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The Journal of Immunology, 2007, 178: 3400-3408.
Copyright © 2007 by The American Association of Immunologists, Inc.

Treatment-Enhanced CD4+Foxp3+ Glucocorticoid-Induced TNF Receptor Family RelatedHigh Regulatory Tumor-Infiltrating T Cells Limit the Effectiveness of Cytokine-Based Immunotherapy1

Aklile Berhanu2,*, Jian Huang2,*, Simon C. Watkins{dagger}, Hideho Okada{ddagger},¶ and Walter J. Storkus3,*,§,¶

* Department of Immunology, {dagger} Department of Cell Biology and Molecular Physiology, {ddagger} Department of Surgery, and § Department of Dermatology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213; and University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Regulatory T cells can suppress activated CD4+ and CD8+ T effector cells and may serve as an impediment to spontaneous or therapeutic type 1 antitumor immunity. In a previous study, we observed minimal therapeutic impact, but significantly enhanced T cell cross-priming and lesional infiltration of tumor-reactive CD8+ T cells into established CMS4 sarcomas after combined treatment of BALB/c mice with rFLt3 ligand (rFL) and recombinant GM-CSF (rGM-CSF). In this study, we show that this cytokine regimen also results in the profound enhancement of CD4+ tumor-infiltrating lymphocytes (TIL) expressing FoxP3, IL-10, and TGF-beta mRNA, with 50 or 90% of CD4+ TIL coexpressing the CD25 and glucocorticoid-induced TNFR family related molecules, respectively. Intracellular staining for Foxp3 protein revealed that combined treatment with rFL plus rGM-CSF results in a significant increase in CD4+Foxp3+ T cells in the spleen of both control and tumor-bearing mice, and that nearly half of CD4+ TIL expressed this marker. In addition, CD4+ TIL cells were of an activated/memory (ICOShighCD62LlowCD45RBlow) phenotype and were capable of suppressing allospecific T cell proliferation and IFN-{gamma} production from (in vivo cross-primed) anti-CMS4 CD8+ T cells in vitro, via a mechanism at least partially dependent on IL-10 and TGF-beta. Importantly, in vivo depletion of CD4+ T cells resulted in the ability of previously ineffective, rFL plus rGM-CSF therapy-induced CD8+ T cells to now mediate tumor regression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The CD8+ T lymphocytes play a critical role in adaptive antitumor immunity by recognizing and attacking tumor cells in an Ag-specific manner (1, 2). Although the mere presence of tumor limits effective CD8+ T cell priming in situ, even when effector CD8+ T cells do become activated during tumor progression, their capacity to mediate clinically meaningful functions within the tumor microenvironment may be negated (3, 4). Recent studies have shown that, in some cases, the suppression of immune responses to self or foreign Ags can be attributed to the presence of regulatory T cells (Tregs)4 that are reactive against self Ags and capable of inhibiting antitumor T effector cell function (5). Consistent with this observation, increased levels of Tregs have been detected in the peripheral blood and tumor tissues of cancer patients (6, 7), with such cellular increases correlating with reduced overall survival (8).

Tregs typically represent 5–10% of peripheral CD4+ T cells in both humans and mice, and the in vivo depletion of this T cell subset results in increased predilection to develop a state of autoreactivity that may culminate in the development of organ-specific autoimmune diseases, such as thyroiditis and gastritis (9, 10). Similarly, the adoptive transfer of purified Tregs prevents the development of autoimmune diseases through enforcement of functional tolerance to self Ags (11). Whereas no single Ag has been accepted as a unique phenotypic marker for Treg discrimination, these cells typically express high levels of CD25 (IL-2R{alpha}-chain), glucocorticoid-induced TNFR family related (GITR) molecule, and CTLA-4 (CD152) (12, 13, 14, 15). Tregs also exhibit an activated/memory cell phenotype in expressing low levels of the CD45RB (CD45RBlow) molecule and high levels of the ICOS (ICOShigh) molecule (11, 16). Perhaps most notably, the forkhead/winged-helix family transcription factor foxp3 seems to be expressed primarily by the Treg subset of CD4+ T cells (17, 18, 19), although a recent report suggests that it may also be expressed by minor populations of CD8+, CD4+CD8+, and CD4–CD8– T cells as well (20). Tregs do not proliferate or produce IL-2 in response to TCR triggering, although they are capable of inhibiting proliferative responses and cytokine production by effector cells, typically in an Ag-nonspecific manner (21, 22) and via mechanisms involving direct cell-to-cell contact or the secretion of the immunosuppressive cytokines IL-10 and TGF-beta (21, 23, 24, 25).

In an earlier study, we observed that the treatment of mice bearing syngeneic CMS4 sarcomas with rFLt3 ligand (rFL) plus recombinant GM-CSF (rGM-CSF) resulted in the markedly enhanced cross-priming of anti-CMS4 CD8+ T cells in vivo (26). However, these responses yielded no meaningful impact on tumor growth, despite the infiltration of significant numbers of tumor-specific CD8+ T cells into tumor lesions. We report in this study that tumors in rFL plus rGM-CSF-treated mice are also infiltrated by high frequencies of CD4+ T cells that exhibit the phenotype and functional characteristics of Tregs, and that in vivo depletion of CD4+ T cells in CMS4-bearing mice results in CD8+ T cell-dependent inhibition of tumor growth and the extended survival of mice if they were treated with (previously ineffective) combined rFL plus rGM-CSF cytokine therapy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Six- to 8-wk-old female BALB/cJ and C57BL/6 mice were purchased from The Jackson Laboratory and maintained in the pathogen-free, central animal facility in the Hillman Cancer Center at the University of Pittsburgh Cancer Institute. All animal work was performed in accordance with an Institutional Animal Care and Use Committee-approved protocol.

