated from H-2K^b gene KO C57BL/6 mice were termed (K^b–/–)DC_OVA/IL-10. Splenic DC_OVA were generated as described previously (15).

Preparation of DC-released exosomes (EXO)

EXO derived from the culture supernatants of BM-derived DC_OVA/IL-10 and (K^b–/–)DC_OVA/IL-10 were prepared as previously described (24) and referred to as EXO and (K^b–/–)EXO, respectively.

Preparation of DC_OVA/IL-10-stimulated CD4⁺ Tr1 cells

Naive OVA-specific CD4⁺ and CD8⁺ T cells were isolated from OT II and OT I mouse spleens or wild-type C57BL/6 mouse spleens, respectively, by lymphocyte enrichment on nylon wool columns (C&A Scientific) and negative selection magnetic sorting using anti-mouse CD8 (Ly2) and CD4 (L3T4) paramagnetic beads, respectively (Dynal Biotech). The purified CD4⁺ and CD8⁺ T cells were >90% CD4⁺V2Vβ5⁺ and CD8⁺V2Vβ5⁺ T cells, respectively. To generate CD4⁺ Tr1 cells, naive CD4⁺ T cells (2 x 10⁵ cells/ml) from OT II/H-2K^b–/–, OT II/IFN-^–/–, and OT II/IL-10^–/– mice were stimulated for 3 days with irradiated (4000 rad) BM-derived DC_OVA/IL-10 (1 x 10⁵ cells/ml) in the presence of IL-2 (20 U/ml) and then purified using CD4 microbeads (Miltenyi Biotec); these T cells are referred to as Tr1, (IFN-^–/–)Tr1, or (IL-10^–/–) Tr1 cells, respectively. To generate analogous Tr cells that did not express OVA pMHC I complexes, CD4⁺ T cells of OT II/H-2K^b–/– mice were similarly incubated with irradiated (K^b–/–)DC_OVA/IL-10 for 3 days; these T cells are referred to as (K^b–/–)Tr1. Except for the designed gene deficiency, the different types of CD4⁺ Tr cells displayed a similar phenotype (i.e., flow cytometric analysis and cytokine profile; data not shown). To generate DC_OVA/IL-10-stimulated CD4⁺ Tr cells in vivo, we transferred naive CD4⁺ T cells (10 x 10⁶ cells/mouse) from OT II/B6.1 mice into C57BL/6 mice. One day later, we immunized these animals with irradiated (4000 rad) DC_OVA/IL-10 and (K^b–/–)DC_OVA/IL-10 (2 x 10⁶ cells/mouse), and 3 days later CD4⁺ T cells were purified from the regional lymph nodes of the immunized mice using CD45.1 microbeads (Miltenyi Biotec). These CD4⁺ Tr cells are referred to as CD4⁺ Tr1(vivo) and (K^b–/–)Tr1(vivo) cells, respectively.

Preparation of CD4⁺25⁺ Tr cells with uptake of EXO

CD4⁺25⁺ Tr cells were prepared as previously described (25). Briefly, CD4⁺25⁺ Tr cells were purified from C57BL/6 and OT II mouse splenocytes by using nylon wool column (C&A Scientific) to enrich the T cell population, CD8 microbeads (Dynal Biotech) to remove CD8⁺ T cells, and then CD25 microbeads (Miltenyi Biotec) to purify CD4⁺25⁺ Tr cells. These purified CD4⁺25⁺ Tr cells were stimulated for expansion by using microbeads coated with anti-CD3/CD28 Ab-coated beads (Dynal Biotec) at 1:1 cell/bead ratio and suspended in DMEM plus 10% FCS and 1500 U/ml IL-2. At day 5 in culture, expanding Tr cells were resorted for CD4⁺ T cells and reselected CD4⁺25⁺ Tr cells were continued culturing until day 10. At the end of culture, the anti-CD3/CD28 Ab-coated beads were removed using AutoMACS (Automagnetic cell sorting). For uptake of EXO, CD4⁺25⁺ Tr cells were cocultured with EXO as previously described (24). CD4⁺25⁺ Tr cells derived from wild-type C57BL/6 and OT II mice were cocultured with EXO and termed B6 Tr/exo and OT II Tr/exo, respectively. Except for OVA-specific TCR, B6 Tr/exo cells are similar to OT II Tr/exo (data not shown). CD4⁺25⁺ Tr cells derived from C57BL/6 mice were cocultured with (K^b–/–)EXO and termed CD4⁺25⁺ Tr/exo(K^b–/–). Except for the respective gene deficiency, these CD4⁺25⁺ Tr/exo cells with gene KO displayed a similar profile of immunologically important cell surface molecule expression and cytokine secretion as the CD4⁺25⁺ Tr/exo cells (data not shown).

Phenotypic characterization of DCs, EXO, CD4⁺ Tr1, and CD4⁺25⁺ Tr cells

For the phenotypic analyses, DC_OVA/IL-10, DC_OVA/IL-10-derived EXO, naive CD4⁺ T cells, CD4⁺ Tr1, CD4⁺25⁺ Tr, Tr/exo, and Tr/exo(K^b–/–) cells were stained with a panel of biotin-conjugated Abs. DCs and CD4⁺ Tr1 cells were also stained with PE-anti-CD4 and FITC-anti-CD11c Abs. For the intracellular cytokines, T cells were permeabilized and stained with biotin-conjugated anti-IL-4, -IL-10, or -IFN- Abs. After washing with PBS, the cells were further stained with PE-conjugated avidin and analyzed by flow cytometry. CD4⁺ Tr1 and CD4⁺ Tr1(vivo) cells were restimulated by culturing with irradiated (4000 rad) OVA II-pulsed LB27 cells for 24 h (15). Their culture supernatants were then analyzed for cytokine expression using ELISA kits (Endogen) as previously described (15).

Ag presentation

RF3370 hybridoma cells (0.5 x 10⁵ cells/well) were cultured with irradiated (4000 rad) DC_OVA, CD4⁺ Tr1, (K^b–/–)Tr1, Tr/exo, and Tr/exo(K^b–/–) (1 x 10⁵ cells/well) for 24 h. The supernatants were then harvested for measurement of IL-2 secretion using an ELISA kit (Endogen) (15).

CD8⁺ T cell proliferation inhibition assays

In vitro assay. In the nonspecific T cell proliferation assay, irradiated (4000 rad) DCs (0.1 x 10⁵ cells/well) or 2-fold dilutions thereof were incubated with naive C57BL/6 CD8⁺ T (0.5 x 10⁵ cells/well) cells in the presence of anti-CD3 Ab (1 µg/ml) for 48 h, then cell division was assessed by [³H]thymidine incorporation as noted (26). In the OVA-specific T cell proliferation assay, irradiated (4000 rad) DC_OVA or DCs (0.1 x 10⁵ cells/well) or 2-fold dilutions thereof were incubated with naive OT I CD8⁺ T (0.5 x 10⁵ cells/well) cells. In some inhibition experiments, irradiated (4000 rad) CD4⁺ Tr1 cells (0.5 x 10⁵ cells/well) and its 2-fold dilutions or anti-IL-10 or -IFN- Abs (10 µg/ml) were added to the above cell cultures. After 48 h, [³H]thymidine incorporation was determined by liquid scintillation counting (15).

