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International Immunology Advance Access originally published online on September 11, 2006
International Immunology 2006 18(11):1549-1562; doi:10.1093/intimm/dxl088
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

TCR transgenic CD8+ T cells activated in the presence of TGFß express FoxP3 and mediate linked suppression of primary immune responses and cardiac allograft rejection

Judith A. Kapp1,2, Kazuhito Honjo2, Linda M. Kapp1, Xiao yan Xu2, Alana Cozier2 and R. Pat Bucy2

1 Department of Ophthalmology, Spain Wallace Building, 619 South 19th Street, University of Alabama at Birmingham, Birmingham, AL 35233-7331, USA
2 Department of Pathology, Spain Wallace Building, 619 South 19th Street, University of Alabama at Birmingham, Birmingham, AL 35233-7331, USA

Correspondence to: J. A. Kapp; E-mail: jkapp{at}uab.edu


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Although CD4+CD25+FoxP3+ regulatory T cells play a role in allograft tolerance, the role of CD8+ cells with immunosuppressive function is less clear. To address this issue, spleen cells from Rag-1-deficient TCR transgenic (Tg) mice expressing a receptor for ovalbumin (OVA) in the context of MHC class I (OT1) were activated with OVA expressing antigen-presenting cell (APC) in the presence or absence of exogenous transforming growth factor ß (TGFß). TGFß inhibited the expression of IFN-{gamma}, granzyme B and the lytic activity of the OT1 T cells while inducing FoxP3 expression in 5–15% of the cells. By contrast, FoxP3 expression was not detected in naive OT-1 T cells or OT-1 T cells activated without exogenous TGFß. TGFß-activated OT1 cells inhibited the activation of Kd-specific CD8+ CTL responses by normal B6 T cells and the proliferation by Kd-specific CD4+ TCR Tg T cells, but only if the OVA epitope was co-expressed by Kd+ APC. This antigen-specific inhibitory activity, referred to as linked suppression, was neither mediated by residual lytic activity within the activated OT1 T cells nor did it depend upon IL-10 or TGFß. Suppression correlated with inhibition of CD86 expression on CD11c+ APC. TGFß-activated OT1 T cells also delayed the rejection of heterotopic, vascularized cardiac allografts mediated by anti-Kd-specific CD4+ TCR Tg T cells, but only if the cardiac allograft expressed both OVA and Kd as transgenes. Prolonged survival of allografts was associated with rapid migration of the FoxP3+ OT1 T cells into the donor heart raising the possibility that suppression may be mediated within the allograft. These data show that TGFß-activated CD8+ T cells mediate antigen-specific, APC-focused patterns of suppression in vitro and in vivo.

Keywords: CTL, transgenic/knockout mice, transplantation


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Over 50 years ago, Medawar and colleagues (1) demonstrated that induction of antigen-specific tolerance in rodents could prolong allograft survival in the absence of pharmacological agents. Although multiple different mechanisms that could contribute to tolerance have been elucidated, the contribution of active suppression mediated by antigen-specific T cells to this process has had variable popularity. The seminal experiments of Gershon and Kondo (2) showing that antigen-specific tolerance could be adoptively transferred with lymphocytes from tolerant to naive mice, a phenomenon they labeled as ‘infectious tolerance’, raised the possibility that tolerance could be maintained by an active process. CD8+ T lymphocytes were shown to mediate antigen-specific tolerance and were referred to as suppressor T cells (Ts) (3, 4). Although the ability of antigen-specific CD8+ T cells to adoptively transfer non-responsiveness was confirmed by numerous investigators in a variety of systems including transplant rejection [reviewed in (5)], the mechanisms involved in suppression were not identified and interest in this pathway waned.

The concept that T cells can mediate immunological tolerance through active suppression has again become widely accepted on the basis of the observations that a distinct subset of naturally occurring CD4+CD25+ T cells (6), referred to as regulatory T cells (Tregs), has the capacity to prevent autoimmune disease mediated by endogenous, self-reactive T cells (7, 8). The presence of CD25 on these resting T cells allowed them to be physically isolated and shown to have specific immunosuppressive activity. Molecular analysis of Tregs identified the unique expression of a transcription factor from the forkhead/winged helix family (FoxP3) (9, 10), which functions as a master switch driving differentiation of naive T cells into the Treg lineage (10–15).

Interest in the role of CD8+ Ts was renewed by the observation that delivery of antigens into the anterior chamber of the eye induced the activation of CD8+ T cells that inhibit responses of primed effector T cells (16) and that the activation of the CD8+ Ts depended upon transforming growth factor ß (TGFß), a normal constituent of the aqueous humor (17). Antigen-specific CD8+CD28– Ts have also been identified in the blood of rejection-free cardiac transplant recipients (18, 19). Moreover, FoxP3 is up-regulated in CD8+CD28– human (20) and rat T cells (21) that have suppressive activity.

Our long-term goal is to determining the mechanisms by which CD8+ Ts can prolong allograft survival. However, the regulation of alloantigen-specific rejection is difficult to dissect because of the complexity of natural alloantigens, the large number of T cells expressing heterogeneous receptors that can recognize even limited alloantigenic differences between the host and the donor animals and multiple pathways of antigen presentation. Consequently, we have used a reductionist approach to develop a model in which the key elements of this complex in vivo response can be isolated. To generate CD8+ Ts, spleen cells from the ovalbumin (OVA)-specific Rag1–/– TCR transgenic (Tg) mice expressing a receptor for OVA in the context of MHC class I (OT1) (22) were incubated with spleen cells from mice expressing a chicken OVA transgene (23) with or without exogenous TGFß. To restrict the specific antigenic epitopes that drive the T cell response, we have generated TCR Tg mice (TCR75) with specificity for a single peptide derived from the H-2Kd MHC class I molecule presented by the I-Ab class II molecule (K54–68d/I-Ab) on the C57BL/6 (B6) background (24). Antigen-presenting cells (APCs) and hearts were obtained from Tg B6 mice, expressing the genomic sequence of H-2Kd (25), the chicken OVA gene or both genes. Here, we report that non-lytic CD8+ OT1 T cells activated in the presence of TGFß expressed FoxP3 and exhibited linked suppression in vitro and in vivo.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Experimental animals
B6 (H-2b) mice were purchased from the National Cancer Institute (Frederick Cancer Research and Development Center, Frederick, MD, USA). C57BL/6-Tg(TcraTcrb)1100Mjb (22) mice, also referred to as OT1, which recognize OVA257–264 in the context of H2Kb were a generous gift of Michael Bevan (University of Washington, Seattle, WA, USA) and were bred and maintained in the animal facilities at the University of Alabama at Birmingham (UAB). The C57BL/6-Tg(TcraTcrb)TCR75Rpb (TCR75) mice, which express TCR{alpha}ß specific for I-Ab/H-2K54–68d epitope, have been previously characterized (24). The OT1 and TCR75 Tg strains were crossed onto the B6.Rag1–/– background to exclude expression of other TCR specificities. B6.Rag1–/–.OT1 mice were also crossed to perforin, or pore-forming protein (pfp), knockout B6 Prf1tm1Sdz (26) obtained from Jackson Laboratory (Bar Harbor, ME, USA), to produce B6.Rag1–/–.OT1.pfp–/– mice (referred to as OT1.pfp–/–). B6.Rag1–/–.OT1 mice were also crossed to IL-10 knockout (B6.129P2-Il10tm1Cgn/J) (27) obtained from Jackson Laboratory, to produce B6.Rag1–/–.OT1.IL-10–/– mice (referred to as OT1.IL-10–/–). Rag1–/– TCR Tg strains were used in all experiments in this study; however, they are referred to by the name of the transgene, without the explicit notation of the Rag genotype, for simplicity. Mice expressing a membrane form of the chicken OVA gene under the chicken actin promoter, C57BL/6-Tg(ACTB-OVA)916Jen, which we will refer to as B6.OVA, were generously provided by Marc Jenkins (23). C57BL/6-Tg(Kd)Rpb (B6.Kd) mice express the full genomic sequence of H-2Kd,which has a tissue distribution indistinguishable from the expression of endogenous H-2Kd in B10.D2 mice (25). B6.OVA, B6.Kd and F1 (B6.Kd x B6.OVA), referred to as B6.Kd.OVA, mice were used as a source of APC or as the cardiac donors in these studies. We have also bred several of these Tg strains to express homozygous polymorphisms for either the CD451/1 (Ly5) or CD901/1 (Thy1) alleles to allow simultaneous tracking of two populations of cells in adoptively transferred B6 (CD452/2CD902/2) recipients. All procedures on animals were conducted with approval by the UAB Institutional Animal Care and Use Committee.