Tumor establishment

CMS4 sarcoma (H-2d) cells were cultured, and s.c. tumors were established and monitored for growth in syngeneic BALB/cJ mice, as previously described (26).

Combinational cytokine therapy and Ab depletions

Mice were injected s.c. in the scruff of the neck with 20 µg each of rFL and rGM-CSF for five consecutive days in a total volume each of 100 µl of PBS, as we have previously shown this protocol to optimize the number of tumor-infiltrating DC (dendritic cells) (TIDC) and consequent priming of anti-CMS4 CD8+ T cells in vivo (26). To deplete CD4+ and/or CD8+ T cells, mice were injected i.p. with 200 µg of anti-CD4 (GK1.5 hybridoma; American Type Culture Collection) and/or 200 µg of anti-CD8 (53-6.72 hybridoma; American Type Culture Collection) ascitic fluid on day 8 (when rFL plus rGM-CSF treatment was initiated), with 100 µg of mAb injected on days 13 and 16 and/or every 3 days thereafter, as indicated. Ab administration resulted in the depletion of >99% of CD4+ or CD8+ T cells, respectively, as assessed by flow cytometry (data not shown). Mice injected with equivalent amounts of rat isotype control Ab (Sigma-Aldrich) were used as controls for depletion experiments.

Preparation of single-cell suspensions from tumor and spleen

Single-cell suspensions were obtained from resected spleens and tumors, as previously reported (26).

CD4+ and CD8+ T cell isolation

Splenocytes or tumor-infiltrating lymphocytes (TIL) were first FcR blocked with anti-CD16/32 Ab (BD Pharmingen) for 15 min at 4°C. To remove DC, anti-CD11c MicroBeads (Miltenyi Biotec) were added to cells and incubated, and CD11c+ cells were adsorbed on MiniMACS magnetic columns, per the manufacturer’s instructions. CD4+ T cells were then isolated by positive selection from the CD11c-negative flow-through fraction after incubating these cells with anti-CD4 MicroBeads (Miltenyi Biotec). Finally, CD8+ T cells are isolated by positive selection from the CD11c-negative, CD4-negative flow-through fraction after incubation with anti-CD8 MicroBeads. In all cases, the isolated T cell populations exceeded 95% purity assessed by flow cytometry (data not shown). Because tumors isolated from untreated mice contained very few CD4+ or CD8+ TIL on day 14, it was not possible to isolate sufficient T cells to serve a control population for studies involving TIL isolated from the rFL plus rGM-CSF-treated mice.

Flow cytometry

After washing cells in FACS buffer (0.1% BSA and 0.05% sodium azide in PBS), the following Abs and their corresponding isotype controls were used for surface staining: CD4 PE, CD4 biotin, CD25 FITC, CD62L FITC, CD45RB PE (all purchased from BD Pharmingen), ICOS FITC (eBioscience), and GITR FITC (R&D Systems). For CD4 biotin detection, the stained cells were further incubated with streptavidin-PerCP for 30 min in the dark at 4°C and washed twice. Cells were then fixed in 1% paraformaldehyde and analyzed using a Coulter EPICS XL flow cytometer and EXPO32 software (both obtained from Beckman Coulter). For Foxp3 intracellular staining, CD4+ T cells were surface stained, as described above, and then further processed using a PE anti-mouse/rat Foxp3 staining kit (eBioscience).

Immunofluorescence staining and imaging

Tumor tissue samples were prepared and sectioned, as previously reported (26). All washing steps were performed using wash buffer (WB; 0.5% BSA in PBS; Sigma-Aldrich). For staining, the sections were fixed in 2% paraformaldehyde (Sigma-Aldrich) at room temperature for 45 min, then incubated with 2% BSA for 45 min, washed, and blocked using goat anti-mouse Fab1 (Jackson ImmunoResearch Laboratories) in WB overnight. For analysis of DC vs T cell subsets, sections were incubated with biotinylated anti-CD11c or anti-CD3 Abs or PE-conjugated anti-CD4 or anti-CD8{alpha} Abs or matching isotype controls (all from BD Pharmingen) for 1 h. This was followed by incubation with streptavidin-Alexa Fluor 488 (Invitrogen Life Technologies) for 1 h. For analysis of the CD11c vs IL-10 vs Foxp3 markers, sections were washed, and were then incubated with rat anti-mouse IL-10 (BD Pharmingen) and hamster anti-mouse CD11c (BD Pharmingen) in WB overnight. After three washes, the sections were incubated with a mixture of goat anti-rat Cy3-conjugated Fab1 (Jackson ImmunoResearch Laboratories) and goat anti-hamster Cy5 (Jackson ImmunoResearch Laboratories) in WB for 2 h. After washing, the sections were treated with Alexa Fluor 488-conjugated rat anti-mouse Foxp3 in WB overnight. For analysis of IL-10 vs CD4 cells, sections were incubated with goat anti-rat cy3-conjugated Fab1 (Jackson ImmunoResearch Laboratories) in WB for 2 h. After washing, the sections were incubated with rat anti-mouse CD4 (BD Pharmingen) in WB for 1 h. The sections were then washed and incubated with goat anti-rat Alexa Fluor 488 (Molecular Probes) in WB for 1 h. After being coverslipped, slide images were acquired using an Olympus 500 Scanning Confocal Microscope (Olympus). Isotype control and specific Ab images were taken using the same level of exposure on the channel settings.