In vivo assay. C57BL/6 mice were immunized i.v. with 0.5 x 10⁶ irradiated (4000 rad) DC_OVA alone or 0.5 x 10⁶ DC_OVA plus 2 x 10⁶ CD4⁺ Tr1, (IFN-^–/–)Tr1, (IL-10^–/–)Tr1 or (K^b–/–)Tr1 cells, or 3 x 10⁶ Tr1(vivo) or (K^b–/–)Tr1(vivo) cells. Six days later, the OVA-specific CD8⁺ T cells in tail blood or splenocyte samples from each mouse were stained by incubating the blood with PE-H-2K^b/OVAI tetramer and FITC-anti-CD8 Ab (PK135) (Beckman Coulter). The erythrocytes were then lysed using a lysis/fixation buffer (Beckman Coulter), and samples were analyzed by flow cytometry according to the company’s protocol (27).

CD8⁺ T cell cytotoxicity assay

To assess the cytotoxic activity of the OVA-specific CD8⁺ T cells induced as above in the proliferation assays, the OVAI-pulsed cells were first strongly (3.0 µM; CFSE^high) and the MutI-pulsed cells weakly (0.6 µM, CFSE^low) labeled with CFSE. Six days following immunization, the above-immunized mice were then injected i.v. with a 1:1 mixture of splenocyte targets that had been pulsed with OVAI or MutI peptides (CFSE^high and CFSE^low). Sixteen hours after target cell delivery, the spleens of the recipient mice were removed and the relative proportions of CFSE^high and CFSE^low target cells remaining in the spleens were analyzed by flow cytometry (27).

Animal studies

Wild-type C57BL/6 mice (n = 8) were immunized s.c. with 0.5 x 10⁶ irradiated (4000 rad) BM-derived DC_OVA, either alone or along with 2 x 10⁶ CD4⁺ Tr1 (wild-type, K^b–/–, IFN-^–/–, or IL-10^–/–) or 3 x 10⁶ CD4⁺ Tr1(vivo) or (K^b–/–)Tr1(vivo) or varying numbers of CD4⁺ (K^b–/–)Tr1 cells (injected i.v.). In another set of animal studies, C57BL/6 mice (n = 8) were immunized with 2 x 10⁶ irradiated (4000 rad) splenic DC_OVA, either alone or along with varying numbers of CD4⁺25⁺ Tr/exo (wild-type or K^b–/–) cells with or without exosomal pMHC I expression (injected i.v.). Seven days later, the mice were challenged s.c. with 0.3 x 10⁶ BL6-10_OVA tumor cells, then tumor growth was monitored daily for up to 6 wk. For humanitarian reasons, all mice with tumors that achieved a size of 1.0 cm in diameter were sacrificed.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
AdV_IL-10-transfected DCs induce differentiation of type 1 regulatory CD4⁺ T cells

To generate tolerogenic DCs, we transfected OVA-pulsed DCs (DC_OVA) with an IL-10-expressing adenoviral vector (AdV_IL-10). These AdV_IL-10-transfected DC_OVA (DC_OVA/IL-10) expressed MHC class II (Ia^b), CD11c (DC marker), CD40, CD54, CD80, and OVA pMHC I (Fig. 1a), and high level of IL-10 (2.2 ng/ml/10⁶ cells per 24 h). Except for deficiency in pMHC I expression, (K^b–/–)DC_OVA/IL-10 derived from AdVIL-10-transfected (K^b–/–)DC_OVA had a similar phenotypic profile as DC_OVA/IL-10 (data not shown). We incubated naive CD4⁺ T cells from OT II/H-2K^b–/– mice with irradiated DC_OVA/IL-10 for 3 days, then purified the CD4⁺ T cells by CD4 microbeads, and analyzed them by flow cytometry. As depicted in Fig. 1b, these T cells expressed cell surface CD4, CD25, CD69, and TCR, indicating that they are activated OVA-specific CD4⁺ T cells. There were no contaminated CD11c-positive residual DCs within the purified T cell population (Fig. 1, b and c). They also expressed intracellular IFN- and IL-10, but not IL-4, as indicated by flow cytometry (Fig. 1b) and ELISA (Fig. 1d). More specifically, they secreted IFN- (2.0 ng/ml/10⁶ cells per 24 h) and IL-10 (2.4 ng/ml/IL-10 per 10⁶ cells per 24 h), but relatively little IL-4 or TNF- and no detectable IL-2 and TGF-β. In addition, they also expressed the cell surface GITR, but not cell surface CD30 and TGF-β nor intracellular CTLA-4 and Foxp3 (Fig. 1b). Taken together, these data indicate that these cells display the phenotype of type 1 CD4⁺ regulatory T (Tr1) cells (28, 29).

View larger version (25K): [in this window] [in a new window]   FIGURE 1. Flow cytometric analysis. DCOVA/IL-10 and DCOVA/IL-10-released EXO (a) and naive CD4+ T and in vitro DCOVA/IL-10-stimulated CD4+ Tr1 cells (b) were stained with a panel of Abs (solid lines) and analyzed by flow cytometry. Isotype-matched irrelevant Abs were used as controls (dotted lines). c, Purified DCOVA/IL-10 and CD4+ Tr1 cells were stained with FITC-anti-CD4 and PE-anti-CD11c Abs and then analyzed by flow cytometry. d, Cytokine expression in CD4+ Tr1 and Tr1(vivo) cell supernatants were measured by ELISA. The values presented represent the means of triplicate cultures. e, The in vitro and in vivo DCOVA/IL-10- and (Kb–/–)DCOVA/IL-10-stimulated CD4+ Tr1, (Kb–/–)Tr1, Tr1(vivo), and (Kb–/–)Tr1(vivo) cells derived from OT II/B6.1 mice were stained with anti-pMHC I Ab (solid lines), and analyzed by flow cytometry. The isotype-matched irrelevant Ab was used as a control (dotted lines). One representative experiment of three in the above experiments is shown.