Induction and assay of antigen-specific CD8+ Ts
We have adapted the system that we previously used to study polyclonal alloreactive Ts (28, 29) to the analysis of TCR Tg T cells in vitro. This is a "two-step" culture system in which the first culture is used to activate naive T cells into a suppressive phenotype and the second-step culture measures their suppressor activity on the induction of a primary CTL response. To activate suppressor activity, spleen cells from OT1 mice (0.5 x 106) were incubated with (0.5 x 106) irradiated (2500 Rad) spleen cells (SpLx) from B6.OVA Tg mice, as a source of APC, with or without 3 ng ml–1 of recombinant TGFß1 (R&D Systems Inc., Minneapolis, MN, USA) in 48-well plates. After 3 days, these cells were harvested, irradiated (2500 Rad), to prevent the TCR Tg cells from overgrowing the second-step culture, and co-cultured with B6 responder spleen cells (2.0 x 106) or enriched splenic T cells (0.7 x 106) and SpLx (1.0 x 106) from either B6.Kd.OVA-double Tg mice or a mixture of spleen cells from B6.Kd and B6.OVA mice. Cultures were harvested after 4 or 5 days and assayed for Kd-specific CTL activity as described below. Blocking antibodies specific for IL-10 (JESS-2A5; BD Biosciences Franklin Lakes, NJ, USA) or TGFß1, -2 and -3 (1D11; R&D Systems Inc.) were added at culture initiation in the indicated experiments.

In some experiments, T cells were enriched from responder B6 spleen cells to enhance the level of CTL responses to Kd stimulators using SpinSepTM, StemCell Technologies (Vancover, BC, Canada), according to the manufacturer's directions. This negative selection methodology was chosen because T cells are enriched by depleting B cells, granulocytes, etc, without introducing the potential problem of cross-linking cell-surface molecules and activating the T cells, which can occur with positive selection methods. The purified T cells routinely were 90–92% CD3+. Moreover, enriched T cells were as sensitive to suppression but the CTL response was higher than unpurified spleen cells (data not shown). Splenic CD11c+ cells were purified by digesting spleens with 0.5 mg ml–1 of collagenase VIII (Sigma–Aldrich, St Louis, MO, USA) in 2 ml of culture medium for 45 min in CO2 incubator. After removal of RBCs with lysing buffer (NH4Cl/Tris–HCl buffer, Sigma–Aldrich), spleen cells were incubated with PE-labeled anti-mouse CD11c antibody (clone HL3, BD Biosciences), re-suspended in MACS buffer (2% FCS/2 mM EDTA in PBS), mixed with anti-PE MACS beads and loaded onto a Miltenyi Biotech (Auburn, CA, USA) miniMACS column attached to a separation magnet. Cells were passed through the column, which was then washed three times, and removed from the magnet. The positively selected cells were flushed out from the MACS column with 1 ml of MACS buffer using a plunger, which typically yielded ≥90% CD11c+ cells.

In some cultures, cell division was determined by incubating cells at 5 x 106 T cells ml–1 with 1 µM 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE, Molecular Probes, Eugene, OR, USA) using the method described by Lyons and Parish (30). Briefly, the cells were incubated at 37°C for 8 min with occasional gentle mixing and the staining reaction was terminated by adding 1/10 volume of FCS to the cell suspension. The geometric mean fluorescence intensity of CFSE staining was measured by CellQuest software (Becton Dickinson), and corrected for linearity by calibration with Sphero Rainbow calibration particles (Spherotech, Inc., Libertyville, IL, USA) to determine the intensity in mean equivalent soluble fluorescence units. The mean cycle number of the population was then determined by the equation [log(CFSEc) – log(CFSEex)] / log(2), where CFSEc is the CFSE intensity of control cells and CFSEex is the CFSE intensity of the experimental cells. Thus, the loss of CFSE with time that is not associated with cellular division is taken into account at each time point.

In other experiments, the proliferative responses were measured in 96-well round-bottom plates containing 5 x 104 TCR75 lymphoid cells and 15 x 104 irradiated (2500 Rad) spleen cells each from the indicated strains of mice as stimulator cells. Various numbers of OT-1 cells, activated for 3 days with irradiated B6.OVA spleen cells with or without exogenous TGFß, were irradiated (2500 Rad) and added to these cultures, which were pulsed with 1 µCi [3H]thymidine ([3H]-TdR) (Amersham, Arlington Heights, IL, USA) 48 h later, and then harvested 16 h later and counted in a ß-counter.

Target cell lines and CTL assay
Two H-2b tumor cell lines [EL4, an MHC class II-negative T cell lymphoma and E.G7-OVA, which is an EL4 cell line that express the chicken OVA gene (31) and P815 (H-2d) a mastocytoma] were used as targets for the CTL assays. These cells were cultured in standard growth media (RPMI 1640 medium supplemented with 5% FBS, 1 mM L-glutamine, 1 mM sodium pyruvate, 50 µM 2-mercaptoethanol, gentamycin, penicillin and streptomycin) at 37°C in a 5% CO2 atmosphere. The E.G7-OVA line was periodically cultured with 1 mg ml–1 of G-418 (Invitrogen, Carlsbad, CA, USA) to maintain expression of the OVA gene. All cell lines were free of mycoplasma.