RT-PCR

mRNA was isolated from cells using TRIzol reagent (Invitrogen Life Technologies), per the manufacturer’s instructions, with cDNA consequently prepared using random hexamer primers (Applied Biosciences). RT-PCR was performed using published primer pairs for foxp3 (27), TGF-beta (27), IFN-{gamma} (28), and IL-10 (29), with PCR products confirmed by gel electrophoresis and ethidium bromide-stained gels documented using a UVP gel camera (Ultraviolet Products) and Lab Works software (PerkinElmer).

Allogeneic MLR

MLR cultures were established using 2 x 105 BALB/cJ normal splenic responder CD4+ T cells and 2 x 105 irradiated (10 Gy) total C57BL/6 stimulator splenocytes in 96-well round-bottom plates. In replicate wells, CD4+ TIL cells freshly isolated from the tumors of rFL plus rGM-CSF-treated mice were added to the MLR cultures in decreasing BALB/c TIL-to-spleen CD4+ T cell ratios to assess TIL-suppressive activity. After 3 days of incubation at 37°C and 5% CO2, the cultures were pulsed with 1 µCi of [3H]thymidine/well (NEN) for the final 16 h of culture, and incorporated radioactivity was quantitated using a liquid beta scintillation counter (Wallac). Data are reported as the mean ± SD of triplicate determinations.

In vitro stimulation of CD8+ T cells

For in vitro stimulation cultures, 2 x 105 CD8+ T cells were cocultured with 5 x 104 irradiated (100 Gy) CMS4 sarcoma cells in 96-well round-bottom plates in a humidified incubator at 37°C and 5% CO2 for 72 h. To assess for the suppression of IFN-{gamma} production by responder CD8+ T cells, freshly isolated CD4+ TIL from mice treated with rFL plus rGM-CSF (or untreated CD4+ splenic T cells) were added to replicate culture wells at various CD4:CD8 ratios, as indicated. In some assays, neutralizing anti-murine IL-10 (final concentration 10 µg/ml; R&D Systems) and/or the TGF-betaR-signaling antagonist IN-1130 (a gift from S.-J. Kim, National Cancer Institute, Frederick, MD; final concentration 1 µM) (30) were added. At the end of the culture period, the plates were centrifuged and the cell-free supernatants were collected and stored at –80°C.

ELISA

Culture supernatants were analyzed using the IFN-{gamma}-specific OptEIA ELISA set (BD Pharmingen), according to the manufacturer’s instructions. The lower limit for the detection of IFN-{gamma} by this assay was 31.3 pg/ml.

Statistical analyses

Statistical analyses were performed using unpaired two-tailed Student’s t test. SPSS for Windows software (SPSS) was used to conduct the analysis. Values of p < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Infiltration of CMS4 tumors by CD4+ T cells is increased after treatment with rFL plus rGM-CSF

In a previous study (26), we observed that treatment of BALB/c mice bearing established s.c. CMS4 sarcomas with rFL plus rGM-CSF resulted in profound tumor infiltration by CD11c+ DC (i.e., TIDC), as well as CMS4-specific CD8+ T cells, although the cumulative immunity promoted by cytokine administration failed to impact tumor progression. Upon further inspection, we now show that treatment with rFL plus rGM-CSF also results in a dramatic increase (vs untreated controls) in CD3+CD4+ TIL (Fig. 1). Notably, CD11c+ TIDC and both CD4+ and CD8+ TIL cell subsets were distributed throughout the lesions and were not anatomically restricted to the periphery of the tumor.


Figure 1
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  		FIGURE 1. Tumors in mice treated with rFL plus rGM-CSF display increased infiltration by CD11c+ DC, CD8+, and CD4+ T cells. BALB/cJ mice bearing day 7-established CMS4 tumors were not treated, or treated with 20 µg/day of each cytokine for five consecutive days and sacrificed at day 12. Five-micron sections of the tumors were stained and analyzed by confocal microscopy. CD11c+ DC (upper panels) or CD3+ T cells are depicted as green-stained cells (lower panels); CD8+ (middle panels) or CD4+ (lower panels) T cells are depicted as red-stained cells, with CD3+CD8+ or CD3+CD4+ T cells appearing yellow due to costaining overlays. These data are representative of three independent experiments performed.

 
A high proportion of CD4+ TIL isolated from rFL plus rGM-CSF-treated mice displays a Treg phenotype

Using flow cytometry, we initially compared the phenotypes of CD4+ TIL vs CD4+ splenic T cells in mice treated with rFL plus rGM-CSF to discern whether specific subpopulations of CD4+ T cells might be preferentially represented within tumor lesions. In particular, given the failure of this cytokine therapy to impact tumor growth (26), we were interested in gauging levels of Tregs in treated animals that might impede anti-CMS4 CD8+ T cell function in situ. Based on prior reports (12, 13), we preliminarily discriminated Tregs as CD4+CD25+ T cells. As shown in Fig. 2A, ~50% (49.3 ± 5.2%) of CD4+ TIL in cytokine-treated, tumor-bearing mice coexpressed CD25 on their cell surface, whereas only ~11% (10.7 ± 2.0%) of T cells isolated from the spleens of these same mice were CD4+CD25+. Notably, we observed the percentages of splenic CD4+CD25+ cells in untreated/nontumor-bearing, treated/nontumor-bearing, and untreated/tumor-bearing mice to be low and equitable (all ~6–11%). An analysis of TIL and splenic CD4+ T cells for coexpression of the GITR marker indicated that ~90% (90.2 ± 1.1%) of CD4+ TIL in treated mice coexpressed this molecule (Fig. 2A). Notably, GITRhigh+CD4+ T cell (consistent with the reported Treg phenotype) (14, 15) frequencies in TIL were greater than those observed in the spleens of cytokine-treated control, tumor-free (i.e., 26.7 ± 2.1%), or tumor-bearing (i.e., 29.8 ± 5.5%) mice. Interestingly, rFL plus rGM-CSF administration alone was associated with elevated frequencies of GITRhigh+CD4+ T cells in the spleens of treated mice, regardless of tumor status (all with p < 0.05 vs untreated, tumor-bearing, or untreated, normal mice). Additional studies suggest that the absolute numbers of CD4+CD25+ T cells were found to be maximally increased 2- to 10-fold in mice treated with rFL plus rGM-CSF vs PBS in spleen, inguinal lymph nodes, and tumor, but not thymus or bone marrow, by day 7 after the initiation of cytokine administration (Fig. 2B).