As noted, these in vitro DC_OVA/IL-10-stimulated CD4⁺ Tr1 cells even derived from OT II/H-2K^b–/– mice expressed low, but nevertheless significant, levels of pMHC I (Fig. 1b), indicating that they would have been acquired from DC_OVA/IL-10 possibly via the pathway of internalization/recycling of synapse-comprised compositions (14). To confirm it, we also incubated these naive CD4⁺ T cells with (K^b–/–)DC_OVA/IL-10 without pMHC I expression and the resulting CD4⁺ Tr1 cells did not express any discernible pMHC I by flow cytometry (Fig. 1e), confirming that CD4⁺ Tr1 cells directly acquire pMHC I from DC_OVA/IL-10. To assess whether it also occurs in vivo, we transferred naive CD4⁺ T cells from OT II/B6.1 mice into C57BL/6 mice, then immunized these animals with either DC_OVA/IL-10 or (K^b–/–)DC_OVA/IL-10. Three days later, we purified the transferred population of CD4⁺ T cells back out of these animals by using CD45.1 microbeads and found that the CD4⁺ Tr(vivo) cells from the mice that had been immunized with DC_OVA/IL-10, but not with (K^b–/–)DC_OVA/IL-10, displayed low, although significant levels of pMHC I (Fig. 1e). These data confirm that naive CD4⁺ T cells do acquire pMHC I from DC_OVA/IL-10 with which they interact, both in vitro and in vivo, indicating that the acquisition of pMHC I by CD4⁺ Tr cells is of physiological significance. In addition, these CD4⁺ Tr(vivo) cells also had a similar cytokine profile as in vitro DC_OVA/IL-10-activated CD4⁺ Tr1 cells (Fig. 1d), indicating that they are also CD4⁺ Tr1 cells.

CD4⁺ Tr1 cells inhibit in vitro CD8⁺ T cell proliferative responses through secretion of IL-10

To assess the functional effect of acquired pMHC I on CD4⁺ Tr1 cells, we performed an IL-2 secretion assay (24). We found that CD4⁺ Tr1 and Tr1(vivo), but not (K^b–/–)Tr1 cells stimulated RF3360 T cell hybridoma cell lines to secrete IL-2 (Fig. 2a), indicating that CD4⁺ Tr1 cells express the functional pMHC I complexes. To assess the suppressive effect of CD4⁺ Tr1 cells, we performed two types of inhibition assays, including the inhibition of nonspecific and OVA-specific T cell proliferation. In the former assay, we added in vitro and in vivo DC_OVA/IL-10-stimulated CD4⁺ Tr1 and Tr1(vivo) cells to the cell culture containing DCs and C57BL/6 CD8⁺ T cells in the presence of anti-CD3 Ab and assessed the CD8⁺ T cell proliferative responses, respectively. We found that these CD4⁺ Tr1 cells inhibited this response in a dose-dependent manner (Fig. 2b). In the later assay, we assessed whether our CD4⁺ Tr1 and Tr1(vivo) cells could inhibit the in vitro DC_OVA-stimulated OVA-specific OT I CD8⁺ T cell proliferation and found that they all inhibited this response in a dose-dependent manner (Fig. 2c), indicating that CD4⁺ Tr1 cells can inhibit both nonspecific and OVA-specific CD8⁺ T cell proliferation in vitro. To confirm it, we performed a similar experiment using the in vitro DC_OVA/IL-10-stimulated CD4⁺ (K^b–/–)Tr1 cells and found that both CD4⁺ Tr1 cells with acquired pMHC I and CD4⁺ (K^b–/–)Tr1 cells without acquired pMHC I inhibited the in vitro DC_OVA-stimulated OVA-specific OT I CD8⁺ T cell proliferation to a similar extent (Fig. 2c), indicating that the acquired pMHC I on CD4⁺ Tr1 does not play any role in inhibition of the in vitro T cell proliferation. Furthermore, this inhibitory effect of CD4⁺ Tr1 cells induced in vitro was 80% inhibited by adding anti-IL-10, but not -IFN-, Ab to the cultures (Fig. 2d), thereby implicating IL-10 as central to the activity of these regulatory T cells.

View larger version (17K): [in this window] [in a new window]   FIGURE 2. Suppressive effect of CD4+ Tr1 cells on in vitro CD8+ T cell proliferation. a, The amount of IL-2 secretion of stimulated RF3370 cells in examining wells was subtracted by the amount of IL-2 in wells containing DCOVA, CD4+ Tr1, Tr1(vivo), (Kb–/–)Tr1, and RF3370 cells, respectively. b, Irradiated DCs and its 2-fold dilutions were incubated with naive C57BL/6 CD8+ T cells. In another set of experiments, irradiated CD4+ Tr1 and Tr1(vivo) cells and their 2-fold dilutions were added to the above cell mixture containing a constant number of DCs and naive CD8+ T cells. c, Irradiated DCOVA and its 2-fold dilutions were incubated with naive OT I CD8+ T cells. In another set of experiments, irradiated CD4+ Tr1, (Kb–/–)Tr1, and Tr1(vivo) cells and their 2-fold dilutions were added to the above cell mixture containing a constant number of DCOVA and CD8+ T cells. d, To assess the role of IL-10, irradiated DCOVA and CD4+ Tr1 cells were cultured with naive OT I CD8+ T cells in the presence of anti-IL-10 or IFN- Ab. After 2 days of incubation, the proliferative responses of CD8+ T cells in the above cultures were determined using a [3H]thymidine uptake assay. One representative experiment of two in the above experiments is shown.

That this suppressive effect was not simply an in vitro artifact was confirmed by demonstrating that these CD4⁺ Tr1 cells could also suppress OVA-specific CD8⁺ T cell proliferative responses in vivo by measurement of OVA-specific CD8⁺ T cells in mouse peripheral blood. Intravenous immunization of DC_OVA-stimulated proliferation of CD8⁺ T cells, such that the OVA-specific CD8⁺ T cells accounted for 1.61% of the total CD8⁺ T cell population in the peripheral blood. However, when we coinjected CD4⁺ Tr1 cells into the DC_OVA-immunized recipients, the percentages of OVA-specific CD8⁺ T cells in mouse peripheral blood significantly dropped to only 0.34% (p < 0.05), suggesting that the pMHC I-carrying CD4⁺ Tr1 cells generated in vitro specifically suppressed the DC_OVA-driven CD8⁺ T cell responses. Importantly, analogous CD4⁺ Tr1 cells that were generated in vivo also significantly suppressed DC_OVA-induced CD8⁺ T cell responses upon passive transfer (0.38% of the blood CD8⁺ T cells in these recipients was OVA specific) (p < 0.05; Fig. 3a), indicating the physiological significance of our observation. To explore the molecular basis of this effect, we repeated these experiments using CD4⁺ Tr1 cells in which either the IL-10 or IFN- gene had been knocked out. The IL-10^–/– Tr1 cells had no significant effect on the CD8⁺ T cell response to DC_OVA vaccination (1.58% of the blood CD8⁺ T cells were OVA specific), indicating that IL-10 is critical for the realization of the CD4⁺ Tr1 cell-suppressive effect, while IFN--deleted CD4⁺ Tr1 cells remained fully functional in terms of significantly ameliorating the CD8⁺ T cells response (0.29% OVA-specific CD8⁺ T cells) (p < 0.05; Fig. 3a). We hypothesized that the pMHC I acquired by Tr1 cells were integral to their suppressive effect. In our next experiment, we used CD4⁺ Tr1 cells generated in vitro by culture of OVA-pulsed, AdV_IL-10-transfected K^b–/– DCs with naive OT II CD4⁺ T cells or in vivo by vaccination of B6.1-positive naive OT II CD4⁺ T cell-transferred C57BL/6 mice with similar DCs. Our data confirmed that neither CD4⁺ (K^b–/–)Tr1 cells generated in vitro nor those generated in vivo were able to discernibly suppress the induction of OVA-specific CD8⁺ T cell proliferation relative to transfer of DC_OVA alone. In mice given DC_OVA plus in vitro-generated (K^b–/–)Tr1, 1.53% of blood CD8⁺ T cells were OVA specific, whereas 1.62% CD8⁺ T cells in the mice treated with in vivo-generated CD4⁺ (K^b–/–)Tr1 cells were OVA specific (Fig. 3a). To confirm that a decrease in OVA-specific CD8⁺ T cell percentage in peripheral blood derived from CD4⁺ Tr cell suppression does represent a reduction in the cell proliferation, we also measured the OVA-specific CD8⁺ T cells in the mouse spleens. We found that the patterns of OVA-specific CD8⁺ T cell number in different groups of mouse spleens were similar to those seen in mouse peripheral blood (Fig. 3b), which is consistent with our previous report (30). Taken together, these data suggest that the expression of both IL-10 and pMHC I is critical to the in vivo regulatory effect of CD4⁺ Tr1 cells.

