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 Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Xia, D. Articles by Xiang, J. Search for Related Content PubMed PubMed Citation Articles by Xia, D. Articles by Xiang, J.

CD8⁺ Cytotoxic T-APC Stimulate Central Memory CD8⁺ T Cell Responses via Acquired Peptide-MHC Class I Complexes and CD80 Costimulation, and IL-2 Secretion¹

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

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
We previously showed that naive CD4⁺ Th cells acquire peptide-MHC class I (pMHC I) and costimulatory molecules from OVA-pulsed dendritic cells (DC_OVA), and act as Th-APCs in stimulation of CD8⁺CTL responses. In this study, we further demonstrated that naive CD8⁺ cytotoxic T (Tc) cells also acquire pMHC I and costimulatory CD54 and CD80 molecules by DC_OVA stimulation, and act as Tc-APC. These Tc-APC can play both negative and positive modulations in antitumor immune responses by eliminating DC_OVA and neighboring Tc-APC, and stimulating OVA-specific CD8⁺ central memory T responses and antitumor immunity. Interestingly, the stimulatory effect of Tc-APC is mediated via its IL-2 secretion and acquired CD80 costimulation, and is specifically targeted to OVA-specific CD8⁺ T cells in vivo via its acquired pMHC I complexes. These principles could be applied to not only antitumor immunity, but also other immune disorders (e.g., autoimmunity).

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
In general, there are two types of CTL responses, Th cell-dependent and Th cell-independent ones. Generation of effective CTL responses to minor histocompatibility or tumor Ags that are not associated with danger signals often requires help from CD4⁺ Th cells by APC cross-priming (1). Sometimes, Th cell-independent CTL immune responses can also be induced, especially in some virus infections (2, 3, 4) or vaccination of peptides emulsified in CFA (5). In these cases, the virus infection and CFA can license dendritic cells (DCs)⁴ to directly activate CD8⁺ CTL without the help effect of CD4⁺ Th cells as described in a model of two cell interactions by APC (Fig. 1A) (6, 7).

View larger version (16K): [in this window] [in a new window]   FIGURE 1. Two models for the Th-independent immune response. A, The model of two-cell interaction, in which APC "licensed" by virus infection or danger signals can directly stimulate effector CTL responses. B, The dynamic model of sequential two-cell interaction, in which the licensed APC stimulate CD8+ T cells to differentiate into effector memory CTL (emCTL). These activated CD8+ T cells acquire pMHC I and costimulatory molecules and act as APC (Tc-APC) to further stimulate the host CD8+ central memory T cell (cmCTL) responses. In contrast, these Tc-APC (emCTL) have also cytotoxic activities to DCOVA and neighboring Tc-APC. Thus, Tc-APC play both positive and negative modulations in immune responses.

In this study, we developed a model system using OVA-transfected EG7 tumor cells and the OVA-specific TCR transgenic OT I and OT II mice with MHC I and II specificities, respectively (6, 14). Based upon this model system, we investigated 1) the specific DC membrane molecules transferred onto OT I CD8⁺ T cells by OVA-pulsed DC (DC_OVA) stimulation and 2) the regulatory effects of these CD8⁺ T cells with acquired APC molecules in immune responses. In addition, we also elucidated the molecular mechanisms responsible for the stimulatory effect and for the targeted delivery of this stimulatory effect of activated CD8⁺ T cells with acquired DC molecules to naive CD8⁺ T cells in vivo.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Reagents, cell lines, and animals

OVA was obtained from Sigma-Aldrich. OVA I (SIINFEKL) and MutI (FEQNTAQP) are OVA-specific and 3LL lung carcinoma-specific peptides, respectively, which were purchased from Multiple Peptide Systems. Monoclonal anti-mouse CD4, CD8, CD11c, CD25, CD44, CD54, CD62L, CD69, CD80, and IL-7R Abs, PE-conjugated anti-perforin, and biotin-conjugated anti-mouse V5.1, 5.2 TCR, anti-CD45.1 Abs were all obtained from BD Pharmingen. The anti-H-2K^b/OVA I (pMHC I) Ab was obtained from Dr. T. Germain (National Institutes of Health, Bethesda, MD) (15). Anti-LFA-1, -IL-2, -IL-4, -IFN- Abs, the CTLA-4/Ig fusion protein, and recombinant mouse GM-CSF, IL-2, IL-7, IL-12, and IL-15 were purchased from R&D Systems. CFSE was obtained from Molecular Probes. OVA I (SIINFEKL) and irrelevant Mut1 (FEQNTAQP) peptides were synthesized by Multiple Peptide Systems. The highly lung metastatic B16 mouse melanoma BL6–10 and OVA-transfected BL6–10 (BL6–10_OVA) cell lines were generated in our own laboratory (13). The mouse thymoma cell line EL4 and the OVA-transfected EL4 cell line EG7 were obtained from American Type Culture Collection. The RF3370 T cell hybridoma cell line bearing TCR specific for pMHC I was a gift from Dr. K. Rock (University of Massachusetts Medical School, Worcester, MA). Female C57BL/6 (B6, CD45.2⁺) C57BL/6.1 (B6.1, CD45.1⁺) mice were obtained from Charles River Laboratories. OVA-specific TCR-transgenic OT I, and IL-2, IFN-, CD54, and CD80 knockout (KO) mice on a C57BL/6 background were purchased from The Jackson Laboratory. Homozygous OT I/IL-2^–/–, OT I/IFN-^–/–, OT I/CD54^–/–, OT I/CD80^–/–, and OT I/B6.1 mice were generated by backcrossing the designated gene KO mice onto the OT I background for three generations; homozygosity was confirmed by PCR according to The Jackson Laboratory’s protocols. All mice were treated according to animal care committee guidelines of the University of Saskatchewan.

Generation of bone marrow (BM)-derived DCs

BM-derived DCs were generated as described previously (16). These DCs were pulsed with 0.1 mg/ml OVA overnight at 37°C, then washed extensively and served as DC_OVA (17)_. DC generated from CD54 and CD80 gene KO mice were referred to as (CD54^–/–)DC_OVA and (CD80^–/–)DC_OVA, respectively.

Preparation of OT I CD8⁺ T cells

Naive OVA-specific CD8⁺ T cells were prepared from splenocytes of OT I-transgenic mice, enriched by passage through nylon wool columns (C&A Scientific), and purified by negative selection using anti-CD4 (L3T4) paramagnetic beads (Dynal) as previously described (13). To generate in vitro DC_OVA-activated CD8⁺ T cells, CD8⁺ T cells (2 x 10⁵ cells/ml) from OT I mice or designated gene-deleted OT I mice were stimulated for 3 days with irradiated (4000 rad) DC_OVA (1 x 10⁵ cells/ml) in the presence of IL-2 (10 U/ml), IL-12 (5 ng/ml), and anti-IL-4 Ab (10 µg/ml) (18). These in vitro DC_OVA-activated CD8⁺ T cells, also referred to herein as CD8⁺ cytotoxic T (Tc)-APC, were then isolated by Ficoll-Paque (Sigma-Aldrich) density gradient centrifugation, or further purified using CD8 microbeads (Miltenyi Biotec). These CD8⁺ T cells derived from OT I mice with respective IL-2 and IFN- gene KO were referred to as Tc (IL-2^–/–)-APC and Tc(IFN-^–/–)-APC, respectively. In vitro (CD54^–/–)DC_OVA- and (CD80^–/–)DC_OVA-activated CD8⁺ T cells derived from OT I mice with respective CD54 and CD80 gene KO were referred to as Tc(CD54^–/–)-APC and Tc(CD80^–/–)-APC, respectively. To prepare in vivo DC_OVA-activated CD8⁺ T cells, OT I mice were i.v. injected with irradiated DC_OVA (5 x 10⁵ cells). Three days later, T cells were purified from immunized mouse splenocytes by nylon wool columns and then positive selection using biotin-conjugated anti-V5.1, 5.2 TCR Ab, and anti-biotin microbeads (Miltenyi Biotec). Con A-stimulated OT I CD8⁺ T (Con A-OT I) cells were similarly generated by incubating splenocytes from OT I mice with Con A (1 µg/ml) and IL-2 (10 U/ml) for 3 days, after which the CD8⁺ T cells were purified on density gradients and then using CD8 microbeads (Miltenyi Biotec).