Figure 2
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  		FIGURE 2. The majority of CD4+ TIL in rFL plus rGM-CSF-treated mice express a GITRhigh+CD4+ Treg phenotype, and half of these cells coexpress CD25. Flow cytometry analyses were performed on CD4+ T cells isolated from the indicated animal treatment cohorts (as described in Materials and Methods). A, CD4+ T cells were analyzed for coexpression of the CD25 and GITR, with the panel numbers inserted reflecting the percentage of CD4+ T cells present in the CD25 (or GITR)+ vs CD25 (or GITR)-negative subpopulations. These data are representative of four independent experiments performed. B, The indicated tissues were harvested from three mice/cohort on day 12 and digested as outlined in Materials and Methods, and single-cell populations were analyzed for CD4+CD25+ frequencies using flow cytometry. Results are representative of two independent experiments performed. C, CD4+ T cells were analyzed for relative expression levels of the CD45RB, ICOS, and CD62L activation/memory markers. The MFI of the isotype controls were similar in all five cases; hence, the control data for the F/G-TIL cohort are shown as a representative plot in the leftmost panels. MFI values are shown for each of the indicated markers. These data are representative of three independent experiments performed. Abbreviations used: F/G, mice treated with rFL plus rGM-CSF; NOR-SP, splenocytes harvested from normal, tumor-free mice; Tu-SP, splenocytes harvested from tumor-bearing mice.

 
CD4+ TIL isolated from rFL plus rGM-CSF-treated mice express an activated/memory phenotype consistent with that of Tregs

Because adoptively transferred CD45RBlowCD4+ T cells isolated from the spleen of normal mice can act as Tregs in preventing organ-specific autoimmune disease in T cell-deficient mice reconstituted with CD45RBhighCD4+ T cells (9, 11), we examined the level of CD45RB expression on CD4+ T cells isolated from the tumors and spleens mice treated with rFL plus rGM-CSF. As shown in Fig. 2C, the median fluorescence intensity (MFI) of the CD45RB molecule on the surface of CD4+ T cells isolated from tumor was (~5-fold) lower than that of CD4+ T cells isolated from the spleen of the same animals. This MFI was also ~15- and 19-fold lower than that observed for splenic CD4+ T cells harvested from untreated/tumor-bearing or untreated/nontumor-bearing mice, respectively. Additionally, in parallel with increased GITR expression, more CD4+ T cells appeared to exhibit a CD45RBlow phenotype in mice treated with rFL plus rGM-CSF vs untreated mice, regardless of tumor status.

We also evaluated CD4+ TIL for coexpression of the ICOS protein, because up-regulated levels of this marker have been detected on Tregs in pancreatic lesions and its blockade rapidly converts early insulitis into diabetes by disrupting the balance of Tregs and T effector cells (16). Contrary to their reduced levels of CD45RB expression, CD4+ TIL from cytokine-treated mice expressed higher levels of ICOS than any splenic CD4+ T cell population examined (Fig. 2C). ICOS expression was virtually nonexistent on splenic CD4+ T cells isolated from untreated, normal, or tumor-bearing mice or from rFL plus rGM-CSF-treated normal controls. However, ICOS expression on splenic CD4+ T cells isolated from cytokine-treated, CMS4-bearing animals was elevated compared with these controls, although not to the degree observed for TIL isolated from these same animals. As also shown in Fig. 2C, CD4+ TIL isolated from rFL plus rGM-CSF-treated mice failed to express the CD62L marker (involved in the recirculation of naive T cells from blood into peripheral lymphoid tissues (31)) on their surface, consistent with phenotype of Tr1-type, IL-10-producing Tregs (32). Splenic CD4+ T cells from these same animals exhibited a phenotype that was intermediate between TIL and control splenocyte populations, with approximately equal levels of CD62L+ and CD62L-negative subpopulations observed.

rFL plus rGM-CSF treatment significantly increases the frequency of CD4+Foxp3+ Tregs in the spleen and results in high levels of CD4+Foxp3+ TIL

The foxp3 gene product is important in the development and function of CD4+ Tregs and is preferentially expressed in this subset of T cells (17, 18). Consistent with the Treg phenotype of CD4+ TIL in rFL plus rGM-CSF-treated mice that we have described to date, approximately half (49.6 ± 6.5%) of CD4+ TIL from these mice expressed intracellular FoxP3 protein (Fig. 3A). Notably, these TIL frequencies were approximately double those observed in the spleens of animals treated with rFL plus rGM-CSF (regardless of tumor status), which in turn were approximately double those observed for splenocytes isolated from nontreated control mice (regardless of tumor status). Additional flow cytometry analyses revealed that the majority of (but not all) CD4+CD25+ and CD4+GITR+ T cells in these cohorts were FoxP3+ (Fig. 3A). FoxP3 expression by CD4+, but not CD8+ (CD4-negative) TIL enriched in rFL plus rGM-CSF-treated animals was confirmed by both flow cytometry and immunofluorescence microscopy analyses (Fig. 3). These data suggest that combined treatment with rFL plus rGM-CSF results in a coordinate increase in peripheral CD11c+ DC (26) and CD4+Foxp3+ T cells in vivo, with further enrichment occurring in the tumors of cytokine-treated animals.


