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Keywords:

  • CD8+ T cells;
  • EBV;
  • human dendric cells;
  • immunosuppression;
  • solid organ transplant patients;
  • T regulatory

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Posttransplantation lymphoproliferative disorders (PTLD) are life-threatening complications of solid organ transplantation, triggered by EBV infection in chronically immunosuppressed (IS) patients. Our goal is to establish DC-based protocols for adoptive immunotherapy of refractory PTLD, while understanding how the immunosuppressive drug environment may subvert DC-EBV-specific T cell interactions. Type-1 CD8+ T cells are critical for efficient immune surveillance and control of EBV infection, whereas type-2 or Treg/type-3 responses may provide an environment conductive to disease progression. We have recently reported that chronic IS inhibits DC function in transplant patients. Here, we have analyzed the comparative ability of mature, type-1 polarized DCs (i.e. DC1) generated from quiescent transplant patients or healthy controls, to boost type-1 EBV-specific CD8+ T cells in vitro. Our results show that unlike healthy controls, where DC1 loaded with MHC class I EBV peptides preferentially reactivate specific type-1 CD8+ T cells, DC1 generated from transplant patients reactivate EBV-specific CD8+ T cells that produce both IFN-γ and IL-10, up-regulate FOXP3 mRNA, and suppress noncognate CD4+ T-cell proliferation via cell–cell contact. These data support a novel regulatory pathway for anti-EBV T-cell-mediated responses in IS transplant patients, with implications for the design of adoptive immunotherapies in this setting.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Solid organ transplantation (SOTx) represents the treatment of choice for end-stage organ failure-associated diseases. In 2005 alone, more than 26 000 patients underwent SOTx worldwide, resulting in a significant improvement in their quality of life (1).The standard immunosuppressed (IS) drug regimens necessary to prevent graft rejection include a combination of calcineurin inhibitors and steroids, with or without adjunctive antiproliferative agents (2). The long-term administration of IS drugs to prevent graft rejection however can lead to untoward complications that include opportunistic infections and malignancies (3,4). Posttransplantation lymphoproliferative disorders (PTLD) are typically B-cell lymphoproliferations, triggered by EBV infection in the context of impaired T-cell immune surveillance, with high rates of morbidity and mortality (5). Approximately 20% of PTLD cases remain refractory to current treatments, mandating the development of novel therapeutic approaches. Restoration of anti-EBV T-cell immunity, by infusion of ex vivo lymphoblastoid cell lines (LCL)-expanded EBV-specific cytotoxic T lymphocytes (CTLs), has been shown to be safe and effective in the prevention or treatment of PTLD in bone marrow transplant recipients (6,7). Here we have hypothesized that DC-based adoptive immunotherapy protocols to treat refractory PTLD may provide a valuable, improved alternative therapeutic approach in the SOTx setting.

DC represents the most powerful antigen (Ag)-presenting cells (APC) for reactivation of memory T cells, but mostly for priming of naïve T cells in healthy control subjects (8). In addition, DC can be loaded ex vivo with tailored Ags, and further manipulated to acquire mature phenotypes and to be type-1 polarized (able to produce high levels of IL-12p70) (9,10). These are important advantages for DC-based immunotherapy protocols, since the selective reconstitution of EBV-specific cellular immune responses (type-1 CD8+ T cells secreting IFN-γ) appears crucial for EBV load control and tumor elimination in PTLD (6,7). However, little mechanistic insight exists as to how chronic administration of IS drugs influences the interactions between DC and T cells that lead to suppression of EBV-specific immunity, and whether indeed DC can be used successfully as APC for the generation of EBV-specific CTLs from naïve precursors in SOTx setting.