Phenotypic characterization of DC_OVA-activated CD8⁺ T cells

For the phenotypic analyses, Tc-APC were stained with a panel of Abs and analyzed by flow cytometry. For the intracellular cytokines, cells were restimulated with 4000 rad-irradiated EG7 tumor cells for 4 h (18), and then processed using a "Cytofix/CytoPerm Plus with GolgiPlug" kit (BD Pharmingen), with R-PE-conjugated anti-perforin Ab (R&D Systems). Culture supernatants of the restimulated Tc-APC and Con A-OT I were analyzed for cytokine expression using ELISA kits (Endogen), as previously reported (17).

In vitro and in vivo membrane molecule transfer assays

In in vitro membrane transfer assay, DC_OVA were incubated with CFSE (0.5 µM) at 37°C for 15 min and washed three times with PBS. CFSE-labeled DC_OVA were incubated with Con A-OT I cells at 37°C for 4 h, then the cell mixtures, the original DC_OVA, and Con A-OT I cells were stained with a panel of PE-Texas Red-X (energy-coupled dye (ECD))-conjugated Abs specific for CD54 and CD80, and analyzed by confocal fluorescence microscopy. The CD8⁺ T cells in the cell mixture were purified by cell sorting and analyzed by flow cytometry. In in vivo membrane transfer assay, naive T cells were isolated from OT I/CD54^–/– and OT I/CD80^–/– mouse spleens, respectively, and enriched by passage through nylon wool columns. The CD8⁺ T cells (5 x 10⁶ cells/mouse) were further purified by negative selection using the anti-mouse CD4 (L3T4) paramagnetic beads (Dynal), and then i.v. injected into wild-type C57BL/6 mice. One day subsequent to the injection, mice were i.v. immunized with irradiated (4000 rad) DC_OVA (0.2 x 10⁶ cells/mouse). Another group of mice remained untreated. Three days after the immunization, mice were sacrificed. T cells were isolated from the spleens of these two groups of mice, and enriched by passage through nylon wool columns. The OVA-specific CD8⁺ OT I T cells were further purified from these T cells by positive selection using the biotin-anti-TCR Ab and anti-biotin microbeads (Miltenyi Biotec), termed CD8⁺ Tc-APCvivo and then stained with FITC-anti-CD54 and FITC-anti-CD80 Abs for flow cytometric analysis, respectively.

Ag presentation

RF3370 hybridoma cells (0.5 x 10⁵ cells/well) were cultured with irradiated (4000 rad) DC_OVA, Tc-APC, Tc-APCvivo, Con A-OT I (1 x 10⁵ cells/well) for 24 h. To investigate the fate of acquired MHC I/peptide expression, Tc-APC alone were cultured for 1, 2, and 3 days in culture medium containing IL-2 (10 U/ml), termed Tc-APC (1, 2, and 3 day), and then harvested for stimulation of RF3370 cells, respectively. The supernatants were harvested for measurement of IL-2 secretion using an ELISA kit (Endogen).

CD8⁺ T cell proliferation assays

For in vitro CD8⁺ T cell proliferation assay, irradiated (4000 rad) stimulators, the Tc-APC, Tc-APCvivo, Con A-OT I cells, DC_OVA (0.4 x 10⁵ cells/well) and their 2-fold dilutions were cultured with a constant number of responders, the naive OT I or C57BL/6 (B6) CD4⁺, CD8⁺ T cells (0.5 x 10⁵ cells/well). In some experiments, each of a panel of neutralizing reagents (anti-IL-2, -H-2K^b, -LFA-1, and -IFN- Abs, and CTLA-4/Ig fusion protein) (each 15 µg/ml; R&D Systems) or a mixture of the above reagents were added to the cell cultures containing Tc-APC and naive CD8⁺ OT I T cells, while control cells received a mixture of isotype-matched irrelevant Abs and fusion protein. After 48 h, thymidine incorporation was determined by liquid scintillation counting (17).

In vitro and in vivo cytotoxicity assays

In in vitro cytotoxicity assay, the above DC_OVA- and Tc-APC-primed OT I CD8⁺ T cells were used as effector (E) cells, while ⁵¹Cr-labeled EG7, DC, DC_OVA, and Tc-APC cells were used as target (T) cells. In another set of cytotoxicity assays, naive CD8⁺ T cells derived from OT I/B6.1 (CD45.1⁺) mice were cocultured with irradiated (4000 rad) DC_OVA and Tc-APC for 2 days. The above active CD8⁺ T cells were harvested and purified on density gradients followed by biotin-conjugated anti-CD45.1 Ab and anti-biotin microbeads (Miltenyi Biotec). These T cells were referred to as DC_OVA/OT I_6.1 and Tc-APC/OT I_6.1, respectively, and used as effector (E) cells, while ⁵¹Cr-labeled EG7 and the control EL-4 tumor cells were used as target (T) cells. Specific killing was calculated as: 100 x ((experimental cpm – spontaneous cpm)/(maximal cpm – spontaneous cpm)), as previously described (17). In in vivo cytotoxicity assay, C57BL/6 mice were i.v. immunized with DC_OVA (0.5 x 10⁶ cells), Tc-APC or Con A-OT I cells (4 x 10⁶ cells). In another group, mice were injected with PBS. Naive mouse splenocytes were incubated with either high (3.0 µM, CFSE^high) or low (0.6 µM, CFSE^low) concentrations of CFSE, to generate differentially labeled target cells. The CFSE^high cells were pulsed with OVA I, whereas the CFSE^low cells were pulsed with Mut1 peptide and served as internal controls. These peptide-pulsed target cells were washed extensively to remove free peptide, and then i.v. coinjected at a 1:1 ratio into the above immunized mice 3 days after the immunization. Sixteen hours after target cell delivery, the spleens were removed and residual CFSE^high and CFSE^low target cells remaining in the recipients’ spleens were sorted and analyzed by flow cytometry.