View larger version (22K): [in this window] [in a new window]   FIGURE 3. Suppressive effect of CD4+ Tr1 cells on in vivo CD8+ T cell proliferation and effector function. In an OVA-specific CD8+ T cell proliferation inhibition assay in vivo, the tail blood samples (a) and splenocytes (b) from mice immunized with irradiated DCOVA, either alone or along with CD4+ Tr1 cells generated in vitro (Tr1) or in vivo (Tr1(vivo)) or (IL-10–/–)Tr1, (IFN-–/–)Tr1, and (Kb–/–)Tr1 cells, were stained with PE-H-2Kb/OVAI tetramers and FITC-anti-CD8 Ab and then analyzed by flow cytometry. The values in each panel represent the percentage of tetramer-positive CD8+ T cells vs the total CD8+ T cell pool in peripheral blood or the total tetramer-positive CD8+ T cells per spleen and, parenthetically, the SD. One representative experiment of three is depicted for each of the above experiments. *, Represents p < 0.05 vs cohorts of immunized mice treated with Tr1, Tr1(IFN-–/–), and Tr1(vivo) cells, respectively (Student’s t test). c, In vivo CD8+ T cell cytotoxicity assay. The CFSE-labeled (CFSEhigh and CFSElow) target cells were i.v. injected into immunized mice. Sixteen hours later, the relative proportions of CFSEhigh and CFSElow cells remaining in the spleens of the recipient mice were assessed by flow cytometry. The values in each panel represent the percentage of CFSEhigh cells (±SD) and CFSElow cells remaining in the spleens. *, Represents p < 0.05 vs cohorts of immunized mice treated with Tr1, Tr1(IFN-–/–), and Tr1(vivo) cells, respectively (Student’s t test). One representative experiment of two in the above experiments is shown.

To determine whether the impact of our pMHC I-expressing CD4⁺ Tr1 cells on CD8⁺ T cell proliferation would translate into effects on their cytotoxic functions, we used an in vivo cytotoxicity assay. We adoptively transferred OVAI peptide-pulsed splenocytes that had been strongly labeled with CFSE (CFSE^high), along with control peptide Mut1-pulsed splenocytes that had been weakly labeled with CFSE (CFSE^low), into recipient mice that had been vaccinated 6 days previously with 0.5 x 10⁶ DC_OVA, and found that 88% of the CFSE^high target cells, but none of the negative control Mut1 peptide-pulsed (i.e., CFSE^low) target cells were killed over 16 h after transfer (Fig. 3c). If, however, we cotransferred 2 x 10⁶ CD4⁺ Tr1 cells with the DC_OVA, only 7% of the CFSE^high target cells were killed (p < 0.05), indicating that these CD4⁺ Tr1 cells also significantly suppressed the OVA-specific CTL activity. Transfer of analogous CD4⁺ Tr1(vivo) cells also effectively suppressed the CTL activity of the OVA-specific CD8⁺ T cells (22% residual CFSE^high target cell killing; p < 0.05). As in the proliferation inhibition assays, we also assessed the roles of IL-10 and pMHC I in this CD4⁺ Tr1 cell suppressive activity. Here too, treatment of the DC_OVA-vaccinated mice with CD4⁺ (IFN-^–/–)Tr1 cells had little impact on the suppressor activity (10% residual CFSE^high target cell killing; p < 0.05), while knocking out the IL-10 gene in these CD4⁺ Tr1 cells largely eliminated their abilities to suppress CTL activity (77% residual killing), as did ablating their expression of pMHC I (i.e., (K^b–/–)Tr1; 74% residual killing). Taken together, these data indicate that the suppressive effect of CD4⁺ Tr1 on in vivo CD8⁺ CTL responses is mainly mediated and specifically targeted by its IL-10 secretion and pMHC I acquisition, respectively.

Acquired pMHC I on CD4⁺ Tr1 cells enhance its efficiency in suppressing antitumor immunity