Our group has shown recently that chronic IS therapy with prednisone and tacrolimus impairs the ex vivo maturation and function of DCs generated from SOTx patients (11). Here we have analyzed the ability of type-1 polarized DC generated from quiescent IS SOTx patients to reactivate EBV-specific CD8+ T cells in vitro. We chose quiescent EBV+ patients as our first attempt to characterize how memory EBV-specific CD8+ T cells are regulated by DC in the SOTx setting, before addressing the EBV SOTx patients. Our results demonstrate significant differences between healthy control and Tx patients' T-cell cultures' outcomes: they confirm that DC1 generated from healthy controls are very effective in reactivating EBV-specific type-1 (IFN-γ) CD8+ T-cell immunity, but also reveal that DC1 from quiescent IS SOTx patients reactivate and expand type-1 ‘regulatory-like’ EBV-specific CD8+ T cells. These EBV-specific CD8+ T cells produce IFN-γ and IL-10, up-regulate FOXP3 mRNA and subsequently suppress noncognate CD4+ T-cell activation and proliferation in a cell-to-cell contact-dependent manner. These results not only provide novel insight into how IS drugs modulate DC/T cell interactions in SOTx patients by creating a microenvironment permissive for skewed anti-EBV immune polarization, but should be considered when planning future experiments designed to prime naïve T cells from IS Tx patients. In addition, our data suggest a general paradigm in which overall T-cell responses may be regulated by DC in the Tx setting: allowing for Ag-specific CD8+ T-cell surveillance, while promoting graft quiescence through inhibition of noncognate CD4+ T-cell activation.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Human subjects and peripheral blood mononuclear cells isolation

HLA-A2+ or HLA-B8+ EBV+ age- and sex-matched healthy volunteers (n = 9) and quiescent IS SOTx patients (multivisceral n = 5 and small bowel n = 4) were recruited following informed consent under IRB-approved protocol. All patients were on daily prednisone and tacrolimus for more than 4 years, and were quiescent at the time of blood donation, without evidence of rejection or signs of EBV disease. Peripheral blood mononuclear cells (PBMCs) were isolated from 30 to 40 mL of heparinized blood by Ficoll-Hypaque density gradient centrifugation as described (12). For some experiments, positive or negative selection of CD4+ or CD8+ T cells from in vitro stimulated (IVS) T cells was performed using magnetic beads (Miltenyi Biotec Inc, Auburn, CA), according to the manufacturer's instructions. The purity of the sorted populations was always ≥90% as confirmed by flow cytometry.

Media, reagents and cell lines

RPMI-1640 was supplemented with 2 mM l-glutamine, 10 mM HEPES, 100 IU/mL penicillin/streptomycin and 10% heat inactivated fetal calf serum (FCS) (all from Gibco BRL, Grand Island, NJ). For IVS T cells generation and maintenance, 10% FCS was replaced by 5% human normal AB serum (HNS) (Nabi, Boca Raton, FL). For DC generation, 10% FCS containing AIM-V media (Gibco BRL) was supplemented with 1000 U/mL GM-CSF (Schering Plough, Kenilworth, NJ) and 1000 U/mL IL-4 (Schering Plough) as described (10). Recombinant human (rh)TNF-α, rhIL-6, rhIL-1β and rhIL-7 were purchased from R&D Systems Inc. (Minneapolis, MN). rhIFNγ was purchased from PeproTech (Rocky Hill, NJ). Ficoll-Paque™ was obtained from Amersham Biosciences AB (Uppsala, Sweden). Bovine serum albumine (BSA), PMA and ionomycin were purchased from Sigma (St. Louis, MO). Carboxyfluorescein diacetate succinimidyl ester (CFSE) was procured from Molecular Probes (Eugene, OR) and Annexin V- fluorescein isothiocyanate (FITC) apoptosis detection kit from BD-PharMingen (San Jose, CA). CD40L-transfected J558L plasmacytoma cells were a gift from Dr. P Lane, University of Birmingham, Birmingham, UK. The TAP-deficient T2 (HLA-A2+) cell line (kindly provided by Dr. Russ Salter, University of Pittsburgh, Pittsburgh, PA) or autologous LCL lines generated in our lab were used as APC in ELISPOT assays.

Synthetic peptides and HLA class I/peptide tetramers

A panel of three HLA-A2-and HLA-B8-restricted EBV peptides derived from the lytic cycle protein BMLF1 (GLCTLVAML) and BZLF1 (RAKFKQLL), and from the latent cycle proteins LMP2 (CLGGLLTMV), EBNA3A (SVRDRLARL) and EBNA3A (FLRGRAYGL) were synthesized at the Peptide Synthesis Facility (University of Pittsburgh) (13). These peptides were employed to load DC (final concentration 10 μg/mL) for T-cell stimulation and functional screening assays. Streptavidin-phycoerythrin (PE)-HLA-A2 or HLA-B8 tetramers incorporating these peptides were generated at the NIAID MHC-Tetramer Core Facility (Emory University, Atlanta, GA) and used to sort the EBV-specific T cells in conjunction with anti-PE beads (Miltenyi).