In vivo CD8⁺ memory T cell (Tm) expansion

Naive C57BL/6 mice were i.v. transferred with in vitro DC_OVA- and Tc-APC-primed CD8⁺ T cells (5 x 10⁶ cells) derived from OT I/B6.1 mice. A tetramer staining assay was performed to examine the presence of OVA-specific CD8⁺ T cells in mouse peripheral blood 5 days and once a week for 3 mo after the adoptive transfer. The tail blood samples were incubated with PE-H-2K^b/OVAI tetramer, FITC-anti-CD8 (PK135), and ECD-anti-CD44 or ECD-anti-CD45.1 Abs. Three months after the adoptive transfer, mice were boosted by i.v. injection of OVA I peptide (50 µl, 0.2 mM) or irradiated (4000 rad) DC_OVA (0.5 x 10⁶ cells), Tc-APC, Con A-OT I, and Tc-APC with various gene KO (3 x 10⁶ cells), respectively. Four days after the boost, the tail blood samples from these mice were stained with PE-H-2K^b/OVAI tetramer, FITC-anti-CD8 and ECD-anti-CD25, CD44, CD62L and CD69 Abs or ECD-anti-CD45.1 Ab.

Animal studies

Wild-type C57BL/6, CD4 KO, or CD8 KO mice (n = 8) were injected i.v. with irradiated (4000 rad) DC_OVA (0.5 x 10⁶ cells/mouse), Tc-APC, Tc(IL-2^–/–)-APC, Tc(IFN-^–/–)-APC, Tc(CD54^–/–)-APC, Tc(CD80^–/–)-APC, or Tc-APCvivo and Con A-OT I cells (4 x 10⁶ cells/mouse), respectively. Six days subsequent to the injection, the mice were i.v. given 0.5 x 10⁶ BL6–10_OVA or BL6–10 tumor cells. The mice were sacrificed 4 wk after tumor cell injection, and the lung metastatic tumor colonies were counted in a blind fashion (19). Metastases on freshly isolated lungs appeared as discrete black pigmented foci that 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.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Active CD8⁺ Tc acquire pMHC I, CD54, and CD80 molecules by DC interaction

To explore membrane transfer acquisition, Con A-stimulated OT I CD8⁺ T (Con A-OT I) cells were cultured with DC_OVA and then analyzed by flow cytometry. The control Con A-OT I cells expressed some CD54 and CD80, but not pMHC I. However, following incubation with DC_OVA, they displayed moderately augmented expression of these molecules (Fig. 2A), suggesting that CD8⁺ T cells acquire these molecules. Because active T cells also express CD54 and some CD80 (20), it is necessary to confirm that the acquisition of these molecules is not due to endogenous up-regulation. Thus, we used Con A-OT I T cells derived from OT I/CD54^–/– and OT I/CD80^–/– mice, which did not express the deleted gene products. However, they did discernibly express CD54 and CD80 after incubation with DC_OVA as determined by flow cytometry (Fig. 2B) and confocal fluorescence microscopy (Fig. 2C). These results indicate that, besides previously reported MHC class I and CD80 molecules (11, 21, 22), CD8⁺ T cells also acquire the immunological synapse-composed CD54 by DC/CD8⁺ T cell interactions (9, 10).

View larger version (37K): [in this window] [in a new window]   FIGURE 2. Transfer of DC membrane molecules to active CD8+ T cells. A, CFSE-labeled DCOVA were incubated with Con A-stimulated OT I CD8+ T cells (Con A-OT I). T cells with (thick solid lines) and without (thick dotted lines) incubation of DCOVA were stained with Abs and analyzed for expression of pMHC I, CD54, and CD80 by flow cytometry, respectively. B, CFSE-labeled DCOVA were incubated with Con A-OT I T cells derived from OT I mice with CD54 and CD80 gene KO, respectively. T cells with (thick solid lines) and without (thick dotted lines) incubation of DCOVA were stained with PE-Abs and analyzed for expression of the above molecules, respectively. The isotype-matched PE Abs were used as controls (thin dotted lines). C, Membrane acquisition analysis by confocal fluorescence microscopy. CFSE-labeled DCOVA were incubated with Con A-OT I T cells from OT I mice with 1) CD54 and 2) CD80 gene KO, stained with ECD-labeled Abs, and analyzed by confocal fluorescence microscopy. Images include DCs (larger cells) alone, T (smaller) cells alone or a mixture of DC and T cells (1) under differential interference contrast, (2) with a cell surface stain consisting of ECD (red) Ab for either CD54 or CD80, (3) with cytoplasmic CFSE stain (green), and (4) with both stains. Our data confirm that 1) DCOVA (larger cells), but not gene-deleted T cells (smaller cells), express CD54 and CD80 molecules (arrows), and 2) during coculture of DCOVA with T cells, the T cells acquire CD54 and CD80 molecules (arrowheads). D, In vitro DCOVA-activated CD8+ T cells (Tc-APC) were stained with a panel of Abs (thick solid lines) and analyzed by flow cytometry. The control CD8+ T cells (thin dotted lines) were only stained with isotype-matched Abs. E, DCOVA, Tc-APC, or Con A-OT I T cells were stained with Abs and analyzed for expression of CD11c and CD8 by flow cytometry. F, Tc-APC from CD54 and CD80 gene KO OT I mice were stained with a panel of Abs (thick solid lines). The control CD8+ T cells (thin dotted lines) were only stained with isotype-matched Abs. G, In vivo membrane transfer assay. The CD8+ T cells purified from OT I/CD54–/– and OT I/CD80–/– mice were transferred into wild-type C57BL/6 mice, respectively. The first group of mice were remained untreated and the second group of mice were immunized with DCOVA. The CD8+ OT I/CD54–/– and OT I/CD80–/– T cells were then purified from the first (thick dotted lines) and the second group (solid lines) of mice and then stained with the FITC-anti-CD54 and FITC-anti-CD80 Abs and the FITC-conjugated isotype-matched Abs (thin dotted lines) for flow cytometric analysis. One representative experiment of three is shown.

We then incubated naive OT I CD8⁺ T cells with DC_OVA for 3 days. The activated CD8⁺ T cells expressed CD8, CD25, and CD69 (Fig. 2D), and secreted IFN- (1.5 ng/ml/10⁶ cells/24 h) and IL-2 (1.8 ng/ml/10⁶ cells/24 h), but not IL-4 by ELISA, indicating that they are active type 1 Tc (Tc1) cells. They also expressed pMHC I (Fig. 2D), indicating they acquire pMHC I by DC activation. In addition, there was no CD11c⁺ DC existing in the purified CD8⁺ T cell population (Fig. 2, D and E). In addition, these Tc1 cells also expressed CD54 and CD80 molecules, and here too they did so whether they were derived from wild-type or homozygous CD54^–/– or CD80^–/– KO mice (Fig. 2F). Thus, we clearly demonstrated that naive CD8⁺ T cells can acquire the synapse-composed pMHC I and costimulatory (CD54 and CD80) molecules by in vitro DC stimulation. To further confirm the membrane acquisition in vivo, C57BL/6 mice with previous transfer of naive CD8⁺ T cells derived from OT I/CD54^–/– or OT I/CD80^–/– mice were immunized with DC_OVA. Three days after the immunization, CD8⁺ OT I T cells purified from immunized mice became slightly CD54 and CD80 positive (Fig. 2G), indicating that naive CD8⁺ OT I T cells can also acquire CD54 and CD80 molecules by in vivo DC_OVA stimulation.