CTL activity was measured using a modification of published flow cytometry methods using CFSE-labeled targets (32, 33) to directly count the number of viable target cells. Briefly, two different cell populations (one that expresses the relevant pMHC epitope and one that did not) were labeled with different concentrations of CFSE that can be discriminated by flow cytometry. The labeled targets were mixed 1:1 and incubated with different concentrations of effector cells, as done in the standard 51Cr release assay. After 4 h incubation, 7-amino-actinomycin D (7-AAD), a membrane impermeable DNA binding dye (BD Biosciences), was added to allow discrimination of dead cells, and the mixture was analyzed by flow cytometry to determine the ratio of viable cells at various effector to target (E:T) ratios. Replicate wells of mixed Ag+ and Ag– cells were also incubated without any effector cells as controls. The ratio of viable Ag+/Ag– cells in each experimental tube divided by the same ratio in the control tubes gives a measure of antigen-specific cytotoxicity by the formula:

Formula
This method gives equivalent results to the standard 51Cr release assay, but avoids the problems of handling and disposal of radioactive material. To measure anti-Kd CTL, P815 (H-2d, mastocytoma) cells were compared with EL4 cells (H-2b, thymoma). OVA-specific CTLs were measured using E.G7-OVA tumor cells and EL4 as a negative control. Addition of 10-µm diameter fluorescent beads (Molecular Probes) as a volume calibrator gave equivalent results substituting the bead count for Ag– cell count (data not shown). As an index of the CTL activity, lytic units were calculated as the inverse of the number of effector cells required for 30% cytotoxicity, by mathematically fitting the curve of percent specific cytotoxicity versus effector cells per 104 targets as adapted from Wunderlich et al. (34). If the highest number of effector cells gave <5% specific cytotoxicity, an estimate of lytic units was calculated by extrapolating this value to 30% lysis using the slope of the positive control cultures.

Adoptive transfer
Spleens from naive OT-I (CD452/2, CD901/1) or TCR75 (CD451/1, CD902/2) mice were removed and single-cell suspensions were prepared. RBCs were removed by hypotonic lysis and then splenocytes were washed and re-suspended in Cellgro® Hanks' balanced salt solution (HBSS) from Mediatech Inc. (Herndon, VA, USA) and injected at the indicated doses into naive B6 (CD452/2, CD902/2) mice via the lateral tail vein.

Heterotopic cardiac transplantation
The method for heterotopic cardiac transplantation was adapted from Ono and Lindsey (35). Briefly, the donor aorta was anastomosed in end-to-side fashion to the recipient abdominal aorta and the pulmonary artery was similarly anastomosed to the vena cava. The heart was palpated daily; no detectable beating was presumed to be rejection, which was confirmed by laparotomy.

Immunofluorescent staining
Single-cell suspensions were stained for cell-surface antigens by incubating with PE-, FITC- or biotin-labeled mAbs specific for CD90.2 (30-H12), CD90.1 (OX-7), CD45.1 (A20), CD11c (HL3), CD86 (GL1) and CD25 (7D4) (BD Biosciences) or GITR (DTA-1) and CTLA-4, (UC10-4B9) (eBioscience, San Diego, CA, USA). After cell-surface staining, cells were fixed, permeabilized and stained for intracellular expression of granzyme B (FITC–16G6) and FoxP3 (PE–FJK-16s), using the staining set reagents, all of which were obtained from eBioscience according to the manufacturer's directions. Biotinylated antibodies were detected with streptavidin (SA)–RED670 obtained from Invitrogen Corporation (Carlsbad, CA, USA) or SA–Cy chrome (BD Biosciences). Flow cytometric analysis was performed using either a FACScan® or FACSCalibur® Instrument (Becton Dickinson, Mountain View, CA, USA). In some experiments, 7-AAD was added to allow gating on viable cells. The data were analyzed using either CellQuestPro (Becton Dickinson) or FlowJo software (Treestar, San Carlos, CA, USA).

For immunohistochemical analysis, 4-µm sections were cut from fresh frozen tissues and stained with the same antibodies that were used for flow cytometry, as previously described (36, 37). Sections were counterstained with 4',6-Diamidino-2-phenylindole to visualize the nuclei.

Real-time quantitative reverse transcription–PCR
TaqMan hybridization detection was used to measure the amount of FoxP3 and IFN-{gamma} mRNA expression in OT1 T cells after antigen activation with and without TGFß. The Roche Lightcycler® instrument was used to compare the fluorescence ‘crossing-point’ for a cDNA plasmid at known number of copies per reaction to random hexamer-primed cDNA made from RNA extracted from known numbers of T cells. By comparing the amount of amplicon produced in a series of reactions with known concentrations of cDNA plasmid for each set of primers and TaqMan probe, the copies per 106 cells were calculated for both FoxP3 and IFN-{gamma} as previously described (38, 39).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Induction of antigen-specific CD8+ Ts
TGFß up-regulates FoxP3-expressing Tregs from CD4+CD25– precursors (40–46) and inhibits the generation of lytic activity (47) while promoting suppressive activity of CD8+ T cells (48). Thus, we asked whether suppressor activity could be induced in TCR Tg OT1 T cells incubated with SpLx from B6.OVA Tg mice, as a source of APC, in the presence or absence of exogenous recombinant TGFß. The cultured cells were harvested after 3 days and assayed for OVA-specific CTL activity using CFSE-labeled E.G7-OVA and EL4 targets. OT1 T cells that were stimulated with B6.OVA spleen cells developed strong OVA-specific CTL activity that was detectable at E:T ratios as low as 1:1 (Fig. 1A), whereas no lytic activity was detected in cultures that did not contain B6.OVA (data not shown). OT1 T cells that were activated in the presence of TGFß developed little if any detectable lytic activity (Fig. 1A), which confirms the studies of Lee and Rich (47). This figure also demonstrates how lytic units (an index of the CTL activity) were calculated as the inverse of the number of effector cells required for 30% cytotoxicity, by mathematically fitting the curve of percent specific cytotoxicity versus effector cells per 104 targets, as adapted from Wunderlich et al. (34). Thus, the lytic unit index is directly proportional to the magnitude of the CTL response. In this experiment, B6.OVA stimulated 160 LU per 106 OT1 T cells, which was reduced to <2 LU per 106 cells by inclusion of TGFß in the culture.


Figure 1
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  		Fig. 1 CTL versus Ts activity of OT1 T cells. OT1 spleen cells were activated by incubation with SpLx from B6.OVA mice as a source of APC ± TGFß (3 ng ml–1). After 3 days, these first-step cultures were harvested and assayed for OVA-specific CTL by mixing with CFSE-labeled E.G7 and EL4 target cells. Lytic activity was expressed as percent specific lysis at various effector to target (E:T) ratios (A). Suppressive activity of these same cells was determined by irradiating and adding them (0.12 x 105) to splenic T cells (7 x 105) from B6 mice stimulated with SpLx (10 x 105) from B6.Kd.OVA mice or a mixture of irradiated B6.Kd and B6.OVA spleen cells. Kd-specific CTLs were measured after 5 days by mixing at various E:T ratios with CFSE-labeled P815 and EL4 target cells (B). Various numbers of first-step cultures were also added the second-step cultures as described in (B) and stimulated with B6.Kd.OVA (C) or B6.Kd and B6.OVA (D) spleen cells and the CTL activity was measured at various E:T ratios. Lytic units were calculated as the inverse of the number of effector cells required for 30% cytotoxicity, by mathematically fitting the curve of percent specific cytotoxicity versus effector cells per 104 targets.