DC generation and EBV-Ag loading

PBMCs were resuspended in AIM-V medium and plated in T75 flasks (Corning, Corning, NY) for 1 h at 37°C, 5% CO2 for monocyte adherence. The nonadherent lymphocytes (NAL) were subsequently removed by decanting, cryopreserved in liquid nitrogen and retained as T-cell responders. The plastic-adherent monocytes were cultured in DC media at 37°C, 5% CO2. On day 6, the iDCs were harvested, using cold Hanks' buffer (Gibco, BRL) (11). iDCs were incubated with rhTNF-((10 ng/mL), rhIL-6 (0.9 μg/mL) and rhIL-1β (10 ng/mL), in the presence of rhIFN-γ (1000 U/mL) for an additional 24 h at 37°C, 5% CO2, to induce DC maturation/polarization (DC1) (10,14). DC1s were then loaded with a mix of either HLA-A2 or -B8-restricted EBV-derived peptides (final concentration 10 μg/mL), as indicated.

CFSE labeling

Lymphocytes were labeled with CFSE, (Molecular Probes), as described (15). Briefly, 10 × 106 NALs or sorted CD8+ or CD4+ were labeled with 0.2 μM CFSE in PBS for 15 min at 37°C, 5% CO2, washed and resuspended in media for cell coculture generation. Cells were harvested on day 5 and analyzed by flow cytometry on gated CD4+ or CD8+ T cells.

Generation of T-cell cocultures

NALs or sorted CD4+ or CD8+T cells (2 × 106/mL) labeled or not with CFSE were incubated in 24-well plates with autologous DC1 (preloaded with EBV-peptides), at a ratio of 20:1, at 37°C, 5% CO2 in the presence of rhIL-7 (10 ng/mL). For CFSE experiments, the labeled cells were harvested on day 5 and analyzed by flow cytometry. For additional experiments, IVS T cells were harvested on day 10 and used in IFNγ/IL-5 ELISPOT analysis, while supernatants were collected for analysis in TGFβ and IL-10 ELISA assays (after 10 days of EBV-specific IVS). In parallel experiments, neutralizing mouse IgG anti-human IL-10 (10 μg/mL) or isotype control Abs (10 μg/mL) (R&D Systems) were added in replicate cultures. Trans-well inserts (0.4-μm pore size; Costar, Corning) were used to separate CFSE-labeled CD8+ (upper chamber) from CD4+ T cells (lower chamber). In these experiments, we used 1 × 106 positively sorted CD8+ or CD4+ T cells and 0.05 × 106 peptide-pulsed DC1 on each side of the membrane.

Intracellular cytokine analysis by fluorescence microscopy

Ten day-IVS T cells were stained with PE-conjugated HLA-A2 or HLA-B8 EBV-specific tetramers and further incubated with anti-PE beads (Miltenyi Biotec Inc) according to the manufacturer's instructions. EBV-specific tetramer+ (TMR+)-sorted CD8+ T cells were stimulated with PMA (5 ng/mL) + ionomycin (100 ng/mL) for an additional 24 h at 37°C, 5% CO2, then exposed to Brefeldin A for the last 4 h of incubation. Aliquots of 105 cells/200 μL were then used to generate cytospins, as described (16). Intracellular cytokines produced in response to PMA + ionomycin were detected by indirect staining with: mouse anti-human IFNγ mAb from BD-PharMingen, Cy2-conjugated donkey anti-mouse F(ab')2 fragment from Jackson Immunoresearch (West Grove, PA), rat anti-human anti-IL-10 mAbs from BD-PharMingen and Alexa Fluor 647-conjugated goat anti-rat F(ab')2 from Molecular Probes (Eugene, OR). Nuclei were counterstained with 4,6-diamidino-2-phenylindole 2HCl (DAPI-Molecular Probes) (16). Microscopic examination was performed with an OLYMPUS microscope model BX40 and Microphoto OM system.