Tc-APC negatively modulate immune responses

Because these CD8⁺ T cells acquired pMHC I and costimulatory molecules, they were referred to as Tc-APC (11). We first assessed their ability to stimulate IL-2 secretion of T cell hybridoma RF3370. As shown in Fig. 3A, RF3370 cells alone did not secret IL-2. However, Tc-APC and Tc-APCvivo stimulated them to secret IL-2 (87 and 48 pg/ml) as did DC_OVA (230 pg/ml), indicating that the acquired pMHC I complexes are functional. The rate of decay was assessed by culturing these Tc-APC for varying time periods in culture. As shown in Fig. 3A, the ability to stimulate IL-2 secretion of RF3370 cells did decay over time. However, readily detectable stimulation was still observed as much as 3 days after in vitro culture, indicating that the acquired pMHC I on Tc-APC is quite stable, which is consistent with another report by Undale et al. (23), showing that the acquired MHC II molecules on human T cells from APC can last for 12 days in culture. Because these active CD8⁺ cells expressed TCR specific for pMHC I complexes, they may kill pMHC I-expressing DC_OVA. As shown in Fig. 3B, Tc-APC exhibited strong cytotoxicity for OVA-expressing EG7 tumor cells (83% specific killing at an E:T cell ratio of 25) and DC_OVA (42% specific killing at an E:T cell ratio of 25), indicating that CD8⁺ T cells when they are activated by DC_OVA and become active Tc-APC can kill DC_OVA. However, they did not show any killing activity to DC without OVA pulsing, indicating that the killing activity is OVA specific. Because these CD8⁺ T cells acquired functional pMHC I, they also became sensitive to neighboring Tc-APC cells expressing TCR (11). Our data displayed that Tc-APC killed ⁵¹Cr-Tc-APC (fratricide) (22% specific killing at an E:T cell ratio of 25), but to a lesser extent, compared with the killing of DC_OVA and EG7 tumor cells, indicating that Tc-APC may negatively modulate immune responses by eliminating pMHC I-expressing DC_OVA and Tc-APC (Fig. 1B).

View larger version (12K): [in this window] [in a new window]   FIGURE 3. Functional analysis of Tc-APC. A, RF3370 hybridoma cells (0.5 x 105 cells/well) were cultured with irradiated (4000 rad) DCOVA, Tc-APC, Tc-APCvivo, and Con A-OT I (1 x 105 cells/well) for 24 h. In addition, Tc-APC which were cultured for 1, 2, and 3 days in culture medium containing IL-2, termed Tc-APC (days 1, 2, and 3) were also used. After 24 h, the supernatants of each well were collected for IL-2 detection. The amount of IL-2 secretions of RF3370 cells stimulated by the above stimulators in examining wells were subtracted by the amounts of IL-2 in wells containing the stimulators alone. *, p < 0.05 (Student t test) vs cohorts of Con A-OT I. B, Cytotoxicity assay. Tc-APC were used as effector (E) cells, whereas 51Cr-labeled EG7, DCs, DCOVA, and Tc-APC cells used as target (T) cells. The data are presented as the percent-specific target cell lysis in 51Cr-release assay. Each point represents the mean of triplicate cultures. One representative experiment of three is shown.

We then assessed their ability to induce in vitro proliferation of CD8⁺ T cells. The positive control DC_OVA strongly induced OT I T cell proliferation (Fig. 4A), whereas Tc-APC and Tc-APCvivo did stimulate the proliferation of OT I CD8⁺ T cells, but to a lesser extent possibly due to its fewer pMHC I and costimulatory molecules, compared with DC_OVA. However, Tc-APC did not stimulate responses of the control naive C57BL/6 (B6) mouse CD8⁺ T cells, nor did Con A-OT I cells (secreting IFN- (1.8 ng/ml/10⁶ cells/24 h) and IL-2 (1.9 ng/ml/10⁶ cells/24 h), but not IL-4 (not detected), but without acquired pMHC I) stimulate OT I CD8⁺ T cell proliferation. Furthermore, as shown in Fig. 4B, adding anti-MHC class I Ab or CTLA-4/Ig fusion protein could significantly inhibit the OT I CD8⁺ T cell proliferative response in the cocultures containing Tc-APC and OT I CD8⁺ T cells by 58 and 48%, respectively, while anti-IL-2 and -LFA-1 Abs, but not the anti-IFN- Ab, had some lesser, but still significant, effect (32, 19, and 27% inhibition), respectively (p < 0.05). Simultaneous addition of all blocking reagents reduced the proliferative response by 92% (p < 0.01). Taken together, our data indicate that this stimulatory response is critically dependent on specific H-2K^b/OVAI/TCR interaction and greatly affected by the nonspecific CD80/CD28 and CD54/LFA-1 interactions, and IL-2 secretion.

View larger version (31K): [in this window] [in a new window]   FIGURE 4. Tc-APC stimulate CD8+ T cell differentiation into central memory T cells. A, In vitro CD8+ T cell proliferation assay. Varying numbers of irradiated Tc-APC, Tc-APCvivo, Con A-OT I, and DCOVA cells were cocultured with naive OT I or B6 CD8+ T cells. After 3 days, the proliferative responses of the CD8+ T cells were determined by [3H]thymidine uptake assays. B, In blocking assay, Tc-APC were cultured with OT I CD8+ T cells in the presence of each of the neutralizing reagents, all neutralizing reagents together (mixed reagents), or all control Abs and fusion proteins (control reagents). *, p < 0.05 (Student t test) vs cohorts of Tc-APC. C, Phenotypic analysis of in vitro Tc-APC-primed CD8+ T cells. CFSE-labeled naive OT I CD8+ T cells were primed with irradiated DCOVA and Tc-APC for 2 days in vitro and stained for CD25, CD44, CD62L, and IL-7R, respectively. Dot plots of CFSE-positive CD8+ T cells are shown indicating that the labeled CD8+ T cells underwent some cycles of cell division, and were sorted (circled) for flow cytometric analysis. One representative experiment of three is shown.

We also conducted phenotypic characterization of in vitro Tc-APC-primed CD8⁺ T cells. Our data showed that DC_OVA and Tc-APC primings resulted in several cycles of CD8⁺ T cell division, and all the primed T cells displayed expression of CD25, CD44 (Tm marker) (24), and CD62L. However, Tc-APC-primed CD8⁺ T cells displayed higher CD62L expression than DC_OVA-primed ones, and also expressed IL-7R (Fig. 4C), indicating they may be central memory CD8⁺ T (cmCTL) cells (CD44⁺CD62L^highIL-7R⁺) (25, 26). To confirm it, DC_OVA- and Tc-APC-primed CD8⁺ T cells derived from OTI/45.1⁺ mice were i.v. transferred into C57BL/6 (B6, CD45.2⁺) mice. The number of detected OVA-specific CD8⁺44⁺ memory T cells derived from DC_OVA- and Tc-APC priming in the mouse blood accounted for 8.2 and 8.4% of the total CD8⁺ T cell population at day 5 after the transfer (Fig. 5, A and B). The numbers then gradually dropped to 2.12 and 5.28% in the first month subsequent to, but stably maintained for at least 3 mo after the transfer (Fig. 5A). Among them, large amounts of OVA-specific CD8⁺ T cells (2.08 and 5.03%) detected in the blood were also CD45.1 positive (Fig. 5B), indicating that they are derived from the originally transferred CD8⁺ T cells. We then examined whether Tc-APC-primed CTL exhibited any other functional traits attributed to typical memory T cells. The functional traits include 1) the enhanced survival and proliferation in response to IL-7 and IL-15 (27), 2) the capacity to generate Ag-specific CTL, and 3) to expand upon Ag stimulation. Our data showed that CTL primed by Tc-APC expanded better in presence of IL-2, IL-7, and IL-15 than those primed with DC_OVA (Fig. 5C). In chromium release assay, Tc-APC-primed CTL (Tc-APC/OT I_6.1) showed cytotoxicity to OVA-expressing EG7, but not to EL4 tumor cells, indicating that the killing activity is OVA specific. However, their cytotoxicity was much lower than DC_OVA-primed ones (DC_OVA/OT I_6.1) (Fig. 5D). In addition, they can be greatly expanded, accounting for 88% of the total CD8⁺ T cell population upon OVA I peptide stimulation, which is 3-fold more than the expanded DC_OVA-primed CD8⁺ Tm cells (28%) (Fig. 5E). The expanded CD8⁺ Tm cells were active CD25⁺44⁺62L⁺69⁺ effector CTL. Taken together, our data demonstrate that DC_OVA- and Tc-APC-primed CTL have high and low cytotoxicity, but low and high survival capacity, behaving as typical effector and central memory T cells, respectively. Therefore, they were referred to as emCTL and cmCTL (Fig. 1B), respectively.