 
Some of the first-step culture cells were irradiated (2500 Rad), to prevent the TCR Tg cells from over growing the second-step culture, and co-cultured with purified splenic T cells from B6 mice stimulated with SpLx from B6.Kd.OVA-double Tg mice. After 5 days, the polyclonal B6 anti-Kd CTL response was measured using P815 and EL4 target cells. The polyclonal B6 Kd-specific CTL response was significantly lower (6 LU per 106 cells) (Fig. 1B) than responses by OT1 T cells (Fig. 1A), which is consistent with their lower precursor frequency. OVA-specific OT1 T cells activated in the presence of TGFß inhibited the specific CTL response (<1 LU per 106 cells), whereas only marginal suppression (4 LU per 106 cells) was induced by OT1 T cells activated in the absence of exogenous TGFß (Fig. 1B). However, this distinction is less apparent at higher concentrations of OT1 T cells as shown by comparing the LU per 106 cells of cultures containing various numbers of activated OT1 T cells (Fig. 1C). OT1 T cells did not suppress the anti-Kd CTL response when Kd- and OVA-expressing APCs were mixed together whether they were activated with or without TGFß (Fig. 1D). This basic experiment has been repeated multiple times with absolutely consistent results demonstrating that CD8+ OT1 T cells suppress responses to unrelated antigens only when the APC express epitopes recognized by both the Ts and the responding T cells.

The requirement for recognition of the same APC by the Ts and the precursors of the effector cells suggests that the APC serves as a ‘bridge’ to mediate functional communication between the two T cells. One mechanism that could account for the requirement for an antigen bridge is that activated OT1 populations contained lytic activity that may eliminate the SIINFEKL/Kb-expressing APC, causing insufficient APC to drive the response by B6 T cells. Although this is a reasonable explanation for the suppressive activity of activated OT1, it is unlikely to account for the activity of OT1 activated in the presence of TGFß because CTL activity was severely reduced by this treatment. However, this is a very important distinction, so this experiment was repeated with T cells from mice in which perforin (pfp) gene had been inactivated. As expected, the OT1.pfp–/– T cells activated in the absence of exogenous TGFß developed little, if any, CTL activity (Fig. 2A) and these cells exhibited very little suppressive activity (Fig. 2B) compared with OT1 T cells. However, when the pfp–/–.OT1 T cells were activated in the presence of exogenous TGFß, they displayed potent suppressive activity but no more lytic activity than those activated in the absence of TGFß (Fig. 2A). These results suggest that the less potent suppressive activity expressed by OT1 T cells that were activated without exogenous TGFß is most likely mediated by lysis of the APC in the second culture, whereas the more potent suppressive activity (generated by antigen activation with TGFß) is mediated by a non-lytic mechanism.


Figure 2
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  		Fig. 2 Ts activity in perforin knockout OT1 T cells. OT1 and OT1.pfp–/–spleen cells were activated and tested as described in Fig. 1.

 
Mechanisms of APC-focused suppression by antigen-specific CD8+ T cells
To determine directly whether antigen-activated OT1 cells induced a loss of APC and/or phenotypic changes in the APC in the second-step culture, activated OT1 (CD452/2CD901/1) T cells were incubated with a mixture of spleen cells expressing different marker alleles from B6.OVA (CD452/2CD902/2) and B6.Kd (CD451/1CD902/2) mice so that each of these populations could be distinguished when mixed together. After 8 h at 37°C, the cells were harvested and stained with various antibodies and analyzed by flow cytometry. Viable cells were gated by the profile of forward versus sidelight scatter and for 7-AAD-negative cells. CD11c antibody was used to identify the dendritic cells (DC) that are the essential APC in the second-step culture (data not shown) and CD86 antibody to identify co-stimulatory molecules. The source of APC was distinguished by gating on either CD11c+CD45.2+ (B6.OVA) or CD11c+CD45.2– cells (B6.Kd). Both sources of CD11c+ cells, incubated for 8 h in the absence of OT1 T cells, up-regulated the expression of the co-stimulatory CD86 molecule compared with either freshly isolated CD11c+ cells prior to culture (Fig. 3) or those held at 4°C for 8 h (data not shown), which is presumably due to activation induced by their adherence to plastic. OT1 T cells activated in the presence of TGFß inhibited the up-regulation, or caused down-regulation, of CD86 by the B6.OVA APC (Fig. 3A), but not B6.Kd APC (Fig. 3B), which lacked the antigen recognized by OT1. OT1 T cells, activated in the absence of TGFß, did not alter the expression of CD86 by APC, whether or not they expressed the SIINFEKL/Kb epitope.


Figure 3
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  		Fig. 3 Phenotype of CD11c+ APC incubated with activated OT1 T cells. OT1 Ts spleen cells were activated by incubation with SpLx from B6.OVA mice as a source of APC ± TGFß (3 ng ml–1). Spleen cells from B6.OVA (CD45.1–) were mixed with B6.Kd (CD45.1+) spleen cells and incubated alone or with the activated OT1 Ts at 4 or 37°C. After 8 h, the cultured cells were stained with CD45.1/CD11c/CD86/7-AAD and the expression of CD86 by CD45.1+ and CD45.1– CD11c + 7-AAD– cells was plotted.

 
The percent specific lysis of the APC in Fig. 3 was calculated using the same formula as in the 4-h CTL assay, by comparing the recovery of OVA+ APC to OVA– APC from the same culture well. This analysis shows that OT1 T cells activated in the absence of TGFß caused 63% specific lysis of CD11c+ cells in 8 h using the same cell ratios as the functional Ts assay, while the Ts activated in the presence of TGFß showed <3% specific lysis under the same conditions (data not shown). Therefore, by several independent criteria, TGFß-activated OT1 TCR Tg T cells mediate specific suppressive activity for a primary CTL response with an APC-focused specificity pattern via a non-lytic mechanism. In the absence of TGFß, these same cells develop CTL activity, and such CTL are also suppressive in this culture system, but this suppression is due to physical clearance of the APC and not a modification of the CD86 co-stimulatory activity of these cells.

Suppression of the B6 anti-Kd CTL response mediated by CD8+ OT1 T cells is strictly dependent upon co-expression of OVA and Kd by the same APC, which suggests that suppression is not mediated by secretion of suppressive cytokines that act at a distance from the APC. However, TGFß is known to up-regulate the expression of TGFß and some regulatory cells express a cell-surface form of TGFß [reviewed in (49)] that requires close contact between the Ts and the responder T cells. Thus, we tested whether neutralizing anti-TGFß antibody altered suppression mediated by activated OT1 T cells. Anti-TGFß augmented the positive control B6 anti-Kd CTL activity by almost 10-fold when added to the control cultures (compare Fig. 4, panels A and B), suggesting that the control response was inhibited by TGFß provided either by the fetal bovine serum in the medium or produced endogenously by the B6 spleen cells. However, anti-TGFß antibody did not abrogate suppression but rather enhanced the suppressive activity of activated OT1 T cells using the lytic and non-lytic pathways. These results suggest that suppression mediated by CD8+ OT1 T cells, activated by APC-expressing OVA as an endogenous protein, does not involve TGFß.