ELISPOT assay for single cell IFNγ and IL-5 release

Ninety six-well nitrocellulose-backed plates (Millipore, Bedford, MA) were precoated with anti-IFNγ (Mabtech, Sweden) or anti-IL-5 mAb (BD, PharMingen) at 10 μg/mL. Fresh NALs or IVS T cells were added to triplicate wells (105 cells/well) and stimulated with either PMA + ionomycin or with T2 cell line or LCL (2 × 104 cells/well) pre-loaded with individual EBV-derived peptides for 24–48 h, at 37°C in 5% CO2. Supernatants were harvested for ELISA measurements, while wells were then washed free of cells and soluble factors, and a second biotinylated anti-IFN-γ (1 μg/mL, Mabtech) or anti-human IL-5 (2 μg/mL, BD, PharMingen) mAb was added for an additional 2–4 h at 37°C, 5% CO2. The reaction was developed as described (17). The spots were counted using an ELISPOT plate reader (CTL, Cleveland, OH).

ELISA for IL-10 and TGF-β1

Culture supernatants from ELISPOT assays (set up as described above) were collected, and levels of IL-10 and TGF-β1 quantified by ELISA (18). Primary and secondary mAbs, and the recombinant cytokine for IL-10 ELISA were purchased from Pierce Endogen (Rockford, IL), while reagents for TGF-β1 ELISA were purchased from BD Bioscience. The lower limits of detection for both these assays were 60 pg/mL.

Analysis of FOXP3 transcripts by quantitative real-time PCR

Total RNA was extracted from fresh or IVS CD4+ and CD8+ sorted T cells, using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. cDNA was prepared from 200 ng total RNA using the SuperScript III First-Strand Synthesis kit (Invitrogen). FOXP3 mRNA levels were quantified using the ABI 7900 Sequence Detection System (Applied Biosystems, Foster City, CA) at the Genomics and Proteomics Core Laboratories at University of Pittsburgh. FOXP3 and 18s primers were purchased from Applied Biosystems. Samples were run in triplicate, and the relative expression of FOXP3 was expressed as fold increase after IVS, by normalizing expression of each target to 18s ribosomal RNA (ΔCT), and then comparing this normalized value to the normalized expression in the baseline sample (ΔΔCT). R.Q. values were calculated as 2ΔΔCT.

Statistical analysis

A paired two-tail Student's t-test was used for statistical analysis of mean ± SD values between groups, where p ≤ 0.05 were considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Decreased EBV-specific IVS T cells yields from SOTx patients are not due to increased cell death, but to decreased CD4+ T-cell proliferation

The absolute number of IVS T cells (Figure 1A) generated from SOTx patients cultures was significantly lower (fold increase = 1.3) than the absolute number observed from healthy controls (fold increase = 2.2). To determine whether the poor IVS T cells yields from SOTx patient cultures was due to increased apoptotic cell death, we analyzed the kinetics of responder T cell Annexin V staining by flow cytometry. As shown in Figure 1B, the kinetics and the levels of CD8+/Annexin V+ T cell in cocultures generated from both healthy controls and SOTx patients were similar, with a maximum level on day 3. In contrast, the % of patients' apoptotic CD4+ T cells was significantly lower than that of normal donor CD4+/Annexin V+ T cells.

image

Figure 1. In vitro-stimulated T cells yields, kinetics of T-cell apoptosis and proliferation from SOTx patients and control cocultures. DC1 loaded with a pool of EBV-derived peptides were cocultured with autologous NALs for 10 days. (A) The activated T-cell yields obtained on day 10 from all cocultures of SOTx patients (n = 9) and controls (n = 9) are shown as mean ± SD. (B) Lymphocytes were harvested at the initiation, and on days 3, 5 and 10 after the start of cocultures. IVS T cells (1 × 105 cells/tube) were stained with Annexin V-FITC, 7ADD-PerCp and CD4-PE or CD8-PE in three-color flow cytometric analysis. Cells were gated on CD4+ or CD8+ T cells, and the percentage of Annexin V+/7ADD staining was measured to determine the level of apoptosis. Results shown as mean ± SD of six (three controls and three patients) experiments performed. (C) NALs from SOTx patients or healthy controls were prestained with CFSE, cocultured with autologous DC1 loaded with a pool of EBV-derived peptides for 5 days and analyzed by flow cytometry. The percentage of proliferating cells was determined in each instance by gating on CFSE-diluted cells out of CD4+ or CD8+ T cells. Results shown are from one experiment representative of eight independent experiments (four controls and four patients).