View larger version (31K): [in this window] [in a new window]   FIGURE 5. Functional analysis of Tc-APC-primed CD8+ T cells. A and B, The in vitro DCOVA- and Tc-APC-primed OT I/B6.1 CD8+45.1+T cells were i.v. injected into C57BL/6 (45.2+) mice. Mouse tail blood cells were stained with PE-H-2Kb/OVAI tetramer (PE-tetramer), FITC-anti-CD8 Ab (FITC-CD8), and ECD-anti-CD44 Ab (ECD-CD44) or -anti-CD45.1 (ECD-45.1), and analyzed by flow cytometry at indicated time points (A) or 30 days (B) after T cell transfer. The value in each panel represents the percentage of PE-tetramer-positive CD8+ T cells vs the total peripheral CD8+ T cell population. The value in parenthesis represents the SD. The PE-tetramer-positive CD44+T cells in the circles are also FITC-CD8+ and ECD-CD45.1+ cells by flow cytometric analysis. C, T cell proliferation assay. OT I CD8+ T cells (0.4 x 105 cells/well) primed on day 0 by irradiated (4000 rad) DCOVA () or Tc-APC () were maintained in cultures for 6 days with the indicated cytokines (IL-2 (50 U/ml), IL-7 (10 ng/ml) and IL-15 (5 ng/ml)) added on days 3 and 5. Live CD8+ T cells with trypan blue exclusion for each culture done in triplicate were counted at the indicated time points. D, In in vitro cytotoxicity assay, naive CD8+ T cells derived from OT I/B6.1 (CD45.1+) mice were cocultured with irradiated (4000 rad) DCOVA and Tc-APC for 2 days. The above active CD8+ T cells were harvested and purified on density gradients followed by biotin-conjugated anti-CD45.1 Ab and anti-biotin microbeads (Miltenyi Biotec). These T cells were referred to as DCOVA/OT I6.1 and Tc-APC/OT I6.1, respectively, and used as effector (E) cells, while 51Cr-labeled EG7 and the control EL-4 tumor cells were used as target (T) cells. Each point represents the mean of triplicate cultures. E, T memory cell expansion. Three months after CD8+ T cell transfer, mice were boosted by i.v. injection of OVA I peptide. Four days subsequent to the boost, mouse tail blood cells were stained with PE-tetramer, FITC-CD8 and ECD-CD25, -CD44, -CD62L, and -CD69 Abs (solid lines) and analyzed by flow cytometry. The ECD-isotype matched control Abs were used as controls (dotted lines). The value in each panel represents the percentage of PE-tetramer-positive CD8+ T cells vs the total peripheral CD8+ T cells. The value in parenthesis represents the SD. One representative experiment of two in the above different experiments is shown.

We then assessed their ability to induce in vivo proliferation of CD8⁺ T cells. As shown in Fig. 6A, Tc-APC and DC_OVA stimulated 0.81 and 2.14% OVA-specific CD8⁺ T cell responses in vivo, respectively. By using Tc-APC with different gene KO, the stimulation of OVA-specific CD8⁺ T cell responses by Tc-APC with IL-2^–/– (0.23%), CD80^–/– (0.19%), but not with IFN-^–/– (0.78%) and CD54^–/– (0.75%) gene KO, was almost lost (Fig. 6B), indicating that the Tc-APC stimulatory effect is mediated by its IL-2 secretion and acquired CD80 costimulation. Interestingly, Con A-OT I cells without acquired pMHC I complexes completely lost their stimulatory effect. To assess Tc-APC-induced CD8⁺ T cell differentiation into CTL, we adoptively transferred OVAI-pulsed/CFSE^high- and Mut1-pulsed/CFSE^low-labeled splenocytes into immunized mice. We found that there was substantial loss of the CFSE^high-labeled cells from the DC_OVA- (90%) and Tc-APC-vaccinated (72%) mice, indicating that both DC_OVA and Tc-APC can stimulate CD8⁺ T cell differentiation into effector CTL. Interestingly, there were also substantial losses in Tc(IFN-^–/–)-APC- (70%) and Tc(CD54^–/–)-APC-immunized (66%) mice, but not from the Tc(IL-2^–/–)-APC- (0%) and Tc(CD80^–/–)-APC-immunized (11%) mice, indicating that the stimulatory effect of Tc-APC in vivo is mediated by its secretion of IL-2 and its acquired CD80 costimulation. Interestingly, the Con A-OT I cell-vaccinated mice did not display any killing activity for the OVA I peptide-pulsed and CFSE^high-labeled target cells (Fig. 6B). These data indicate that the acquired pMHC I complexes play a critical role in targeting the stimulatory effect of the CD8⁺ Tc-APC on OVA-specific CD8⁺ T cell proliferation and cytotoxicity in vivo.

View larger version (44K): [in this window] [in a new window]   FIGURE 6. Tc-APC stimulate CD8+ CTL responses in vivo. A, In tetramer staining assay, mice were i.v. immunized with irradiated DCOVA, Tc-APC, Con A-OT I, and Tc-APC with various gene KO, respectively. Six days after the immunization, the tail blood samples of immunized mice were stained with PE-tetramer and FITC-CD8 Ab, then analyzed by flow cytometry. The value in each panel represents the percentage of tetramer-positive CD8+ T cells vs the total CD8+ T cells. The value in parenthesis represents the SD. B, In in vivo cytotoxicity assay, the above immunized mice were i.v. coinjected at 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 the immunization. Sixteen hours after target cell delivery, the residual CFSEhigh and CFSElow target cells remaining in the recipients’ spleens were sorted and analyzed by flow cytometry. The value in each panel represents the percentage of CFSEhigh cells vs CFSElow cells remaining in the spleens. The value in parenthesis represents the SD. One representative experiment of three in the above different experiments is shown.