Figure 4
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  		Fig. 4 Effect of anti-TGFß on suppression by activated OT1 T cells. OT1 spleen cells were activated with or without 3 ng ml–1 TGFß and tested for suppressive activity by adding to responder B6 T cells and Kd.OVA spleen cells as a source of APC, as described in the legend for Fig. 1. Suppressive activity was tested in the absence (left panel) or presence of 10 µg ml–1 anti-TGFß1, -2 and -3 antibody (right panel).

 
APC-focused suppression might involve elaboration of immunosuppressive cytokines such as IL-10 that act across small distances to inhibit only T cells binding to the same APC. To test this possibility, the effect of TGFß on lytic and suppressive activity of T cells from OT1.IL-10–/– mice was compared with T cells from OT1 mice. IL-10-deficient OT1 T cells developed levels of lytic activity that was similar to the IL-10-sufficient OT1 T cells and they were equally inhibited by TGFß (Fig. 5A). When added to the second-step culture, the TGFß-treated IL-10-deficient T cells were somewhat more suppressive than the TGFß-treated IL-10-sufficient T cells (Fig. 5B), suggesting that IL-10 production by the CD8+ T cells is not essential for suppression of the B6 anti-Kd CTL response. To determine whether IL-10 production by cells other than the OT1 T cells might contribute to suppression, neutralizing anti-IL-10 antibody was added to the second-step culture. Anti-IL-10 very slightly augmented the B6 anti-Kd CTL response but it did not inhibit the suppressive activity of IL-10-sufficient or IL-10-deficient OT1 T cells (Fig. 5). Thus, IL-10 does not appear to play an essential role in suppression mediated by TGFß-activated CD8+ T cells under these culture conditions.


Figure 5
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  		Fig. 5 Role of IL-10 in suppression by activated OT1 T cells. Spleen cells from OT1 and OT1.IL-10–/– mice were activated with or without 3 ng ml–1 TGFß and tested for CTL activity after 3 days (A) or added to B6 T cells and Kd.OVA spleen cells as a source of APC (B), as described in the legend for Fig. 1. Suppressive activity was tested in the absence (left panel) or presence of 10 µg ml–1 anti-IL-10 antibody (right panel).

 
TGFß activates FoxP3 expression in TCR Tg T cells
Since the expression of FoxP3 has been identified as a unique marker of endogenous Tregs (13–15) and Tregs induced by activation in the presence of TGFß (40), we asked whether OT1 T cells activated in the presence of TGFß might also express FoxP3. First, real-time quantitative reverse transcription–PCR using with TaqMan hybridization probes was used to measure the amount of FoxP3 and IFN-{gamma} mRNA, in which absolute copies per 106 cells were determined by comparison with amplification of known amounts of plasmid DNA containing the relevant amplicon as previously described (38, 39). Approximately 6 x 104 copies FoxP3 mRNA and 15 x 104 copies IFN-{gamma} mRNA per 106 cells were detected in lymphocytes from naive mice that contained ~4% FoxP3+ CD4+ Tregs as determined by flow cytometry (data not shown). The results of multiple independent experiments show that OT1 activated in the presence of TGFß consistently expressed two orders of magnitude higher levels of FoxP3 and one order of magnitude lower levels of IFN-{gamma} mRNA than OT1 T cells activated in the absence of TGFß (Fig. 6A). The low level of FoxP3 mRNA detected in the OT1 T cells activated with antigen but no TGFß is the same order of magnitude as expressed by the endogenous CD4+ Tregs and was most likely derived from the Tregs contained in the B6.OVA splenocytes used as the source of APC because the OT1 mice were Rag1–/– and have no appreciable CD4+CD25+ T cells (data not shown).


Figure 6
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  		Fig. 6 FoxP3 expression in OT1 T cells. Expression of IFN{gamma} and FoxP3 mRNA (copies per 106 cells) by activated OT1 (±TGFß) was measured by real-time quantitative reverse transcription–PCR with TaqMan hybridization probes (A). OT1 Ts (±TGFß) were stained for intracellular FoxP3 and granzyme B (B).

 
Polyclonal antibodies (50) and a recently developed mAb (clone FJK-16s) (51, 52) to murine FoxP3 have been reported to identify these cells by both flow cytometry and in tissue sections. Therefore, we examined the intracellular protein expression by OT1 T cells that had been activated by B6.OVA with or without exogenous TGFß by flow cytometry with antibodies specific for granzyme B (16G6, eBioscience) and FoxP3 (FJK-16s, eBioscience). Granzyme B was expressed in activated OT1 T cells and this was diminished by the addition of TGFß (Fig. 6B). A discrete subset of FoxP3-positive cells was detected in cultured OT1 cells that were activated in the presence of TGFß, whereas none were detectable in the absence of exogenous TGFß (Fig. 6B). Therefore, the development of non-lytic Ts function in OT1 T cells activated by antigen in the presence of TGFß is associated with a coordinate pattern of differentiation involving activation of FoxP3 expression and inhibition of IFN-{gamma} mRNA, intracellular granzyme B expression and CTL activity.

Natural Tregs express CD25, GITR, CTLA-4, FoxP3 [reviewed in (53)] and cell-surface TGFß, in some (54, 55), but not in all systems (53). Thus, we asked whether naive or activated T cells from OT1 mice expressed these markers. CD8+ T cells from freshly isolated spleens of naive OT1 mice displayed no significant numbers of CD25+, CTLA-4+, GITR+ or FoxP3+ cells (data not shown), suggesting that there are few, if any, CD8+ T cells that are equivalent to the natural Treg in Rag–/– OT1 mice. OT1 T cells activated by incubation with B6.OVA stimulators in the absence of exogenous TGFß uniformly expressed high levels of CD25 but no detectable FoxP3 as shown above and only low levels of CTLA-4 and intermediate levels of GITR (Fig. 7). A subset (~8%) of the OT1 T cells activated in the presence of exogenous TGFß expressed FoxP3 and they expressed high levels of CD25 but only intermediate levels of GITR and no CLTA4 (Fig. 7). The majority of the OT1 T cells activated in the presence of exogenous TGFß were FoxP3– but these cells expressed high levels of CD25 and intermediate levels of GITR, indicating that these reagents cannot be used to separate activated FoxP3+ from FoxP3– OT1 T cells. Cell-surface expression of TGFß was not detected on resting or activated CD8+ OT1 T cells (data not shown), which further supports the conclusion that TGFß does not play a role in the inhibitory activity of the TGFß-activated CD8+ Ts.


Figure 7
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  		Fig. 7 Cell-surface antigens expressed by FoxP3+ OT1 T cells. OT1 Ts spleen cells were activated by incubation with SpLx from B6.OVA mice as a source of APC without or with exogenous TGFß (3 ng ml–1). The cultures were harvested after 3 days and stained with anti-CD8 and -CD25, GITR or CTLA4 followed by intra-cellular staining for FoxP3. CD8+ cells were gated and displayed as FoxP3 versus CD25, GITR or CTLA4.