Figure 3
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  		FIGURE 3. rFL plus rGM-CSF treatment significantly increases the population of CD4+Foxp3+ T cells in the spleens and tumor lesions of treated mice. A, CD4+ T cells described in Fig. 2 were surface stained with isotype control, CD25, or GITR Abs, and intracellular stained with isotype control or Foxp3 Abs and analyzed by flow cytometry. The data shown for CD4/isotype and CD4/Foxp3 are gated on lymphocytes by forward/side scatter, whereas the data shown for CD25/Foxp3 and GITR/Foxp3 are gated on CD4+ T cells. The numbers in the quadrants of the panels represent the percentage of CD4+ T cells or CD25+ T cells or GITR+ T cells that are positive/negative for Foxp3 expression. B, Tumor sections from nontreated vs rFL plus rGM-CSF-treated mice (as in Fig. 1) were analyzed for coexpression of the CD4 or CD8 vs FoxP3 markers by immunofluorescence microscopy. Data are representative of two independent experiments performed.

 
In addition to the intracellular staining of Foxp3 protein, we also performed RT-PCR using foxp3-specific primers on total RNA isolated from CD4+ T cells to determine whether the increase in Foxp3 expression observed in rFL plus rGM-CSF-treated tumor-bearing mice was reflected at the transcriptional level (Fig. 4A). Consistent with our flow cytometry data, high levels of foxp3 mRNA transcripts were detected in CD4+ TIL and splenic CD4+ T cells of tumor-bearing mice treated with rFL plus rGM-CSF, and this level of expression was higher than that observed for CD4+ T cells isolated from untreated, normal, or tumor-bearing mice. We also observed that transcripts for the Tr1-associated cytokine IL-10 and the (nominally) Th1-associated cytokine IFN-{gamma} were also enriched in TIL vs splenic T cell populations isolated from cytokine-treated mice, and that CMS4 cells themselves were unlikely contributing to the observed levels of foxp3, IL-10, or IFN-{gamma} mRNA transcripts from CD4+ TIL (Fig. 4A). Consistent with these RT-PCR results for IL-10 mRNA expression, immunofluorescence staining of CMS4 tumor tissue sections isolated from rFL plus rGM-CSF-treated, but not control, untreated mice revealed extensive production of IL-10 protein in situ, with confocal immunofluorescence imaging supporting IL-10 association with CD4+ or FoxP3+ TIL, but not with CD8+ TIL or CD11c+ TIDC in situ (Fig. 4B). Whereas TGF-beta transcripts also appeared somewhat elevated in TIL (vs splenocytes) harvested from rFL plus rGM-CSF-treated animals, TGF-beta mRNA expression was also detected in splenic CD4+ T cells isolated from untreated, normal, or tumor-bearing mice and in cultured CMS4 tumor cells (Fig. 4A). Given the latter finding, immunofluorescence analyses for TGF-beta in situ were not performed.


Figure 4
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  		FIGURE 4. rFL plus rGM-CSF treatment significantly enhances IL-10 production in situ within tumor lesions that is associated with CD4+ (FoxP3+) T cells, but not CD11c+ TIDC. A, Total RNA was isolated from the indicated CD4+ T cell populations (see Fig. 2), as outlined in Materials and Methods. RNA (1 µg) was reverse transcribed, and PCR amplification using specific primers was performed. beta2-microglobulin (beta2-m) was used as a reaction and loading control. B, Tumor sections from control or rFL plus rGM-CSF-treated mice were stained with Abs against CD4 (a and e), CD8 (b and f), CD11c (c, d, g, h, and i), foxP3 (c, d, g, h, and i), and IL-10 (all panels; see Materials and Methods) and analyzed by confocal immunofluorescence microscopy. Images (x60 magnification) reflect mid-plane section analyses of untreated (a–d) and rFL plus rGM-CSF-treated CMS4 tumors (e–i). Insets in a, b, e, and f, as well as d, h, and i, reflect higher power (x160–240 magnification). Data are representative of two independent experiments performed.

 
CD4+ TIL isolated from rFL plus rGM-CSF-treated mice suppress alloreactive CD4+ T cell proliferation and IFN-{gamma} secretion by tumor-specific CD8+ T cells in vitro

The hallmark of CD4+ Tregs is their functional ability to suppress responder/effector T cell proliferation and cytokine production (21, 22, 33). As a result, we wanted to determine whether CD4+ TIL isolated from rFL plus rGM-CSF-treated mice suppressed the in vitro proliferation of responder naive CD4+ T cells in an allogeneic MLR. In accordance with their Treg phenotype, we observed that the CD4+ TIL effectively suppressed the proliferation of alloreactive responder T cells in a dose-dependent manner (Fig. 5A). We further evaluated whether these TIL could suppress IFN-{gamma} production from in vivo primed anti-CMS4 CD8+ T cells. We previously reported that rFL plus rGM-CSF treatment of CMS4-bearing mice results in the enhanced cross-priming of tumor-specific CD8+ T cells in situ (26). For the current studies, we cocultured splenic CD8+ T cells harvested from rFL plus rGM-CSF-treated CMS4-bearing mice with irradiated CMS4 tumor cells in vitro in the absence or presence of CD4+ TIL isolated from the same cytokine-treated animals. CD4+ T cells isolated from the spleens of rFL plus rGM-CSF-treated, nontumor-bearing mice were used as controls for TIL. As shown in Fig. 5B, CD4+ TIL, but not control CD4+ splenocytes significantly reduced IFN-{gamma} production from in vivo primed CD8+ T cells against CMS4 tumor cells in vitro. CD4+ TIL-mediated suppression of antitumor CD8+ T cell production of IFN-{gamma} was partially blocked by addition of neutralizing anti-IL-10 Ab or the TGF-betaR-signaling antagonist IN-1130, with addition of both reagents normalizing these in vitro responses (Fig. 5C). These data suggest that CD8+ TIL may be functionally impeded from providing therapeutic benefit in rFL plus rGM-CSF-treated animals due to the suppressive impact of CD4+ Treg TIL, potentially via their secretion of IL-10 and/or TGF-beta.