We i.v. immunized mice with in vitro or in vivo DC_OVA-stimulated Tc-APC and then challenged the immunized mice with OVA-expressing BL6–10_OVA tumor cells. Our data showed that all mice (eight of eight) immunized with PBS (experiment I of Table I) or naive OT I T cells also died of lung metastasis (data not shown). However, 75% (six of eight) mice immunized with in vitro DC_OVA-stimulated Tc-APC had no lung tumor metastasis (experiment I of Table I). The average number of lung metastasis (18 ± 17) in this group of mice was significantly reduced compared with that in the PBS control group. DC_OVA immunization was the most effective, resulting in 100% (eight of eight) of mice being protected from lung metastasis. The specificity of the protection was confirmed with the observation that Tc-APC did not protect against BL6–10 tumors that did not express OVA, with all mice having large numbers (>100) of lung metastatic tumor colonies. Interestingly, 62.5% (five of eight) mice immunized with in vivo DC_OVA-stimulated Tc-APCvivo also had no lung tumor metastasis with a significantly reduced average number of lung metastasis (37 ± 19) (experiment II of Table I), indicating that both in vitro or in vivo DC_OVA-stimulated Tc-APC were able to induce effective OVA-specific antitumor immunity in vivo.

To study the cellular mechanism, CD4 and CD8 KO mice were used for immunization. As shown in experiment III of Table I, 75% (six of eight) wild-type C57BL/6 or 63% (five of eight) CD4 KO mice were still protected from BL6–10_OVA tumor challenge, indicating that activation of CD8⁺ CTL response by Tc-APC is independent on the host CD4⁺ T cells. However, all CD8 KO mice (eight of eight) had numerous lung tumor metastases, indicating that the Tc-APC-driven antitumor immunity is CD4⁺ Th cell independent and is mediated by the host CD8⁺ CTLs rather than the irradiated Tc-APC injection. To further elucidate the molecular mechanism, Tc-APC with respective gene deficiency were used for immunizations. Our data showed that Tc(IL-2^–/–)-APC and Tc(CD80^–/–)-APC-immunized mice, but not Tc(IFN-^–/–)-APC and Tc(CD54^–/–)-APC-immunized mice, substantially lost their antitumor immunity compared with Tc-APC-immunized mice (experiment III of Table I), indicating that the acquired CD80 costimulation and Tc-APC-secreted IL-2 play an important role in stimulation of CD8⁺ CTL responses in vivo. Interestingly, all mice immunized with Con A-OT I cells with a similar cytokine profile as Tc-APC, but without acquired pMHC I, had large numbers (>100) of lung metastatic tumor colonies compared with the Tc-APC-treated group, indicating that the acquired pMHC I complexes are responsible for not only providing signal 1 (pMHC/TCR) stimulation, but also targeting its secreted cytokine and acquired costimulation to OVA-specific CD8⁺ CTL responses in vivo.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Cross-priming has been thought to be an exclusive feature of specialized APC (28) and has given rise to some controversy with respect to its biological significance when Ag concentrations are not excessively high (29). T-T Ag presentation, dependent upon activated T cells first acquiring MHC class and CD80 molecules from APC and then stimulating other T cells, is increasingly attracting attention (21, 22). However, the results of the relevant studies were disparate, in part because multiple experimental systems have been used. In some reports, CD4⁺ T-APC were found to induce IL-2 production and proliferative responses among naive responder T cells (30, 31). In other reports, these T-APC have been shown to induce apoptosis in activated CD4⁺ T cells or anergization of CD4⁺ T cell lines or immunosuppressive effects in the context of autoimmune responses (22, 32, 33). In these studies, the T-APC used were derived from rather uncharacterized Con A-stimulated allogeneic or Ag-pulsed CD4⁺ T cell lines. Therefore, it is difficult to assess the extent to which they are representative of T-APCs as they would be generated in vivo.

Recently, Brandes et al. (34) have demonstrated that human T cells expressing MHC II and costimulatory molecules can act as APC and stimulated induction of naive T cell proliferation and differentiation. More recently, Kennedy et al. (25) have reported that the active Th lymphocytes when pulsed with Ag peptides can cross-prime memory CTL responses. In consistent with these two reports regarding the T cell priming, we have recently demonstrated that CD4⁺ Th cells were able to acquire the immunological synapse-composed MHC II and costimulatory molecules (CD54 and CD80) as well as the bystander MHC I by DC activation. These CD4⁺ Th cells secreting IL-2 and IFN-, but not IL-4 and IL-10 and with acquired pMHC I and costimulatory molecules, acted themselves as Th1-APC in stimulation of OVA-specific CD8⁺ T cell responses and antitumor immunity (13). In this study, we provide evidence that CD8⁺ Tc1-APC-secreting IL-2 and IFN-, but not IL-4 and IL-10 and with acquired pMHC I and costimulatory molecules, can also stimulate OVA-specific CD8⁺ T cell responses and antitumor immunity in vivo. Furthermore, we demonstrated that the in vivo DC_OVA-activated CD8⁺ T cells (Tc-APCvivo) can also stimulate CD8⁺ T cell proliferation in vitro and in vivo, and induce antitumor immunity, indicating the physiological significance of this new concept of Tc-APC. Dynamic imaging of T cell-DC interactions in lymph nodes has been reported by using two-photon microscopy (35, 36). To further confirm the in vivo membrane transfer from DC onto T cells, dynamic imaging study is underway in the laboratory of J. Xiang by using the newly generated DC cell line, DC2.4/H-2K^b-GFP expressing fusion protein H-2K^b-GFP and two-photon microscopy.

DC are powerful APC that are critical for the initiation of T cell responses. In some viral and bacterial infections (2, 3, 4), DC are licensed under danger signals to directly activate CD8⁺ CTL responses without the help effect of CD4⁺ Th cells. The mechanism and regulation of DC survival and death (37, 38) are likely to be important in maintaining the homeostatic balance of the immune system. A few reports have linked extended survival of DC to lymphoproliferative diseases (39, 40). In contrast, reduced survival of DC has been reported to be associated with impaired immune responses (38). It has been shown that DC loaded with the Ag and s.c. injected into immune mice are rapidly eliminated (41). However, it is presently unclear whether T cells also influence the lifespan of DC in an Ag-specific fashion. In this study, we demonstrated that CD8⁺ T cells, when activated by Ag-loaded DC, become active Tc-APC and capable of killing the Ag-loaded DC. This finding is consistent with a recent report by Yang et al. (42), showing that DC are susceptible to immediate perforin-dependent elimination of CTL. In addition, we also demonstrated that active Tc-APC with acquired pMHC I complexes can also kill neighboring Tc-APC (fratricide), which is consistent with a previous report by Huang et al. (11, 12). Taken together, our results indicate that Tc-APC may negatively modulate immune responses by eliminating pMHC I-expressing DC_OVA and Tc-APC, especially when the initial viral or bacterial stimulation is too strong.

According to the progressive linear differentiation hypothesis (7), T cell differentiation involves a phase of proliferation preceding the acquisition of fitness and effector function. Primed CD8⁺ T cells reach a variety of differentiation stages that contain effector cells as well as cells that have been arrested at intermediate levels of differentiation. Thus, they retain a flexible gene imprinting. T cells that may survive after retraction phase of an immune response can be resolved into distinct subsets of either cmCTL cells representing cells at intermediate levels of differentiation or fully differentiated memory CTL (emCTL) cells with effector capacity (43, 44). It has been shown that strong Ag presentations stimulate the development of effector CTL, whereas less efficient Ag presentation can lead to the generation of cmCTL (45). In this study, we demonstrated that CD8⁺ Tc-APC were able to stimulate naive CD8⁺ T cell differentiation into central memory CD44⁺CD62^highIL-7R⁺ T cells with less cytotoxicity and longer survival capacity in vivo, compared with DC_OVA-primed CD44⁺CD62^lowIL-7R^– effector CTLs with high cytotoxicity and shorter survival capacity. Thus, it seems reasonable to conclude that due to the lower level of activation/costimulation signals provided by Tc-APC to the naive CD8⁺ T cells as compared with DC, Tc-APC-primed CTL would preferentially differentiate into central memory T cells (cmCTL).