 
OT1 T cells activated in the presence of TGFß suppressed Kd-specific responses by TCR75 Tg T cells in vitro and in vivo
Next, we wanted to verify that the suppressive activity exhibited by OT1 T cells was not an in vitro artifact by testing them in vivo. Because of our expertise in cardiac transplantation, we decided to test whether OT1 T cells could suppress cardiac rejection mediated by CD4+ TCR Tg T cells. Before undertaking the in vivo experiment, it was necessary to determine whether responses by naive CD4+ TCR75 Tg T cells that recognize a peptide derived from the H-2Kd molecule (K54–68d) presented by I-Ab (24) were susceptible to suppression in vitro. Thus, OT1 T cells were added to cultures containing spleen cells from TCR75 mice, and responses measured both by incorporation of [3H]-TdR (Fig. 8A) and by dilution of CFSE (Fig. 8B). Despite the fact that the Kd peptide can be cross-presented by APC that do not express the Kd protein, antigen-activated OT1 T cells suppressed the proliferation of TCR75 cells driven by double Tg B6.Kd.OVA APC, but showed only marginal inhibition of the same response driven by a mixture of B6.Kd and B6.OVA APC in the same culture well (Fig. 8). Thus, the same APC-focused ‘linked-suppression’ specificity pattern is detected for inhibition of either naive CD4+ or CD8+ T cells. Also, activated OT1 T cells suppressed the proliferative response in vitro, whether they were activated with or without exogenous TGFß.


Figure 8
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  		Fig. 8 Effect of OT1 Ts on proliferation of TCR75 T cells. OT1 Ts were co-cultured with 104 naive TCR75 T cells and B6.Kd.OVA SpLx and proliferation was measured on day 3 by [3H]-TdR incorporation (A). Naive TCR75 T cells (CD90.1+) where labeled with CFSE and co-cultured with OT1 Ts (CD90.1–) with either no antigen (dark line) or with B6.Kd.OVA SpLx (light line). The profile of CFSE dilution by TCR75 T cells (CD90.1+CD4+ cells) was analyzed on day 3 (B).

 
One of the key advantages of this set of TCR Tg and ‘antigen’ Tg mice is that graded numbers of the effector T cells and Ts can be transferred into a normal animal to study their activity in an authentic in vivo context of transplantation rejection and tolerance. Furthermore, the combinations of allelic markers for CD45 and CD90 on the TCR Tg cells allow both populations of TCR Tg cells to be physically tracked in vivo. To utilize these tools to probe the mechanisms of antigen-specific regulation in allograft rejection, we have assessed the ability of OT1 T cells activated by antigen in the presence or absence of exogenous TGFß to inhibit transplant rejection. Since rapid rejection of B6.Kd hearts can be induced by adoptively transferred, naive TCR75 T cells (25, 37), we chose to use this model to investigate the role of OT1 Ts so that the potential effects of the Ts could be measured directly on the effector T cells as well as on overall allograft survival.

To verify the requirement that antigens recognized by both the Ts and the target effector T cells must be present on the same APC, B6 mice were adoptively transferred with both populations of T cells and transplanted with either B6.Kd.OVA or B6.Kd hearts. Positive controls showed that 105 naive TCR75 T cells induced rapid rejection of double Tg B6.Kd.OVA hearts in the absence of OT1 Ts (Fig. 9), which demonstrates that the presence of the OVA Tg per se does not substantially decrease the immunogenicity of the Kd Tg since B6.Kd hearts were previously shown to be rejected with the same kinetics (25). Co-injection of 106 OT1 Ts activated in the presence of TGFß significantly prolonged rejection time of B6.Kd.OVA hearts mediated by TCR75 T cells, but only minimally prolonged rejection time of B6.Kd hearts and this was not statistically significant. However, if the dose of TCR75 was increased by 10-fold, OT1 T cells were unable to prolong graft survival (data not shown), suggesting that graft survival depends upon a critical balance of effector T cells and Tregs. Suppression of graft rejection by OT1.IL-10–/– activated in the presence of TGFß was somewhat better than that mediated by IL-10-sufficient OT1 T cells in that half of the grafts survived longer than 60 days. By contrast, OT1 T cells activated in vitro in the absence of exogenous TGFß did not significantly inhibit rejection of B6.Kd.OVA hearts by TCR75 T cells, suggesting that cytolytic T cells are inefficient suppressors in vivo, at least under these experimental conditions.


Figure 9
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  		Fig. 9 Effect of activated OT1 T cells on allograft survival. Normal B6 mice were given 105 naive TCR75 cells with or without 106 OT1 Ts activated in vitro (±TGFß) or 106 OT1.IL-10–/– T cells activated with TGFß in vitro. The OT1 Ts were not irradiated. The next day, these mice were transplanted with B6.Kd or B6.Kd.OVA hearts and graft survival determined by palpation of the orthotopic heartbeat. The difference between survival times for the TGFß-induced OT1 cells in mice that got B6.Kd.OVA hearts is statistically significant (P < 0.05) by the Whitney–Mann rank-sum test compared with either the survival of B6.Kd hearts with the same T cell transfer or those that were transferred with OT1 T cells activated in the absence of TGFß, but with a B6.Kd.OVA heart.

 
The spleens and hearts from B6 mice adoptively transferred with OT1 Ts and naive TCR75 T cells, which had been transplanted with a B6.Kd.OVA heart, were also examined by immunofluorescence staining of tissue sections. Three days after transplantation, both OT1 and TCR75 T cells were found in the spleen but only OT1 T cells were found in the heart (Fig. 10). FoxP3+ OT1 T cells localized in the allograft rather than in the spleen (Fig. 10) or lymph nodes (data not shown) and their frequency was higher in B6.Kd.OVA allografts than in B6.Kd (data not shown). CD90.1-negative FoxP3+ cells (the endogenous B6 Tregs) were detected in the spleens of the transplanted mice (Fig. 10) at levels that were similar to those observed in non-transplanted mice (data not shown). However, all the FoxP3+ cells detected in the allograft 3 days after transplantation were CD8+ OT1 T cells, indicating that endogenous CD4+ Tregs have not migrated into the allograft at this time point. This is not surprising since the CD8+ OT1 T cells had been activated in vitro before adoptive transfer, whereas the TCR75 T cells were freshly isolated, naive T cells that are activated in the spleen but do not migrate from the spleen into the heart until 4 days after cardiac transplantation (37).