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To further examine mechanisms leading to the poor IVS T-cell yields from SOTx patient cocultures, we analyzed T cell proliferation using CFSE dilution assays and flow cytometry. As shown in Figure 1C, we noted significant differences in the patterns of T cell proliferation between patient and healthy control cell cocultures. Both EBV-specific CD8+ T cells (50 ± 15%) and Ag noncognate CD4+ T cells [(45 ± 20)%] proliferated in healthy controls (Figure 1C). In contrast, for SOTx patients, significant proliferation of EBV-specific CD8+ T cells [(55 ± 15)%] in response to Ag stimulation occurred, but this was not the case for CD4+ T cells [(20 ± 10)%], suggesting that these cells may be anergized (Figure 1C).

Immune-polarization of EBV-specific CD8+ T cells generated from SOTx patients

To further evaluate the type-1 versus type-2 versus type-3/Treg immune-polarization of EBV-specific CD8+ T cells in SOTx patients and normal controls, we performed cytokine ELISPOT and ELISA analyses. The frequencies of resting peripheral blood CD8+ T cells that produced IFN-γ upon EBV-peptide stimulation were on average lower in SOTx patients than in normal controls (data not shown), as reported previously (19). In addition, the pattern of peripheral blood CD8+ T-cell immune polarization against all EBV epitopes evaluated (lytic-BZLF-1/BMLF-1 and latent-EBNA-3A/LMP2-a) in patients was similar to that of normal controls. A type-1 predominance was observed, with an approximate IFN-γhigh/IL-5low ratio of 5:1 (data not shown). General T-cell immunocompetence was evaluated in response to PMA + ionomycin (Figure 2A) and the frequency of EBV-specific CD8+ T cells secreting IFN-γ was expanded significantly in both patients and normal controls to similar levels, with expanded Type-1 polarization (IFN-γhigh/IL-5low ratio of 6:1) against each EBV peptide tested individually (Figure 2B). The levels of IL-10 and TGF-β1 in supernatants from ELISPOT assays were assessed by ELISA. Interestingly, the supernatants generated from SOTx patients exhibited significantly higher levels of IL-10 versus normal controls in response to either nonspecific (PMA + ionomycin) or EBV-specific stimulation (Figure 3A), and CD8+ T cells were the source of IL-10 (Figure 3B). In addition, soluble TGF-β1 was not detected above media background levels in any of the samples evaluated (data not shown).

image

Figure 2. Type-1/type-2 immune polarization of EBV-specific CD8+ T cells expanded from SOTx patients and controls. NALs were cocultured with autologous DC1 loaded with a pool of EBV peptides for 10 days. IVS T cells (1 × 105 cells/well) from control subjects (n = 9) and SOTx patients (n = 9) were harvested and tested in ELISPOT assays after PMA + ionomycin nonspecific stimulation (A) or against the T2 cell line (2 × 104 cells/well) pulsed with individual EBV peptides (B). Results are expressed as number of IFN-γ/IL-5 secreting cells/105 cells of nine experiments performed.

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image

Figure 3. Treg/type-3 immune polarization of EBV-specific CD8+ T cells expanded from SOTx patients and controls. (A) NAL and IVS T cells from controls or from SOTx patients were stimulated with PMA + ionomycin or EBV-peptides in ELISPOT for 48 h and supernatants were harvested and tested in ELISA to determine IL-10 levels. Results are reported as means ± SD of nine experiments performed. (B) Bulk, sorted CD4+ or CD8+ T cells from IVS T cells generated from SOTx patients were stimulated with PMA + ionomycin or with EBV-peptides, and supernatants were harvested and tested in ELISA to determine IL-10 levels. Results shown are means ± SD of three experiments performed. (C) Positively sorted EBV-TMR-PE CD8+ T cells (red) were stimulated with PMA + ionomycin and stained for intracellular IFN-γ-Cy2 (green) or IL-10-Alexa Fluor 647 (deep red). Colocalization of IFN-γ and IL-10 is shown in yellow (overlay). Nuclei were counterstained with DAPI. The slides were analyzed by fluorescent microscopy ×400. Results shown are from one experiment representative of five performed.