Adoptively transferred active Tc cells cannot only eradicate established tumors, but they can also induce endogenous recipient-derived Tc-mediated antitumor responses and long-lived recipient CD8⁺ Tc-derived Tm cells (46, 47). However, the corresponding immune mechanism behind this finding has not been disclosed. In this study, we clearly elucidated that the in vitro DC-stimulated CD8⁺ Tc-APC with acquired DC molecules can become long-lived Tm and these cells can also stimulate the host CD8⁺ cmCTL responses, thus leading to the formation of long-term host-derived Tm cells. More importantly, we have also elucidated the molecular mechanism involved in Tc-APC-induced memory T cell responses and antitumor immunity. Our data demonstrated that the Tc-APC-induced cmCTL responses and antitumor immunity are mediated by its IL-2 secretion and acquired CD80 costimulation. Interestingly, the acquired pMHC I complexes on Tc-APC are responsible for not only providing the signal 1 (pMHC/TCR) in stimulation of CD8⁺ T cells, but also targeting its stimulatory effect to OVA-specific CD8⁺ T cells in vivo.

Taken together, this study demonstrated that CD8⁺ T cells can acquire pMHC I and costimulatory molecules by DC stimulation, and act as Tc-APC. These Tc-APC can play both negative and positive modulations in antitumor immune responses by eliminating DC and neighboring Tc-APC, and stimulating OVA-specific CD8⁺ central memory T responses and antitumor immunity. The stimulatory effect of Tc-APC is mediated via its IL-2 secretion and acquired CD80 costimulation, and is specifically targeted to CD8⁺ T cells in vivo via its acquired pMHC I complexes. These principles could be applied to not only antitumor immunity, but also other immune disorders (e.g., autoimmunity).

	Acknowledgments

 
We thank X. Wu and M. Boyd for help in confocal fluorescence microscopy and flow cytometry, respectively.

	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 work was supported by Research Grants (MOP 79415 and 67230) from the Canadian Institute of Health Research (to J.X.).

² D.X. and S.H. made the same contribution in this study.

³ Address correspondence and reprint requests to Dr. Jim Xiang, Saskatoon Cancer Center, 20 Campus Drive, Saskatoon, Saskatchewan S7N 4H4, Canada. E-mail address: jxiang{at}scf.sk.ca

⁴ Abbreviations used in this paper: DC, dendritic cell; pMHC, peptide-MHC; pMHC II, pMHC class II; pMHC I, pMHC class I; DC_OVA, OVA-pulsed DC; KO, knockout; BM, bone marrow; Tc, cytotoxic T; Tc1, type 1 Tc; ECD, energy-coupled dye; Tm, memory T cell.