Our final affirmation of the functional relevance of CD4⁺ Tr1 cell pMHC I was an assessment of their impact on development of antitumor immunity in mice vaccinated with DC_OVA and challenged with OVA-transfected BL6–10_OVA tumor cells. All untreated tumor-bearing mice succumbed to BL6-10_OVA tumor cell inoculation, whereas all DC_OVA-immunized mice (eight of eight) survived tumor challenge (Fig. 4a). Cotreatment at the time of DC_OVA vaccination with 2 x 10⁶ CD4⁺ Tr1 cells ablated the protective effects of vaccination (eight of eight mice died of their tumors). Most (75%) of the CD4⁺ (IL-10^–/–)Tr1 cell-treated, DC_OVA-immunized mice (six of eight) maintained their immune protection against the BL6-10_OVA tumor cell challenge, while the deaths of the two unprotected mice in this group was delayed relative to the wild-type CD4⁺ Tr1 cell-treated group (Fig. 4b). These data confirm that the CD4⁺ Tr1 cell suppressive effects on DC_OVA-induced antitumor immunity is mainly mediated by IL-10 secretion. Again, IFN- gene KO in the CD4⁺ Tr1 cells had no significant effect on their activity. To disclose the critical role of the acquired pMHC I in CD4⁺ Tr1 cell-mediated suppression, in another set of experiments, all DC_OVA-immunized mice were injected i.v. with 2 x 10⁶ CD4⁺ Tr1 or (K^b–/–)Tr1 cells. As above, all (sight of eight) Tr1 cell-treated mice succumbed to their tumor burdens, whereas all (eight of eight) mice given 2 x 10⁶ CD4⁺ (K^b–/–)Tr1 cells survived the tumor cell challenge (Fig. 4b), indicating that the acquisition of pMHC I contributes importantly to CD4⁺ Tr1 cell activity. As alluded to above, we hypothesized that IL-10 was the effector molecule for suppression of the protective CTL response by the CD4⁺ Tr1 cells, but that expression by these cells of pMHC I allowed for cognate, and therefore much more efficient, interactions between the IL-10-expressing CD4⁺ Tr1 cells and the CD8⁺ CTL. This suggests that if sufficiently large numbers of pMHC I-deficient CD4⁺ Tr1 cells were present in the system, then the targeting deficiency of these cells could perhaps be overcome. Thus, in our next set of experiments, we titrated increasing numbers of (K^b–/–)Tr1 cells (2–7 x 10⁶ cells/mouse) into the DC_OVA-immunized mice and assessed the impact of these treatments on tumor protection as above. We found that six of eight mice given 7 x 10⁶ (K^b–/–)Tr1 cells succumbed to their tumors and that as the numbers of CD4⁺ (K^b–/–)Tr1 cells administered were reduced, then recipient survival increased in a dose-dependent fashion (Fig. 4c). Thus, 7 x 10⁶ (K^b–/–)Tr1 cells performed as well as 1 x 10⁶ K^b+/+ Tr1 cells in terms of inducing tumor tolerance. This suggests that acquisition of Ag-specific pMHC I by CD4⁺ Tr1 cells, which would increase efficiency of cognate CD8⁺ T cell targeting, increased the efficiency of tolerance induction by the CD4⁺ Tr1 cells by 700%. Finally, just as was observed with suppression of CTL activity, we found that treatment of immunized mice with CD4⁺ Tr1 cells generated in vivo also inhibited DC_OVA-induced antitumor immunity, inasmuch as only one (13%) eight of these mice survived (Fig. 4d). Here too, treatment of the mice with 3 x 10⁶ CD4⁺ (K^b–/–)Tr1(vivo) cells had no impact on tumor immunity, indicating again the physiological significance of pMHC I acquisition by CD4⁺ Tr1 cells.

View larger version (31K): [in this window] [in a new window]   FIGURE 4. Suppressive effect of CD4+ Tr1 cells on antitumor immunity. Wild-type C57BL/6 mice were either injected s.c. with irradiated DCOVA alone or in conjunction with i.v. injected Tr1 (a), Tr1 with respective gene KO (b), varying numbers of (Kb–/–)Tr1 cells (c), or Tr1(vivo) or (Kb–/–)Tr1(vivo) (d). Six days later, the mice were s.c. inoculated with BL6-10OVA tumor cells, then mouse survival was monitored daily for up to 6 wk. One representative experiment of two is depicted.

Next, we assessed whether the non-Ag-specific thymus-derived CD4⁺25⁺ Tr cells (2, 3) can obtain the Ag specificity via acquired pMHC I. Since the nonspecific CD4⁺ T cells can acquire pMHC I via uptake of Ag-specific DC-released EXO through the CD54-LFA-1 interaction pathway (31), but not via interactions with Ag-specific DCs. To confirm the Ag-specific targeting role of pMHC I, we conducted another set of experiments by using the nonspecific CD4⁺25⁺ Tr cells with uptake of DC_OVA/IL-10-released EXO. Naive CD4⁺25⁺ Tr cells purified from C57BL/6 or OT II mouse spleens were in vitro expanded using anti-CD3 and anti-CD28 Ab-coated beads. These expanded Tr cells derived from OT II mice expressed CD4, TCR, active T cell markers (CD25 and CD69) (Fig. 5a), and Foxp3 (Fig. 5b), indicating that they are active Tr cells. Except for TCR expression, CD4⁺25⁺ Tr cells derived from C57BL/6 mice had a similar phenotype as those derived from OT II mice (data not shown). We then purified EXO, (K^b–/–)EXO, and (Ia^b–/–)EXO from DC_OVA/IL-10, (K^b–/–)DC_OVA/IL-10, and (Ia^b–/–)DC_OVA/IL-10 culture supernatants, respectively (24). Similar to DC_OVA/IL-10, EXO also expressed MHC class I (H-2K^b) and class II (Ia^b), CD11c, CD40, CD54, CD80, and pMHC I, but in less content, compared with DC_OVA/IL-10 (Fig. 1a) (24). Except for lacking the pMHC I expression, (K^b–/–)EXO had a similar phenotype as EXO (data not shown). To assess acquisition of exosomal pMHC I, CD4⁺25⁺ Tr cells were incubated with EXO for 4 h and then analyzed by flow cytometry. CD4⁺25⁺ Tr cells originally without pMHC I expression did express some pMHC I after incubation with EXO, indicating that they acquire the exosomal pMHC I (Fig. 5c). This was further confirmed by the evidence that Tr cells failed to express any discernible pMHC I when incubated with (K^b–/–)EXO lacking pMHC I expression (Fig. 5c). To assess the functional effect of exosomal pMHC I, we performed an IL-2 secretion assay (24). We found that CD4⁺25⁺ Tr/exo, but not Tr/exo(K^b–/–), stimulated RF3360 T cell hybridoma cell lines to secrete IL-2 (Fig. 5d), indicating that CD4⁺25⁺ Tr/exo cells express the functional pMHC I complexes.