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To further identify the cellular source of IFN-γ and IL-10, we used HLA-A2 or HLA-B8 TMRs to positively sort IVS EBV-specific CD8+ T cells using anti-PE magnetic beads. As shown in Figure 3C, the control EBV-TMR+ cells produced high levels of IFN-γ (type-1 polarization), while only very few cells produced both IFN-γ and IL-10 (Figure 3C overlay). In contrast, SOTx patients' EBV-TMR+ cells produced equally high levels of both IFN-γ and IL-10 (type-1-like Treg polarization).

EBV-specific type-1-like CD8+ Treg actively suppress Ag noncognate CD4+ T-cell proliferation in an Ag-specific, cell-to-cell contact-dependent manner

We further investigated the mechanism by which EBV-specific CD8+ T cells from SOTx patients suppressed CD4+ T-cell proliferation. The proliferation of CD4+ T cells was inhibited exclusively when patient DC1 presented specific EBV-peptides to autologous CD8+ T cells (Figure 4B), and not due to patient DC alone (Figure 4A). Moreover, CD8+ type-1-like Treg-mediated suppression was not due to IL-10, since addition of anti-IL-10 blocking mAb at the initiation of cocultures did not restore the level of CD4+ T-cell proliferation (Figure 4C). To further test whether cell–cell contact was required, we separated CD4+ and CD8+ T cells using trans-well membranes, with DC1 loaded with EBV peptides on each side of the trans-well membrane at the same total number as in the other conditions. CD8+ T cells proliferated actively in response to EBV stimulation as expected, as they interacted exclusively with the APCs (Figure 4D). In addition, CD4+ T cells were released from the CD8+ T effect, and their proliferation was restored when CD8+ cells and CD4+ T cells were separated (Figure 4D), indicating that cell–cell contact was required for CD4+ T-cell regulation by CD8+ type-1-like Treg.

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Figure 4. EBV-specific CD8+ type-1 like Treg generated from SOTx patients inhibit noncognate CD4+ T-cell proliferation in a cell–cell contact-dependent manner. NALs from SOTx patients or healthy controls were stained with CFSE, and then cocultured with autologous DC1 either unloaded or loaded with a pool of EBV-derived peptides. In selected wells, IL-10 blocking mAb was added at the initiation of the cocultures. Alternatively, immune magnetic bead sorted resting CD4+ or CD8+ T cells were stained with CFSE and incubated with autologous DC1 pulsed with EBV peptides in wells separated by trans-well membranes. IVS T cells were harvested on day 5, and CD4+- or CD8+-gated events were examined for CFSE dilution. Results are from one experiment representative of eight performed.

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EBV-specific type-1-like CD8+ Treg generated from SOTx patients up-regulate FOXP3 mRNA

We further examined whether the EBV-specific CD8+ T cells expanded from SOTx patients expressed FOXP3, a transcription factor known to be associated with CD4+CD25+ naturally occurring Treg, and more recently also with adaptative Ag-specific Treg (20). Results obtained by RT-PCR on all subjects tested are shown in Table 1. As indicated in Figure 5, IVS CD8+ T cells expanded from SOTx patients, significantly up-regulated FOXP3 mRNA levels as compared with baseline values of freshly-isolated CD8+ T cells (fold increase = 39 ± 26). In contrast, the CD8+T cells expanded in vitro from controls only modestly up-regulated expression of FOXP3 mRNA (fold increase = 6 ± 2) (Figure 5). Furthermore, FOXP3 mRNA expression was detected also in CD4+ T cells in both patients (fold increase = 8 ± 10) and controls (fold increase = 2 + 1), but they were not significant different from each other.