Received for publication March 2, 2006. Accepted for publication May 4, 2006.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Bennett, S. R., F. R. Carbone, F. Karamalis, R. A. Flavell, J. F. Miller, W. R. Heath. 1998. Help for cytotoxic-T-cell responses is mediated by CD40 signalling. Nature 393: 478-480. [Medline]
2. Buller, R. M., K. L. Holmes, A. Hugin, T. N. Frederickson, H. C. Morse, 3rd. 1987. Induction of cytotoxic T-cell responses in vivo in the absence of CD4 helper cells. Nature 328: 77-79. [Medline]
3. Ke, Y., J. A. Kapp. 1996. Oral antigen inhibits priming of CD8⁺ CTL, CD4⁺ T cells, and antibody responses while activating CD8⁺ suppressor T cells. J. Immunol. 156: 916-921. [Abstract]
4. Hou, S., X. Y. Mo, L. Hyland, P. C. Doherty. 1995. Host response to Sendai virus in mice lacking class II major histocompatibility complex glycoproteins. J. Virol. 69: 1429-1434. [Abstract]
5. Bennett, S. R., F. R. Carbone, F. Karamalis, J. F. Miller, W. R. Heath. 1997. Induction of a CD8⁺ cytotoxic T lymphocyte response by cross-priming requires cognate CD4⁺ T cell help. J. Exp. Med. 186: 65-70. [Abstract/Free Full Text]
6. Hogquist, K. A., S. C. Jameson, W. R. Heath, J. L. Howard, M. J. Bevan, F. R. Carbone. 1994. T cell receptor antagonist peptides induce positive selection. Cell 76: 17-27. [Medline]
7. Lanzavecchia, A., F. Sallusto. 2002. Progressive differentiation and selection of the fittest in the immune response. Nat. Rev. Immunol. 2: 982-987. [Medline]
8. Lenschow, D. J., T. L. Walunas, J. A. Bluestone. 1996. CD28/B7 system of T cell costimulation. Annu. Rev. Immunol. 14: 233-258. [Medline]
9. Grakoui, A., S. K. Bromley, C. Sumen, M. M. Davis, A. S. Shaw, P. M. Allen, M. L. Dustin. 1999. The immunological synapse: a molecular machine controlling T cell activation. Science 285: 221-227. [Abstract/Free Full Text]
10. Viola, A., S. Schroeder, Y. Sakakibara, A. Lanzavecchia. 1999. T lymphocyte costimulation mediated by reorganization of membrane microdomains. Science 283: 680-682. [Abstract/Free Full Text]
11. 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]
12. 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]
13. 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]
14. Foulds, K. E., L. A. Zenewicz, D. J. Shedlock, J. Jiang, A. E. Troy, H. Shen. 2002. Cutting edge: CD4 and CD8 T cells are intrinsically different in their proliferative responses. J. Immunol. 168: 1528-1532. [Abstract/Free Full Text]
15. 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]
16. Chen, Z., S. Dehm, K. Bonham, H. Kamencic, B. Juurlink, X. Zhang, J. R. Gordon, J. Xiang. 2001. DNA array and biological characterization of the impact of the maturation status of mouse dendritic cells on their phenotype and antitumor vaccination efficacy. Cell. Immunol. 214: 60-71. [Medline]
17. 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]
18. Nishimura, T., K. Iwakabe, M. Sekimoto, Y. Ohmi, T. Yahata, M. Nakui, T. Sato, S. Habu, H. Tashiro, M. Sato, A. Ohta. 1999. Distinct role of antigen-specific T helper type 1 (Th1) and Th2 cells in tumor eradication in vivo. J. Exp. Med. 190: 617-627. [Abstract/Free Full Text]
19. Xiang, J., H. Huang, Y. Liu. 2005. A new dynamic of CD8⁺ T effector cell response via CD4⁺ T helper-antigen-presenting cells. J. Immunol. 174: 7497-7505. [Abstract/Free Full Text]
20. Sansom, D. M., N. D. Hall. 1993. B7/BB1, the ligand for CD28, is expressed on repeatedly activated human T cells in vitro. Eur. J. Immunol. 23: 295-298. [Medline]
21. Tatari-Calderone, Z., R. T. Semnani, T. B. Nutman, J. Schlom, H. Sabzevari. 2002. Acquisition of CD80 by human T cells at early stages of activation: functional involvement of CD80 acquisition in T cell to T cell interaction. J. Immunol. 169: 6162-6169. [Abstract/Free Full Text]
22. Tsang, J. Y., J. G. Chai, R. Lechler. 2003. Antigen presentation by mouse CD4⁺ T cells involving acquired MHC class II:peptide complexes: another mechanism to limit clonal expansion?. Blood 101: 2704-2710. [Abstract/Free Full Text]
23. Undale, A. H., P. J. van den Elsen, E. Celis. 2004. Antigen-independent acquisition of MHC class II molecules by human T lymphocytes. Int. Immunol. 16: 1523-1533. [Abstract/Free Full Text]
24. Dutton, R. W., L. M. Bradley, S. L. Swain. 1998. T cell memory. Annu. Rev. Immunol. 16: 201-223. [Medline]
25. Kennedy, R., A. H. Undale, W. C. Kieper, M. S. Block, L. R. Pease, E. Celis. 2005. Direct cross-priming by Th lymphocytes generates memory cytotoxic T cell responses. J. Immunol. 174: 3967-3977. [Abstract/Free Full Text]
26. Kaech, S. M., J. T. Tan, E. J. Wherry, B. T. Konieczny, C. D. Surh, R. Ahmed. 2003. Selective expression of the interleukin 7 receptor identifies effector CD8 T cells that give rise to long-lived memory cells. Nat. Immunol. 4: 1191-1198. [Medline]
27. Tan, J. T., B. Ernst, W. C. Kieper, E. LeRoy, J. Sprent, C. D. Surh. 2002. Interleukin (IL)-15 and IL-7 jointly regulate homeostatic proliferation of memory phenotype CD8⁺ cells but are not required for memory phenotype CD4⁺ cells. J. Exp. Med. 195: 1523-1532. [Abstract/Free Full Text]
28. Rock, K. L., S. Gamble, L. Rothstein. 1990. Presentation of exogenous antigen with class I major histocompatibility complex molecules. Science 249: 918-921. [Abstract/Free Full Text]
29. Zinkernagel, R. M.. 2002. On cross-priming of MHC class I-specific CTL: rule or exception?. Eur. J. Immunol. 32: 2385-2392. [Medline]
30. Mauri, D., T. Wyss-Coray, H. Gallati, W. J. Pichler. 1995. Antigen-presenting T cells induce the development of cytotoxic CD4⁺ T cells. I. Involvement of the CD80-CD28 adhesion molecules. J. Immunol. 155: 118-127. [Abstract]
31. Taams, L. S., W. van Eden, M. H. Wauben. 1999. Antigen presentation by T cells versus professional antigen-presenting cells (APC): differential consequences for T cell activation and subsequent T cell-APC interactions. Eur. J. Immunol. 29: 1543-1550. [Medline]
32. Mannie, M. D., S. K. Rendall, P. Y. Arnold, J. P. Nardella, G. A. White. 1996. Anergy-associated T cell antigen presentation: a mechanism of infectious tolerance in experimental autoimmune encephalomyelitis. J. Immunol. 157: 1062-1070. [Abstract]
33. Nisini, R., A. Fattorossi, C. Ferlini, R. D’Amelio. 1996. One cause for the apparent inability of human T cell clones to function as professional superantigen-presenting cells is autoactivation. Eur. J. Immunol. 26: 797-803. [Medline]
34. Brandes, M., K. Willimann, B. Moser. 2005. Professional antigen-presentation function by human T Cells. Science 309: 264-268. [Abstract/Free Full Text]
35. Bousso, P., E. Robey. 2003. Dynamics of CD8⁺ T cell priming by dendritic cells in intact lymph nodes. Nat. Immunol. 4: 579-585. [Medline]
36. Miller, M. J., O. Safrina, I. Parker, M. D. Cahalan. 2004. Imaging the single cell dynamics of CD4⁺ T cell activation by dendritic cells in lymph nodes. J. Exp. Med. 200: 847-856. [Abstract/Free Full Text]
37. Hou, W. S., L. Van Parijs. 2004. A Bcl-2-dependent molecular timer regulates the lifespan and immunogenicity of dendritic cells. Nat. Immunol. 5: 583-589. [Medline]
38. Hon, H., E. B. Rucker, 3rd, L. Hennighausen, J. Jacob. 2004. Bcl-x_L is critical for dendritic cell survival in vivo. J. Immunol. 173: 4425-4432. [Abstract/Free Full Text]
39. Nopora, A., T. Brocker. 2002. Bcl-2 controls dendritic cell longevity in vivo. J. Immunol. 169: 3006-3014. [Abstract/Free Full Text]
40. Ashcroft, A. J., S. M. Cruickshank, P. I. Croucher, M. J. Perry, S. Rollinson, J. M. Lippitt, J. A. Child, C. Dunstan, P. J. Felsburg, G. J. Morgan, S. R. Carding. 2003. Colonic dendritic cells, intestinal inflammation, and T cell-mediated bone destruction are modulated by recombinant osteoprotegerin. Immunity 19: 849-861. [Medline]
41. Hermans, I. F., D. S. Ritchie, J. Yang, J. M. Roberts, F. Ronchese. 2000. CD8⁺ T cell-dependent elimination of dendritic cells in vivo limits the induction of antitumor immunity. J. Immunol. 164: 3095-3101. [Abstract/Free Full Text]
42. Yang, J., S. P. Huck, R. S. McHugh, I. F. Hermans, F. Ronchese. 2006. Perforin-dependent elimination of dendritic cells regulates the expansion of antigen-specific CD8⁺ T cells in vivo. Proc. Natl. Acad. Sci. USA 103: 147-152. [Abstract/Free Full Text]
43. Geginat, J., A. Lanzavecchia, F. Sallusto. 2003. Proliferation and differentiation potential of human CD8⁺ memory T-cell subsets in response to antigen or homeostatic cytokines. Blood 101: 4260-4266. [Abstract/Free Full Text]
44. Schwendemann, J., C. Choi, V. Schirrmacher, P. Beckhove. 2005. Dynamic differentiation of activated human peripheral blood CD8⁺ and CD4⁺ effector memory T cells. J. Immunol. 175: 1433-1439. [Abstract/Free Full Text]
45. van Faassen, H., M. Saldanha, D. Gilbertson, R. Dudani, L. Krishnan, S. Sad. 2005. Reducing the stimulation of CD8⁺ T cells during infection with intracellular bacteria promotes differentiation primarily into a central (CD62L^highCD44^high) subset. J. Immunol. 174: 5341-5350. [Abstract/Free Full Text]
46. Dobrzanski, M. J., J. B. Reome, J. A. Hollenbaugh, R. W. Dutton. 2004. Tc1 and Tc2 effector cell therapy elicit long-term tumor immunity by contrasting mechanisms that result in complementary endogenous type 1 antitumor responses. J. Immunol. 172: 1380-1390. [Abstract/Free Full Text]
47. Wang, L. X., J. Kjaergaard, P. A. Cohen, S. Shu, G. E. Plautz. 2004. Memory T cells originate from adoptively transferred effectors and reconstituting host cells after sequential lymphodepletion and adoptive immunotherapy. J. Immunol. 172: 3462-3468. [Abstract/Free Full Text]

This article has been cited by other articles:

				S. Hao, J. Yuan, and J. Xiang 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 J. Leukoc. Biol., October 1, 2007; 82(4): 829 - 838. [Abstract] [Full Text] [PDF]

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 Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Xia, D.