View larger version (38K): [in this window] [in a new window]   FIGURE 5. Nonspecific CD4+25+ Tr cells acquire Ag specificity via uptake of Ag-specific DC-released EXO. a, Purified T cells were stained with a panel of Abs (solid lines) and analyzed by flow cytometry. b, CD4+25+ Tr cells were stained with FITC-anti-CD4, PE-anti-CD25, and energy-coupled dye (ECD)-anti-Foxp3 Abs and then analyzed by flow cytometry. The FITC-CD4 and PE-CD25-positive cells were sorted (circled) for analysis of Foxp3 expression (solid line). c, CD4+25+ Tr, Tr/exo, and Tr/exo(Kb–/–) cells were stained with biotin-anti-pMHC I Ab followed by FITC-avidin (solid lines) and analyzed by flow cytometry. In the above experiments, isotype-matched irrelevant Ab was used as control (dotted lines). d, The amount of IL-2 secretion of stimulated RF3370 cells in examining wells was subtracted by the amount of IL-2 in wells containing DCOVA, CD4+25+ Tr/exo, Tr/exo(Kb–/–), and RF3370 cells, respectively. e, In OVA-specific CD4+ T cell proliferation assay, the tail blood samples from wild-type C57BL/6 mice immunized with irradiated DCOVA, either alone or along with CD4+25+ Tr cells derived from C57BL/6 (B6 Tr) or OT II (OT II Tr) mice or B6 Tr/exo, Tr/exo(Kb–/–), and Tr/exo(Iab–/–) cells, respectively, were stained with PE-H-2Kb/OVAI tetramers and FITC-anti-CD8 Ab and then analyzed by flow cytometry. The values in each panel represent the percentage of tetramer-positive CD8+ T cells vs the total CD8+ T cell pool and, parenthetically, the SD. *, Represents p < 0.05 vs cohorts of immunized mice treated with C57BL/6 (B6) and OT II mouse Tr cells or with B6 mouse Tr/exo and Tr/exo(Iab–/–) cells, respectively, or representing p > 0.05 vs cohorts of immunized mice treated with B6 mouse Tr/exo(Kb–/–) cells (Student’s t test). f, Wild-type C57BL/6 mice (n = 8) were immunized s.c. with irradiated splenic DCOVA, either alone or along with varying numbers of CD4+25+ Tr/exo and Tr/exo(Kb–/–). Seven days later, the mice were challenged s.c. with BL6-10OVA tumor cells, then tumor growth was monitored daily for up to 6 wk. One representative experiment of two is depicted for each of the above experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The Ag specificity of CD4⁺ regulatory T (Tr) cells has been evidenced in CD4⁺ Tr cell-mediated immune suppression in autoimmune diseases (4, 5), infectious diseases (6, 7), and antitumor immunity (8, 9). However, the natural target Ags recognized by these cells remain largely unknown. Our current knowledge of the Ag specificity of Tr cells has come largely from studies with Ag-specific TCR-transgenic mice (5, 10, 11). The important role for Ag-specific CD4⁺ Tr cells has also been documented using nontransgenic mouse systems. For example, McGuirk et al. (7) provided the first demonstration of pathogen-specific Tr cells at the clonal level. Zhang et al. (32) demonstrated that in (2C x dm2)_F1 mice, which express a transgenic TCR specific for MHC I L^d, double-negative (i.e., CD4^–CD8^–) Tr cells acquire allo-MHC peptides from APCs and use these for recognition and trapping of allo-specific CD8⁺ T cell, which they then eliminate. However, the molecular mechanism by which the suppressive effects of CD4⁺ Tr cells are specifically targeted to the Ag-specific CD8⁺ effector T cells following Ag presentation has been somewhat unclear. In this study, for the first time, we clearly elucidate that it is the acquired pMHC I that delivers the DC_OVA/IL-10-stimulated CD4⁺ Tr1 cell’s regulatory effect directly to Ag-specific CD8⁺ T cells, which specifically engages the TCR of CD8⁺ T cells recognizing the same Ag peptides and thereby suppresses the CD8⁺ T cell response in situ. This augmentation of CD4⁺ Tr1 cell-CD8⁺ effector T cell interactions was associated with an 700% increase in the efficiency with which the CD4⁺ Tr1 cells inhibited tumor Ag-specific immunity, relative to that observed with otherwise equivalent CD4⁺ (K^b–/–)Tr1 cells. It is well known that the suppressor function of thymus-derived CD4⁺25⁺ Tr cells is Ag nonspecific (33). In this study, however, we further demonstrated that CD4⁺25⁺ Tr cells expressing CTLA4, GITR, and Foxp3 can also acquire the Ag specificity via uptake of Ag-specific DC-released EXO. Coincidentally, CD4⁺25⁺ Tr/exo cells with acquired exosomal pMHC I (Ag specificity) (although with suppressive mechanisms distinct from CD4⁺ Tr1 cells (2)) also displayed enhanced inhibitory efficiency on tumor Ag-specific immunity by 8- to 10-fold compared with CD4⁺25⁺ Tr/exo(K^b–/–) cells without exosomal pMHC I expression, thus further supporting our above conclusion.

CD4⁺ Tr cells can suppress the proliferation of CD4⁺ and CD8⁺ T cells (34, 35), in part, via inhibition of IL-2 production (36) or reduce the effector function of T cells (37). They can transfer suppressive properties to other CD4⁺ T cells, resulting in new CD4⁺ suppressive T cells (38) or T cell anergy (39, 40). They can also down-regulate MHC class II, CD80, and CD86 expression on DCs (35, 41) and induce IDO expression (42). In this study, we elucidated the molecular mechanism for the DC_OVA/IL-10-stimulated CD4⁺ Tr1 cell suppressive effect in vivo. We demonstrated that its suppressive effect on OVA-specific CD8⁺ CTL responses is mainly mediated by its IL-10 secretion, probably leading to induction of CD8⁺ T cell anergy (43, 44) by altering the CD28 costimulation pathway (45). It has been shown that Ag-specific CD4⁺ Tr cells derived from TCR-transgenic mice more efficiently suppress immune responses than do polyclonal CD4⁺ Tr T cells from nontransgenic mice (5, 10). However, in this study, we demonstrated that CD4⁺25⁺ Tr/exo cells derived from either the transgenic OT II or wild-type C57BL/6 mice (i.e., with or without OVA-specific TCR) exhibited a similar level of suppression in DC_OVA-stimulated CD8 CTL responses, indicating that the OVA-specific TCRs on CD4⁺25⁺ Tr cells do not make any contribution to the immune suppression, which is consistent with a recent report by Li et al. (46).

Taken together, our results suggest a new model for acquisition of Ag specificity by adoptive CD4⁺ Tr cells, that of acquiring pMHC I from the Ag-presenting DCs (Fig. 6). According to this model, 1) naive CD4⁺ T cells, when stimulated with Ag-pulsed and IL-10-secreting DCs, become Ag-specific CD4⁺ Tr cells secreting IL-10 that carry pMHC I transferred from the DCs and 2) the acquired pMHC I significantly increase the efficiency with which CD4⁺ Tr cells target the suppressive IL-10 to Ag-specific CD8⁺ T cell responses and thereby suppress these responses and antitumor immunity. The principles elucidated in this study may have significant implications not only in antitumor immunity, but also in other sectors of immunology (e.g., autoimmunity and transplantation).

View larger version (15K): [in this window] [in a new window]   FIGURE 6. A model for augmentation of Ag-specific CD8+ T cell suppression by CD4+ Tr cells that have acquired pMHC I complexes from APC. According to this model, IL-10-expressing the tolerogenic DC expressing IL-10 1) induces CD4+ T cell differentiation into a regulatory phenotype (Tr) by virtue of their IL-10 secretion and 2) transfers its bystander pMHC I complexes onto naive Ag-specific CD4+ T cell-expressing Ag-specific TCR through the pMHC class I-/TCR interaction between the DC and the CD4+ T cell by DC activation. The IL-10-expressing CD4+ Tr cell with acquired pMHC I complex then specifically interacts with the cognate Ag-specific CD8+ T cells through the latter cell’s TCR, suppressing their responses to the Ag challenge.

	Acknowledgments

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This study was supported by a research grant (MOP 79415) from the Canadian Institutes for Health Research. S.H. was supported by a Postdoctoral Fellowship from the Saskatchewan Health Research Foundation.

² Address correspondence and reprint requests to Dr. Jim Xiang, Saskatoon Cancer Center, 20 Campus Drive, Saskatoon, Saskatchewan S7N 4H4, Canada. E-mail address: jim.xiang{at}saskcancer.ca

³ Abbreviations used in this paper: Tr, regulatory T; DC, dendritic cell; pMHC I, peptide MHC class I; AdV, adenovirus; GITR, glucocorticoid-induced TNFR; KO, knockout; BM, bone marrow; EXO/exo, exosome.