Table 1.  FOXP3 mRNA levels after IVS of CD8+ and CD4+ T cells
ControlsFold increasePatientsFold increase
CD4+CD8+CD4+CD8+
C13.29.1P15.644
C21.26.9P22267.7
C31.44.3P31.519.3
C41.25.3P42.611.1
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Figure 5. EBV-specific CD8+ type-1-like Treg generated from SOTx patients up-regulate FOXP3 mRNA. Expression level of FOXP3 mRNA was measured by real time RT-PCR on CD4+ and CD8+ T cells sorted from NALs or from IVS T cells. Results are means ± SD of eight independent experiments (four controls and four patients) and shown as comparative fold increase in FOXP3 mRNA expression.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Detailed functional studies of the DC-T cell interaction in SOTx patients receiving chronic IS are scarce. Here we analyzed the comparative efficacy of type-1-polarized DC loaded with a pool of MHC class I-restricted EBV-derived peptides to reactivate specific CD8+ T cells from blood of quiescent SOTx patients and normal controls. The EBV peptides selected for the analysis provided antigenic material for CD8+ T cells to address both viral replication and latency control.

Our results show, as expected, that CD8+ T cells isolated from EBV+ healthy controls could be easily boosted with mature DC loaded with EBV Ags and that reflected, in most cases, the reactivation of existing EBV-specific memory CD8+ T cells (21). IVS-expanded CD8+ T cells proliferated well and this involved the expansion of both Ag-specific CD8+ T cells and Ag nonspecific CD4+ T-cell proliferation. In response to EBV peptide challenge, specific CD8+ T cells produced high levels of IFN-γ, and maintained a type-1 immune polarization (IFN-γhigh/IL-5low) (Figure 2), while producing only low levels of IL-10 (Figure 3).

In contrast, SOTx patients' T cells stimulated with DC1 loaded with EBV-peptides generated disparate results. Although EBV-specific CD8+ T cells expanded in cocultures, the overall yields were modest compared with normal controls. Upon further analysis, we found that while CD8+ T cells proliferated well after cognate EBV-specific activation, and exhibited a IFN-γhigh/IL-5 low functional phenotype, they also released high levels of IL-10. These cells suppressed noncognate CD4+ T-cell activation and proliferation, mediated largely by direct cell–cell contact. Since CTLA4 up-regulation was not detected on the EBV-specific CD8+ T cells (data not shown), we are currently investigating the potential involvement of other inhibitory molecules (i.e. programmed death-1) that might be mediating CD4+ T cell inhibition of proliferation. Moreover, EBV-specific CD8+ T cells up-regulated FOXP3 mRNA, a transcription factor reported to be restricted to CD4+CD25+ natural-occurring Treg. These findings prompted us to designate these patient CD8+ T cells as type-1-like EBV-specific CD8+ Treg. To our knowledge, this is the first study to report that DC presentation of EBV Ags via MHC class I in SOTx patients promotes the expansion of memory CD8+ T cells expressing a FOXP3+ type-1-like Treg (IFN-γhigh/IL-10high/IL-5low) phenotype able to suppress Ag noncognate CD4+ T cells. Interestingly, previous reports have described the induction of Ag-specific CD8+ T cells with ‘veto’ activity, that trigger apoptosis in bystander T cells that recognize Ags on the veto cells, a phenomenon facilitated by IS drugs (i.e. rapamycin) (22,23). However, results presented here describe the generation of Ag-specific CD8+ T cells with regulatory capacity that induce hypo-responsiveness rather than apoptosis in CD4+ T cells (Figure 1B).

Regulatory T cells have been identified in mice and humans, and have been typically described as CD4+CD25+ T cells. They appear to be involved in maintaining immunologic homeostasis and selftolerance, while preventing pathologic conditions such as chronic inflammatory disease/autoimmunity or allograft rejection (24–26). Certain subsets of Treg, called natural regulatory cells, are generated in the thymus during T-cell development (27). Other Treg subsets such as Tr1 (IL-10) or T helper type 3 (TGF-β) referred to as Treg can develop from conventional T cells exposed to specific stimulatory conditions, such as blockade of costimulatory signals, anti-inflammatory cytokines or IS drugs (28–30). More recent studies have demonstrated that conversion to a FOXP3+ phenotype and Treg function can be exhibited by both CD4+CD25+ and CD8+CD25+ T cells from FOXP3 cells (31–33). Ziegler has postulated that in humans exclusively, FOXP3 behaves like an activation-induced gene in CD4+ T cells following in vitro TCR stimulation, emphasizing that human and mouse FOXP3 are regulated differently (34). These de novo generated FOXP3+ Tregs from healthy controls actively proliferate upon Ag-cognate stimulation, and suppress bystander cells by a cell contact-dependent and cytokine independent manner (34,35). Our novel CD8+ T cell results (Figure 5) are therefore in good agreement with Ziegler's findings.