Figure 10
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  		Fig. 10 Localization of adoptively transferred T cells in cardiac recipients. Representative photomicrographs of sections from normal B6 mice given 105 naive TCR75 (CD451/1, CD902/2) cells and 106 OT1 (CD452/2, CD901/1) Ts activated in vitro with TGFß and transplanted with a B6.Kd.OVA heart the next day. Animals were sacrificed 3 days after transplantation. The left hand panels were stained with CD90.1 (red) to identify OT1 Ts and CD45.1 (green) to identify TCR75 T cells. The right hand panels are stained with CD90.1 (red) to identify OT1 Ts and FoxP3 (green). These sections were counterstained with the blue nuclear dye, 4',6-Diamidino-2-phenylindole.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
These data demonstrate that stimulation of CD8+ T cells from the TCR Tg OT1 mice with B6.OVA APC in the presence or absence of TGFß induced T cells that could suppress the proliferative response by CD4+ T cells and development of CD8+ CTL responses in vitro when stimulated with APC expressing both OVA and Kd antigens. These results support the observations published by Kezuka and Streilein (56) demonstrating that OT1 T cells activated by OVA-pulsed APC, which had been treated with TGFß, suppress OVA-specific delayed-type hypersensitivity responses. Our results extend these findings by showing that CD4+ proliferative responses and CD8+ CTL responses are also suppressed by TGFß-activated OT1 T cells if the APC express OVA and the stimulating antigen. This observation is similar to that reported for mixed lymphocyte response (MLR)-induced Ts that inhibit responses to third-party H-2 alloantigens, provided these are expressed on the same cell surface as the antigens used for activation of the Ts, a phenomena termed linked suppression (57, 58). APCs are hypothesized to serve as a bridge to mediate functional communication between the two T cells. The observation that suppression of irrelevant responses does not occur when separate APC bearing the antigens recognized by the Ts and responder T cells are mixed together is taken as evidence that suppression is not mediated by the elaboration of inhibitory cytokines that act at a distance from the APC. Thus, the TGFß-activated CD8+ T cells are antigen specific in their induction and in their suppressor effector function.

There are several potential mechanisms that could account for the pattern of linked suppression that was observed in these experiments. First, activated OT1 populations that contain lytic activity might eliminate the SIINFEKL/Kb-expressing APC, causing insufficient APC to drive the response by B6 T cells. This is the most likely explanation for the suppressive activity of OT1 T cells activated in the absence of TGFß. This interpretation is supported by the observation that antigen-activated OT1 T cells up-regulated the expression of granzyme B and demonstrated substantial cytolytic activity for OVA-transduced tumor cells in a standard 4-h CTL assay. Furthermore, these cells substantially depleted CD11c+ APC that expressed the relevant epitope during the first 8 h of culture and further reduced these cells by >98% by 15 h of culture (data not shown). In addition, OT1 T cells lacking a functional perforin gene could not mediate either classical CTL activity or the Ts activity measured when they were activated in the absence of exogenous TGFß. Together, these data indicate that antigen-activated OT1 T cells can suppress immune responses by direct elimination of the antigen-expressing APC via the classical granule-dependent CTL pathway.

The lytic mechanism is unlikely to explain the suppressive activity of OT1 activated in the presence of TGFß based on several observations. First, naive OT1 T cells activated by antigen in the presence of TGFß displayed virtually no lytic activity as measured in the standard 4-h assay. Second, the CD8+ Ts induced in the presence of TGFß did not substantially clear the APC from culture, but rather inhibited their expression of CD86, a critical co-stimulatory molecule. Third, TGFß reduced the expression of IFN-{gamma} and granzyme B by the majority of the OT1 T cells, while it induced the expression of FoxP3 in a sub-population of OT1 cells. Fourth, OT1 T cells lacking a functional perforin gene (and lytic activity) developed potent suppressive activity upon activation in the presence of exogenous TGFß. Moreover, perforin-deficient OT1 T cells activated in the presence of TGFß also up-regulated expression of FoxP3 and down-regulated expression of granzyme B (data not shown).

The non-lytic mechanism involved in suppression mediated by TGFß-activated OT1 T cells is not yet well defined but it correlates with a decrease in the expression of the CD86 co-stimulatory molecule by CD11c+ APC. There are, of course, a myriad of other molecules expressed by APC that might also be altered by the OT1 Ts, such as: MHC class I and MHC class II antigens, TNFR members such as CD40 (59), adhesion molecules such as ICAM (60) and other B7 family members such as CD80, B7-DC and B7-H1 (61, 62). To date, we have found no differences in the level of expression of MHC class I, MHC class II or CD80 in DC incubated with OT1 activated in the presence or absence of TGFß but other molecules have yet to be tested. The change in co-stimulatory molecules expressed by APC may directly result in a less vigorous stimulation of the effector precursor population of either CD4 or CD8 T cells, but may also induce further rounds of Tregs as shown by other investigators (20, 21, 56). Suppression by OT1 T cells activated in the presence of TGFß was not due to the carryover of TGFß into the test culture since inclusion of excess neutralizing antibody to TGFß1, -2 and -3 did not abrogate suppression. This experiment also demonstrates that suppression by activated OT1 T cells does not require TGFß in the second-step culture system, suggesting that their mechanism of action is different than that of CD4+CD25+ Treg reported by some investigators (54, 55). IL-10 is another immunosuppressive cytokine that has been frequently reported to be secreted by CD4+ Tr1 T cells [reviewed in (63)]; however, IL-10 does not appear to play a significant role in suppression mediated by CD8+ T cells in this system. In fact, the observation that suppression of unrelated responses by CD4+ and CD8+ T cells by TGFß-activated OT1 T cells occurs only if the APC expresses both OVA and the stimulating antigen (linked suppression) suggests that direct cell contact between the Ts and the effector cells may be brought about by close interaction of both T cells on the surface of an APC. Alternatively, the Ts and effector T cells may act sequentially with the APC such that the OT1 T cells reduce the expression of CD86 on the APC, which in turn renders the APC less able to activate responder T cells that subsequently bind to the APC. The latter mechanism was elegantly shown to explain linked recognition between helper T cells, effector T cells and APC by Ridge et al. (64) but it remains to be determined which pathway is used by CD8+ Ts.

The observation that TGFß inhibited the expression of granzyme B and IFN-{gamma} by CD8+ OT1 T cells agrees with a recent report by Thomas and Masague (65) using polyclonal CD8+ T cells. The observation that TGFß also up-regulated the expression of FoxP3 in a subset of the activated CD8+ OT1 T cells, as it does in CD4+CD25– T cells (40–46), suggests that the induction of immunosuppressive CD4 and CD8 T cells by activation in the presence of TGFß constitutes a paradigm of immunoregulation. Except for one report that endogenous CD8+ T cells express auto-antigen-specific suppressive activity (53), CD8+ Ts are more akin to the induced CD4+CD25+ Tregs, which are activated by antigens in the peripheral immune system from CD4+CD25– precursors cells [reviewed in (66)], than they are to natural Tregs. The observations that FoxP3 is not expressed by naive or antigen-activated CD8+ T cells and only occurs when stimulated with antigen under conditions that lead to tolerance may explain the failure to detect significant FoxP3 expression in CD8+ T cells in the FoxP3 gene expression reporter strain (Foxp3gfp) and the failure of pathogen-driven immune responses to up-regulate FoxP3 in CD4+ T cells (67). Like Tregs (66), the data in this report demonstrate that cell contact is required for CD8+ Ts to inhibit immune responses. TGFß-activated OT1 T cells, like MLR-induced CD8+ Ts (28) and donor-specific CD8+ Ts from transplant patients (18, 68), inhibit responses only to antigens that are linked to the inducing antigens, suggesting that suppression is mediated by direct cell contact rather than the release of cytokines. Thus, the requirement for direct cell contact is a common feature of CD8+ Ts and CD4+ Tregs.