Received for publication January 15, 2008. Accepted for publication June 17, 2008.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Hori, S., T. Nomura, S. Sakaguchi. 2003. Control of regulatory T cell development by the transcription factor Foxp3. Science 299: 1057-1061. [Abstract/Free Full Text]
2. Shevach, E. M.. 2002. CD4⁺ CD25⁺ suppressor T cells: more questions than answers. Nat. Rev. Immunol. 2: 389-400. [Medline]
3. Cozzo, C., J. Larkin, III, A. J. Caton. 2003. Cutting edge: self-peptides drive the peripheral expansion of CD4⁺CD25⁺ regulatory T cells. J. Immunol. 171: 5678-5682. [Abstract/Free Full Text]
4. Huang, X., J. Zhu, Y. Yang. 2005. Protection against autoimmunity in nonlymphopenic hosts by CD4⁺CD25⁺ regulatory T cells is antigen-specific and requires IL-10 and TGF-β. J. Immunol. 175: 4283-4291. [Abstract/Free Full Text]
5. Sanchez-Fueyo, A., S. Sandner, A. Habicht, C. Mariat, J. Kenny, N. Degauque, X. X. Zheng, T. B. Strom, L. A. Turka, M. H. Sayegh. 2006. Specificity of CD4⁺CD25⁺ regulatory T cell function in alloimmunity. J. Immunol. 176: 329-334. [Abstract/Free Full Text]
6. Doetze, A., J. Satoguina, G. Burchard, T. Rau, C. Loliger, B. Fleischer, A. Hoerauf. 2000. Antigen-specific cellular hyporesponsiveness in a chronic human helminth infection is mediated by T_h3/T_r1-type cytokines IL-10 and transforming growth factor-β but not by a T_h1 to T_h2 shift. Int. Immunol. 12: 623-630. [Abstract/Free Full Text]
7. McGuirk, P., C. McCann, K. H. Mills. 2002. Pathogen-specific T regulatory 1 cells induced in the respiratory tract by a bacterial molecule that stimulates interleukin 10 production by dendritic cells: a novel strategy for evasion of protective T helper type 1 responses by Bordetella pertussis. J. Exp. Med. 195: 221-231. [Abstract/Free Full Text]
8. Wang, H. Y., D. A. Lee, G. Peng, Z. Guo, Y. Li, Y. Kiniwa, E. M. Shevach, R. F. Wang. 2004. Tumor-specific human CD4⁺ regulatory T cells and their ligands: implications for immunotherapy. Immunity 20: 107-118. [Medline]
9. Wang, H. Y., R. F. Wang. 2005. Antigen-specific CD4⁺ regulatory T cells in cancer: implications for immunotherapy. Microbes Infect. 7: 1056-1062. [Medline]
10. Hori, S., M. Haury, A. Coutinho, J. Demengeot. 2002. Specificity requirements for selection and effector functions of CD25⁺4⁺ regulatory T cells in anti-myelin basic protein T cell receptor transgenic mice. Proc. Natl. Acad. Sci. USA 99: 8213-8218. [Abstract/Free Full Text]
11. Scalapino, K. J., Q. Tang, J. A. Bluestone, M. L. Bonyhadi, D. I. Daikh. 2006. Suppression of disease in New Zealand Black/New Zealand White lupus-prone mice by adoptive transfer of ex vivo expanded regulatory T cells. J. Immunol. 177: 1451-1459. [Abstract/Free Full Text]
12. Huang, J. F., Y. Yang, H. Sepulveda, W. Shi, I. Hwang, P. A. Peterson, M. R. Jackson, J. Sprent, Z. Cai. 1999. TCR-mediated internalization of peptide-MHC complexes acquired by T cells. Science 286: 952-954. [Abstract/Free Full Text]
13. Hwang, I., J. F. Huang, H. Kishimoto, A. Brunmark, P. A. Peterson, M. R. Jackson, C. D. Surh, Z. Cai, J. Sprent. 2000. T cells can use either T cell receptor or CD28 receptors to absorb and internalize cell surface molecules derived from antigen-presenting cells. J. Exp. Med. 191: 1137-1148. [Abstract/Free Full Text]
14. He, T., S. Zong, X. Wu, Y. Wei, J. Xiang. 2007. CD4⁺ T cell acquisition of the bystander pMHC I colocalizing in the same immunological synapse comprising pMHC II and costimulatory CD40, CD54, CD80, OX40L, and 41BBL. Biochem. Biophys. Res. Commun. 362: 822-828. [Medline]
15. Xiang, J., H. Huang, Y. Liu. 2005. A new dynamic model of CD8⁺ T effector cell responses via CD4⁺ T helper-antigen-presenting cells. J. Immunol. 174: 7497-7505. [Abstract/Free Full Text]
16. Shi, M., S. Hao, T. Chan, J. Xiang. 2006. CD4⁺ T cells stimulate memory CD8⁺ T cell expansion via acquired pMHC I complexes and costimulatory molecules, and IL-2 secretion. J. Leukocyte Biol. 80: 1354-1363. [Abstract/Free Full Text]
17. Faulkner, L., G. Buchan, M. Baird. 2000. Interleukin-10 does not affect phagocytosis of particulate antigen by bone marrow-derived dendritic cells but does impair antigen presentation. Immunology 99: 523-531. [Medline]
18. Akbari, O., R. H. DeKruyff, D. T. Umetsu. 2001. Pulmonary dendritic cells producing IL-10 mediate tolerance induced by respiratory exposure to antigen. Nat. Immunol. 2: 725-731. [Medline]
19. Kim, S. H., E. R. Lechman, N. Bianco, R. Menon, A. Keravala, J. Nash, Z. Mi, S. C. Watkins, A. Gambotto, P. D. Robbins. 2005. Exosomes derived from IL-10-treated dendritic cells can suppress inflammation and collagen-induced arthritis. J. Immunol. 174: 6440-6448. [Abstract/Free Full Text]
20. Mitchell, D. A., S. K. Nair, E. Gilboa. 1998. Dendritic cell/macrophage precursors capture exogenous antigen for MHC class I presentation by dendritic cells. Eur. J. Immunol. 28: 1923-1933. [Medline]
21. Porgador, A., J. W. Yewdell, Y. Deng, J. R. Bennink, R. N. Germain. 1997. Localization, quantitation, and in situ detection of specific peptide-MHC class I complexes using a monoclonal antibody. Immunity 6: 715-726. [Medline]
22. Xing, Z., Y. Ohkawara, M. Jordana, F. L. Graham, J. Gauldie. 1997. Adenoviral vector-mediated interleukin-10 expression in vivo: intramuscular gene transfer inhibits cytokine responses in endotoxemia. Gene Ther. 4: 140-149. [Medline]