The biology of Treg in the context of SOTx has recently attracted significant interest, regarding understanding of their function, and more importantly, of how to generate allo-reactive Tregs for immunotherapeutic purposes to eliminate graft rejection. Experimental murine models using anti-CD4 mAb and donor-specific transfusion pre-conditioning have demonstrated that, while in vivo-induced allospecific CD4+CD25+ Treg require allo Ag-specific activation, they can regulate bystander cells in an Ag-independent manner that relies on IL-10 and CTLA4 (36). Conversely, Karim et al. showed recently that reactivation of CD4+CD25+ Treg specific for unrelated, nongraft Ags, allows these Treg to suppress alloreactive T cells in a bystander manner (37).

IS drugs used to inhibit allospecific immune responses in the Tx setting also induce Treg during the development of the allogeneic T-cell response, but the mechanisms responsible for Treg-mediated suppression in this setting have not been fully characterized. Glucocorticoids inhibit the ability of DC to acquire a mature phenotype, to drive recall Ag- or alloAg-induced T-cell proliferation, or to secrete IL-12p70 (38), while inducing the generation of IL-10-producing Treg (30,39). Our published results have shown that chronic in vivo administration of tacrolimus and prednisone to SOTx patients has an ex vivo carry-over effect on DC differentiation and function in short-term cultures (11). On the other hand, other reports have also shown that glucocorticoids can directly affect CD4+ and CD8+ T cells, promoting their ability to produce IL-10, an anti-inflammatory cytokine that inhibits Th1- or Th2-type immunity (40). Although the effects of tacrolimus on DC are controversial, its strong inhibitory influence on T-cell function is well recognized (41). Therefore, we must acknowledge that endogenous levels of these drugs may influence both DC and T cell functions in our setting. In this context, we have demonstrated the critical role of Ag presentation by ‘semi-mature’ DCs in activating Ag-specific, type-1-like CD8+ Treg. These T cells produce IFN-γ in response to Ag restimulation, inhibit Ag nonspecific CD4+ T-cell activation by cell–cell contact, and secrete IL-10 that may have an inhibitory effect on T cells as well as on DC. Although additional analysis will be required to further delineate the mechanisms by which DC interact with CD8+ and CD4+ T cells and how this multi-layered suppressive network functions in vivo, we present a hypothetical model in Figure 6.

image

Figure 6. Hypothetical model of interactions between EBV-specific CD8+ T cells, DC and Ag nonspecific CD4+ T cells in SOTx patients.

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In conclusion, our results show that EBV-specific CD8+ T cells can be expanded ex vivo from SOTx peripheral blood using DC1-based protocols, but they display both type-1 effector (IFN-γhigh) and regulatory (cell surface receptor/IL-10high) features, suggesting that future immunotherapeutic approaches will likely be successful. However, these approaches will have to consider means by which to subvert systemic type-1-like regulatory EBV-specific CD8+ T-cell responses to afford optimal clinical benefit. In addition, our results suggest possible mechanism(s) by which T-cell immune surveillance is regulated in quiescent SOTx patients receiving prednisone and tacrolimus maintenance therapy. By extrapolation from our findings and in accordance with published information relevant to Ag-specific CD4+CD25+ Treg, it appears that reactivation of any Ag-specific memory CD8+ T cells may subsequently regulate Ag-unrelated T cell (including allospecific and EBV-specific T cell) activation, either as a desired outcome (i.e. graft acceptance) or as an unwanted side-effect (i.e. impaired protective Ag cognate immune response).

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The authors thank our research coordinators Sheila Fedorek, Clyde Harris and Darleen Koritsky for their support in collecting patients' blood samples. This work was supported by grants from the American Cancer Society (CRTG-02-043-01-CCE), American Heart Association (0230165N) and National Institutes of Health (5P5OHLO74732) (DM).

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  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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