There have been many studies showing that Tregs that arise during a variety of tolerance-inducing conditions are inhibitors of graft rejection [reviewed in (69–71)]. Much less is known about the role of CD8+ Ts in preventing graft rejection but FoxP3 is up-regulated in human (20) and rat (21) CD8+CD28– Ts. Likewise, TGFß-activated OT1 T cells uniformly expressed CD25, developed a sub-population of FoxP3-expressing cells and mediated suppression in vitro and in vivo, suggesting that suppression may be mediated by the FoxP3+ OT1 T cells. CD8+CD25+ cells expressing FoxP3 have recently been reported to be induced by treatment of human T cells in vivo or in vitro with a modified, non-depleting anti-CD3 mAb (hOKT3{gamma}1) (72). The anti-CD3-induced CD8+CD25+ T cells arose from CD8+CD25–FoxP3– T cells and inhibited responses by syngeneic CD4+ T cells stimulated by mitogens or antigens. Although it is not clear whether these polyclonal CD8+CD25+ T cells inhibit responses specifically or non-specifically, they required contact with the responding CD4 population. These studies suggest that FoxP3 may serve as a critical regulator of CD8+ T cell function, as it does in CD4+ Tregs; however, it has yet to be determined whether the suppressive activity of CD8+ Ts is exclusively the property of the FoxP3+ cells in any of these systems. Because antibodies to intracellular proteins, such as FoxP3, cannot be used to purify viable cells, we have searched for surrogate markers (including CD25, GITR and CTLA-4) but so far have failed to identify any that are differentially expressed by the TGFß-activated FoxP3+ but not the FoxP3– cells. Thus, definitive identification of the CD8+ Ts awaits breeding of the OT1 TCR Tg mice to FoxP3 green fluorescent protein (GFP) knockin (Foxp3gfp) mice (67), which have been generously provided to us by Alex Rudensky (University of Washington, Seattle, WA, USA).

To verify that the suppressive activity of TGFß-activated OT1 T cells was not an in vitro artifact, OT1 T cells, which had been activated in vitro, were adoptively transferred to B6 mice that received TCR75 T cells and cardiac grafts. Our results showed that OT1 T cells suppressed allograft rejection, mediated by Kd-specific CD4+ TCR75 T cells, but only if the Ts had been activated in the presence of TGFß. Suppression of cardiac rejection occurred only when the Kd hearts also expressed OVA, suggesting that linked suppression is also manifest in vivo. CD8+ T cells expressing FoxP3 were reported by Liu et al. (73) to be activated in rats by donor-specific transfusions with UVB-irradiated blood and these T cells prolonged cardiac transplant survival when transferred to naive syngeneic hosts. Like the OT1 Ts, the CD8+ Ts from tolerant rats were antigen specific and inhibited proliferative responses to the tolerizing, but not unrelated, alloantigens in a contact-dependent fashion. Moreover, suppression of cardiac graft rejection by adoptively transferred CD8+ Ts was limited to the tolerizing, but not an unrelated, heart donor. Collectively, these data suggest that the CD8+ Ts are antigen specific in their activation and in their suppressive activity, which is distinct from CD4+ Tregs, which are generally thought to inhibit responses non-specifically after activation by specific antigen (74, 75).

The mechanisms by which the CD8+ OT1 Ts inhibit responses in vitro and in vivo are not yet understood; however, neither IL-10 nor TGFß is essential. Although our in vitro studies demonstrated that suppressive activity of TGFß-activated OT1 correlated with the failure to up-regulate CD86 on DC, the possibility that inhibitory receptors, such as PIR-B (73, 76), the inhibitory enzyme, indoleamine 2,3-dioxygenase (77), or other inhibitory pathways might also be up-regulated, is currently under investigation (73, 76). Both Tregs and CD8+ Ts can prevent the activation of naive T cells but their ability to inhibit effector functions by activated T cells has been less well studied. Our observation that adoptively transferred, TGFß-activated CD8+ Ts migrated into the allograft is in agreement with the reports that FoxP3 mRNA is detectable in tolerated allografts that are infiltrated by CD4+CD25+ Tregs (78, 79) and T cells expressing the FoxP3 protein have been shown by immunohistochemistry to be present in lymphoid tissues, tumors (50) and tolerated allografts (80, 81). These observations raise the possibility that one site where Tregs and CD8+ Ts inhibit rejection is within the allograft. Whether FoxP3+ CD8+ T cells could develop and mediate suppression at the level of the secondary lymphoid tissues is not addressed by our studies because the CD8+ T cells were activated prior to the adoptive transfer. Hence, their preferential migration to the heart rather than the lymphoid tissue most likely reflects the activation state of these cells, as previously reported for CD4+ TCR Tg T cells (37). Inhibition of rejection within the allograft could also be mediated by OT1 T cells at the level of DC because TCR75 T cells, which are activated primarily in the spleen after cardiac transplantation, migrate to the heart where they undergo additional rounds of proliferation and up-regulation of cytokines (37). By contrast, activated TCR75 T cells also migrate to the small intestine of graft recipients but they do not undergo proliferation, presumably due to the absence of antigen (37). Our data also show that the TGFß-activated FoxP3+ OT1 Ts migrated into the heart but not into lymphoid tissues 3 days after transfer to cardiac recipients, whereas FoxP3– OT1 cells migrated to both the heart and the spleen. If the suppressive activity of the OT1 Ts is a function of the FoxP3+ cells, then these results further support the hypothesis that suppression may be mediated within the transplanted organ rather than in the lymphoid tissues when activated Ts are adoptively transferred.


   Acknowledgements
 
This research was supported by grants from National Institutes of Health, HL50724 (R.P.B.), EY014877 (J.A.K.), Research to Prevent Blindness (J.A.K.) and the Foundation for Fighting Blindness (J.A.K.). The authors thank Marc Jenkins for the B6.OVA mice and Michael Bevan for the OT-1 mice.


   Abbreviations
 
APC, antigen-presenting cell
7-AAD, 7-amino-actinomycin D
B6, C57Bl/6
CFSE, carboxyfluorescein succinimidyl ester
DC, dendritic cell
E:T, effector to target
GFP, green fluorescent protein
[3H]-TdR, [3H]thymidine
MLR, mixed lymphocyte response
OT1, TCR transgenic mice expressing a receptor for ovalbumin in the context of MHC class I
OVA, ovalbumin
pfp, pore-forming protein or perforin
SA, streptavidin
SpLx, irradiated spleen cells
Tg, transgenic
TGFß, transforming growth factor ß
Ts, suppressor T cell
Treg, regulatory T cell
UAB, University of Alabama at Birmingham

   Notes
 
Transmitting editor: K. Okumura

Received 10 May 2006, accepted 8 August 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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