23. Zhang, W., Z. Chen, F. Li, H. Kamencic, B. Juurlink, J. R. Gordon, J. Xiang. 2003. Tumour necrosis factor- (TNF-) transgene-expressing dendritic cells (DCs) undergo augmented cellular maturation and induce more robust T-cell activation and anti-tumour immunity than DCs generated in recombinant TNF-. Immunology 108: 177-188. [Medline]
24. Hao, S., O. Bai, F. Li, J. Yuan, S. Laferte, J. Xiang. 2007. Mature dendritic cells pulsed with exosomes stimulate efficient cytotoxic T-lymphocyte responses and antitumour immunity. Immunology 120: 90-102. [Medline]
25. Xia, G., J. He, Z. Zhang, J. R. Leventhal. 2006. Targeting acute allograft rejection by immunotherapy with ex vivo-expanded natural CD4⁺ CD25⁺ regulatory T cells. Transplantation 82: 1749-1755. [Medline]
26. Pace, L., S. Rizzo, C. Palombi, F. Brombacher, G. Doria. 2006. Cutting edge: IL-4-induced protection of CD4⁺CD25^– Th cells from CD4⁺CD25⁺ regulatory T cell-mediated suppression. J. Immunol. 176: 3900-3904. [Abstract/Free Full Text]
27. Xia, D., S. Hao, J. Xiang. 2006. CD8⁺ cytotoxic T-APC stimulate central memory CD8⁺ T cell responses via acquired peptide-MHC class I complexes and CD80 costimulation, and IL-2 secretion. J. Immunol. 177: 2976-2984. [Abstract/Free Full Text]
28. Battaglia, M., S. Gregori, R. Bacchetta, M. G. Roncarolo. 2006. Tr1 cells: from discovery to their clinical application. Semin. Immunol. 18: 120-127. [Medline]
29. Zhang, X., H. Huang, J. Yuan, D. Sun, W. S. Hou, J. Gordon, J. Xiang. 2005. CD4^–8^– dendritic cells prime CD4⁺ T regulatory 1 cells to suppress antitumor immunity. J. Immunol. 175: 2931-2937. [Abstract/Free Full Text]
30. Shi, M., J. Xiang. 2006. CD4⁺ T cell-independent maintenance and expansion of memory CD8⁺ T cells derived from in vitro dendritic cell activation. Int. Immunol. 18: 887-895. [Abstract/Free Full Text]
31. Hao, S., Y. Liu, J. Yuan, X. Zhang, T. He, X. Wu, Y. Wei, D. Sun, J. Xiang. 2007. Novel exosome-targeted CD4⁺ T cell vaccine counteracting CD4⁺25⁺ regulatory T cell-mediated immune suppression and stimulating efficient central memory CD8⁺ CTL responses. J. Immunol. 179: 2731-2740. [Abstract/Free Full Text]
32. Zhang, Z. X., L. Yang, K. J. Young, B. DuTemple, L. Zhang. 2000. Identification of a previously unknown antigen-specific regulatory T cell and its mechanism of suppression. Nat. Med. 6: 782-789. [Medline]
33. Thornton, A. M., E. M. Shevach. 2000. Suppressor effector function of CD4⁺CD25⁺ immunoregulatory T cells is antigen nonspecific. J. Immunol. 164: 183-190. [Abstract/Free Full Text]
34. Piccirillo, C. A., E. M. Shevach. 2001. Cutting edge: control of CD8⁺ T cell activation by CD4⁺CD25⁺ immunoregulatory cells. J. Immunol. 167: 1137-1140. [Abstract/Free Full Text]
35. Levings, M. K., R. Sangregorio, M. G. Roncarolo. 2001. Human CD25⁺CD4⁺ T regulatory cells suppress naive and memory T cell proliferation and can be expanded in vitro without loss of function. J. Exp. Med. 193: 1295-1302. [Abstract/Free Full Text]
36. Thornton, A. M., E. M. Shevach. 1998. CD4⁺CD25⁺ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J. Exp. Med. 188: 287-296. [Abstract/Free Full Text]
37. Trzonkowski, P., E. Szmit, J. Mysliwska, A. Dobyszuk, A. Mysliwski. 2004. CD4⁺CD25⁺ T regulatory cells inhibit cytotoxic activity of T CD8⁺ and NK lymphocytes in the direct cell-to-cell interaction. Clin. Immunol. 112: 258-267. [Medline]
38. Jonuleit, H., E. Schmitt, H. Kakirman, M. Stassen, J. Knop, A. H. Enk. 2002. Infectious tolerance: human CD25⁺ regulatory T cells convey suppressor activity to conventional CD4⁺ T helper cells. J. Exp. Med. 196: 255-260. [Abstract/Free Full Text]
39. Vanasek, T. L., S. L. Nandiwada, M. K. Jenkins, D. L. Mueller. 2006. CD25⁺Foxp3⁺ regulatory T cells facilitate CD4⁺ T cell clonal anergy induction during the recovery from lymphopenia. J. Immunol. 176: 5880-5889. [Abstract/Free Full Text]
40. Qiao, M., A. M. Thornton, E. M. Shevach. 2007. CD4⁺CD25⁺ [corrected] regulatory T cells render naive CD4⁺CD25^– T cells anergic and suppressive. Immunology 120: 447-455. [Medline]
41. Cederbom, L., H. Hall, F. Ivars. 2000. CD4⁺CD25⁺ regulatory T cells down-regulate co-stimulatory molecules on antigen-presenting cells. Eur. J. Immunol. 30: 1538-1543. [Medline]
42. Munn, D. H., M. D. Sharma, J. R. Lee, K. G. Jhaver, T. S. Johnson, D. B. Keskin, B. Marshall, P. Chandler, S. J. Antonia, R. Burgess, et al 2002. Potential regulatory function of human dendritic cells expressing indoleamine 2,3-dioxygenase. Science 297: 1867-1870. [Abstract/Free Full Text]
43. Seewaldt, S., J. Alferink, I. Forster. 2002. Interleukin-10 is crucial for maintenance but not for developmental induction of peripheral T cell tolerance. Eur. J. Immunol. 32: 3607-3616. [Medline]
44. Sharif, O., V. N. Bolshakov, S. Raines, P. Newham, N. D. Perkins. 2007. Transcriptional profiling of the LPS induced NF-B response in macrophages. BMC Immunol. 8: 1-17. [Medline]
45. Joss, A., M. Akdis, A. Faith, K. Blaser, C. A. Akdis. 2000. IL-10 directly acts on T cells by specifically altering the CD28 co-stimulation pathway. Eur. J. Immunol. 30: 1683-1690. [Medline]
46. Li, J., M. Bracht, X. Shang, J. Radewonuk, E. Emmell, D. E. Griswold, L. Li. 2006. Ex vivo activated OVA specific and non-specific CD4⁺CD25⁺ regulatory T cells exhibit comparable suppression to OVA mediated T cell responses. Cell. Immunol. 241: 75-84. [Medline]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Hao, S. Articles by Xiang, J. Search for Related Content PubMed PubMed Citation Articles by Hao, S. Articles by Xiang, J.