Figure 5
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  		FIGURE 5. CD4+ TIL from cytokine-treated mice suppress alloantigen-specific T cell proliferation and IFN-{gamma} secretion by anti-CMS4 CD8+ T cells isolated from rFL plus rGM-CSF-treated, tumor-bearing mice. A, CD4+ T cells isolated from normal BALB/cJ mice were cocultured with total C57BL/6 splenocytes at a ratio of 1:1 in MLR cultures. CD4+ TIL isolated from rFL plus rGM-CSF-treated, CMS4-bearing mice were added to replicate cultures at the indicated TIL-to-normal splenocyte CD4+ T cell ratios. After 3 days, [3H]thymidine incorporation was determined by beta scintillation counting. Background proliferation associated with BALB/cJ CD4+ responder T cells only, CD4+ TIL only, or TIL plus allogeneic C57BL/6 splenocytes was <3200 cpm. Data are representative of three independent experiments performed. B, CD8+ T cells from rFL plus rGM-CSF-treated tumor-bearing mice spleen were cocultured with irradiated CMS4 cells (10:1 CD8+ T cell-to-tumor ratio) in the absence or presence of CD4+ TIL isolated from rFL plus rGM-CSF-treated mice or CD4+ normal splenocytes (NOR-SP), at the indicated CD4+ T cell-to-CD8+ T cell ratios. After 72 h of culture, supernatants were collected for, and analyzed by, IFN-{gamma} ELISA. C, Studies in B were repeated at a 1:1 CD4+ T cell-to-CD8+ T cell ratio, in the absence or presence of neutralizing anti-IL-10 Ab (10 µg/ml) or TGF-betaR-signaling inhibitor (1 µM). CD8+ TIL cocultured with tumor alone produced 5.1 ± 0.2 ng of IFN-{gamma}/ml. All data are representative of three independent experiments performed. *, p < 0.05 vs control (i.e., NOR-SP).

 
In vivo depletion of CD4+ T cells results in significantly improved antitumor efficacy of rFL plus rGM-CSF therapy that is CD8+ T cell dependent

To address whether removal of Tregs would reveal therapeutic immunity in rFL plus rGM-CSF-treated mice bearing established day 8 s.c. CMS4, tumors were injected with depleting anti-CD4 and/or anti-CD8 Abs. As shown in Fig. 6A, the injection of anti-CD8 Ab alone or an isotype-matched control rat IgG failed to impact CMS4 tumor growth in either untreated or rFL plus rGM-CSF-treated mice. Similarly, with very few exceptions, injection of anti-CD4 Ab alone failed to slow CMS4 tumor progression in animals that did not receive cytokine treatment. The only treatment group in which CMS4 growth was significantly inhibited was the cohort treated with rFL plus rGM-CSF and coinjected with the depleting anti-CD4 mAb. We repeatedly observed that this effect required chronic administration of anti-CD4-depleting Ab and that progressive tumor growth could be re-established within 9 days of discontinuation of administering anti-CD4 mAb (in animals that had not been rendered tumor free using this approach; data not shown). Based on the results obtained from animals receiving coinjections of anti-CD4 and anti-CD8 mAbs, the therapeutic benefit observed with CD4+ T cell depletion alone required the participation of CD8+ T cells, because tumor grew progressively (at control rates) when both CD4+ and CD8+ T cells were depleted in the rFL plus rGM-CSF-treated mice (Fig. 6A). Cumulative data from four independent experiments (involving 15 total animals/cohort) support the ability of rFL plus rGM-CSF administration to provide significant prolongation of survival, but only when combined with CD4+ T cell depletion (Fig. 6B).


Figure 6
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  		FIGURE 6. In vivo depletion of CD4+ T cells reveals therapeutic benefit from treatment with rFL plus rGM-CSF, which is CD8+ T cell dependent. A, BALB/cJ mice bearing established day 8 CMS4 tumors were either untreated or treated with rFL plus rGM-CSF for five consecutive days. On days 8, 13, and every 3 days thereafter for up to 49 days posttumor inoculation, the mice received i.p. injections of either rat isotype (control), anti-CD4, and/or anti-CD8-depleting mAbs. Tumor areas (mm2) were calculated every 3 days, with the data obtained for individual animals (5 mice/cohort) depicted. Data are representative of four experiments performed. B, Data from all experiments (15 mice/cohort) are presented in a Kaplan-Meier plot. Values of p < 0.05 for rFL plus rGM-CSF treated/CD4 depleted vs all other cohorts. The differences between the untreated/CD4-depleted vs untreated, control Ab-injected cohort were not significant (p = 0.83). Symbols used: •, untreated/control Ab injected; {circ}, untreated/CD4 depleted; {blacktriangledown}, untreated/CD8 depleted; {blacktriangledown}, untreated/CD4 plus CD8 depleted; {blacksquare}, rFL plus rGM-CSF treated/control Ab injected; {square}, rFL plus rGM-CSF treated/CD4 depleted; {diamondsuit}, rFL plus rGM-CSF treated/CD8 depleted; 224, rFL plus rGM-CSF treated/CD4 plus CD8 depleted.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we have shown that rFL plus rGM-CSF treatment of tumor-bearing mice results in increased numbers of CD4+ TIL cells exhibiting Treg phenotype and function in CMS4 tumor lesions in vivo. CD4+ TIL from rFL plus rGM-CSF-treated mice were uniformly CD45RBlow and ICOShigh, with nearly half of these T cells coexpressing the Treg markers CD25 and FoxP3, and 90% of them coexpressing high level GITR on their cell surfaces. Of the CD4+FoxP3+ TIL, 97% coexpressed GITR, whereas only 73% coexpressed CD25. CD8+Foxp3+ regulatory TIL were not identified in our analyses. The lack of CD25 expression by some of the CD4+FoxP3+ TIL is perhaps not surprising, as it confirms data reported by Fontenot et al. (20) that in mice expressing a GFP-Foxp3 fusion-protein reporter knockin allele (Foxp3gfp mice), only ~50–60% of CD4+FoxP3+ splenic or pulmonary T cells coexpressed CD25, supporting the notion that not all Tregs can be identified based on constitutively high expression of CD25 on their cell surface. Of greater therapeutic importance, CD4+ TIL isolated from rFL plus rGM-CSF-treated animals mediated suppressor activity and inhibited both allogenic CD4+ T cell proliferation and the ability of therapy-induced CD8+ TIL cells to secrete IFN-{gamma} in response to CMS4 tumor cells in vitro.

The ability of CD4+ Tregs to inhibit effector CD8+ T cell activity is a significant clinical concern and could well explain the lack of therapeutic impact that we previously observed for combined rFL plus rGM-CSF administration in three H-2d transplantable tumor models (CMS4 sarcoma, 4TI breast carcinoma, and murine renal adenocarcinoma cell line renal cell carcinoma), despite noting the ability of this regimen to promote profound tumor infiltration by seemingly semimature/mature DC and the effective cross-priming of antitumor CD8+ T effector cells (26). Indeed, this appears well justified based on our current studies in which depletion of CD4+ T cells in concert with provision of rFL plus rGM-CSF therapy results in virtually uniform tumor regression and prolonged survival, as long as CD8+ T cells are not codepleted. Overall, our results suggest that augmented numbers and function of Tregs (at least within the tumor microenvironment) in the CMS4 therapy model is a collateral penalty associated with rFL plus rGM-CSF-based treatment, and that Treg depletion resulting from administration of anti-CD4 mAb does not effect therapeutic benefit unless combined with an immune potentiating regimen (such as rFL plus rGM-CSF).

Given these considerations, one is left with the need to provide a rationale as to the following: how this combined cytokine protocol leads to the promotion of Tregs (even in treated tumor-free animals); how Tregs turn off otherwise therapeutic immunity mediated by CD8+ TIL after treatment with rFL plus rGM-CSF in vivo; and within the somewhat heterogenous population of rFL plus rGM-CSF-induced CD4+ T cells, which are the bona fide Tregs. In the first case, based on studies in mice deficient for FL or GM (34, 35, 36, 37), there are very limited data to support a direct effect of either of these cytokines on Treg expansion. Instead, the overriding weight of evidence suggests that Tregs may be invoked indirectly via the effects of FL and/or GM on DC development and differentiation (38, 39, 40, 41). Our previous report (26) suggests that rFL plus rGM-CSF administration results in peripheral increases in each of the three major (CD11b+CD11c+, CD8{alpha}+CD11c+, and B220+CD11c+) DC subsets in the spleens of treated mice, but that TIDC in these animals are almost uniformly of at least a semimature, myeloid CD11b+CD11c+ phenotype. These TIDC were efficient in acquiring and processing particulate/soluble Ags in vitro and in vivo and expressed intermediate to high levels of T cell costimulatory molecules, and the only quality noted that would be consistent with a nonstimulatory phenotype was the greater propensity of these APCs to secrete IL-10 in response to TLR ligands (when compared with control DC in the spleens of rFL plus rGM-CSF-treated animals or untreated mice) (26). Given their high expression levels of I-Ad and costimulatory molecules, as well as their IL-10 production capabilities, our described TIDC are similar to semimature DC populations previously reported to induce CD4+CD25+IL-10+ Tregs (Tr1) in vivo (38, 39). However, our immunofluorescence data suggest that TIDC do not appear to be making IL-10 in situ, and that the ability of therapy-induced TIDC to promote Treg responses most likely occurs in regional lymph nodes, where cognate interaction/costimulation with responder T cells could activate these APCs to produce and secrete IL-10. A further conundrum is associated with the overlap in the kinetics of Treg and CD11c+ DC populational increases observed in mice receiving cytokine therapy, in which a simple cause/effect relationship in the expansion/differentiation of these two cell types is difficult to envision. We are currently evaluating the role of chemokine/chemokine receptor pathways in Treg recruitment into tissues (including tumors) and whether tissue Tregs are actively proliferating in situ in our therapy model to further refine the mechanism(s) of action associated with our combined cytokine therapy.

With regard to how Tregs turn off otherwise therapeutic immunity mediated by CD8+ TIL after treatment with rFL plus rGM-CSF in vivo, and based on our previous documentation of effective anti-CMS4 CD8+ T cell cross-priming and the requirement for such immunity in mediating tumor regression in CD4-depleted animals (26), we believe that the rate-limiting impact of Tregs occurs at the level of suppressing the CD8+ T effector population in vivo in this model. Treg functional paradigms (40) suggest that such suppression would most likely be dependent on secreted IL-10 or TGF-beta, or due to direct Treg:CD8+ T cell interactions (41, 42, 43). Our current in vitro data would suggest that both IL-10 and TGF-beta most likely play important roles in Treg-mediated suppression of antitumor CD8+ T cell effector functions, with our in situ analyses supporting CD4+FoxP3+ TIL as major producers of IL-10 (and the major population of suppressor cells) within the tumor microenvironment. These data are consistent with recent studies for the potent inhibitory impact of IL-10 on CD8+ T effector cell functions, including the secretion of IFN-{gamma} (41, 44).

Because anti-CD4 Ab administration depletes both Th and regulatory CD4+ T cells, it will be interesting to observe in future experiments whether complete tumor regression can be achieved by selective targeting of Tregs or their immunosuppressive cytokines. Although one strategy could involve the application of depleting anti-CD25 Ab, there are clearly conceptual disadvantages to using this strategy because rFL plus rGM-CSF treatment-activated effector CD4+CD25+ (non-Treg) and CD8+CD25+ Tc1-type cells could be indiscriminately codepleted. An alternative treatment strategy would be to inject anti-GITR (DTA-1) mAb, which does not appear to deplete Tregs, but may neutralize their function (14, 45), or alternatively, costimulate non-Treg T effector cells (46). Clearly, given our preliminary indictment of IL-10 and TGF-beta as important in vitro mediators of Treg suppression of CD8+ T cell function in the current model, we will also prospectively investigate the in vivo impact of their neutralization on the efficacy of rFL plus rGM-CSF therapy by coadministration of specific antagonists (i.e., anti-IL-10/IL-10R Abs, anti-TGF-beta Abs, TGF-beta soluble receptors, and TGF-betaR-signaling inhibitors).

Overall, our findings demonstrate that Tregs may represent rate-limiting inhibitors to therapy-associated clinical benefit in the cancer setting. Our results reinforce the need to consider not only resident Tregs in tumor-bearing hosts at the time of therapy, but also the potential for therapy-induced augmentation of Treg numbers/function in situ that may limit treatment efficacy. Clearly, in our model, the promotion of tumor-specific CD8+ Tc1 cells was not sufficient to guarantee clinical success, given that our therapy also directly or indirectly supported Tregs in situ. As a consequence, these findings suggest that an assessment of Treg function should be required before a clinically ineffective protocol is empirically discarded, particularly if immune monitoring supports the ability of the regimen to enhance the activation of tumor-specific CD8+ Tc1 effector cells. In such a case, combinational approaches targeting the silencing of Tregs should be seriously contemplated.


   Acknowledgments
 
We thank Sean Alber for assistance with immunofluorescence imaging and Dr. Amy Wesa for careful review and constructive comments provided during the preparation of this manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


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

1 This work was supported by National Institutes of Health R01 Grant CA63350 (to W.J.S.). Back

2 A.B. and J.H. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Walter J. Storkus, Departments of Dermatology and Immunology, W1041 Biomedical Sciences Tower, University of Pittsburgh School of Medicine, 200 Lothrop Street, Pittsburgh, PA 15213. E-mail address: storkuswj{at}upmc.edu Back

4 Abbreviations used in this paper: Treg, regulatory T cell; DC, dendritic cell; GITR, glucocorticoid-induced TNFR family related; MFI, median fluorescence intensity; rFL, rFLt3 ligand; rGM-CSF, recombinant GM-CSF; TIDC, tumor-infiltrating DC; TIL, tumor-infiltrating lymphocyte; WB, wash buffer. Back

Received for publication November 10, 2006. Accepted for publication December 20, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Boon, T., J. C. Cerottini, B. Van den Eynde, P. van der Bruggen, A. Van Pel. 1994. Tumor antigens recognized by T lymphocytes. Annu. Rev. Immunol. 12: 337-365. [Medline]
  2. Celluzzi, C. M., J. L. Mayordomo, W. J. Storkus, M. T. Lotze, L. D. Falo. 1996. Peptide-pulsed dendritic cells induce antigen-specific CTL-mediated protective tumor immunity. J. Exp. Med. 183: 283-287. [Abstract/Free Full Text]
  3. Anichini, A., A. Molla, R. Mortarini, G. Tragni, I. Bersani, M. Di Nicola, A. M. Gianni, S. Pilotti, R. Dunbar, V. Cerundolo, G. Parmiani. 1999. An expanded peripheral T cell population to a cytotoxic T lymphocyte (CTL)-defined, melanocyte-specific antigen in metastatic melanoma patients impacts on generation of peptide-specific CTLs but does not overcome tumor escape from immune surveillance in metastatic lesions. J. Exp. Med. 190: 651-667. [Abstract/Free Full Text]
  4. Marincola, F. M., E. M. Jaffee, D. J. Hicklin, S. Ferrone. 2000. Escape of human solid tumors from T-cell recognition: molecular mechanisms and functional significance. Adv. Immunol. 74: 181-273. [Medline]
  5. Sakaguchi, S.. 2005. Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-self. Nat. Immunol. 6: 345-352. [Medline]
  6. Liyanage, U. K., T. T. Moore, H. G. Joo, Y. Tanaka, V. Herrmann, G. Doherty, J. A. Drebin, S. M. Strasberg, T. J. Eberlein, P. S. Goedegebuure, D. C. Linehan. 2002. Prevalence of regulatory T cells is increased in peripheral blood and tumor microenvironment of patients with pancreas or breast adenocarcinoma. J. Immunol. 169: 2756-2761. [Abstract/Free Full Text]
  7. Wolf, A. M., D. Wolf, M. Steurer, G. Gastl, E. Gunsilius, B. Grubeck-Loebenstein. 2003. Increase of regulatory T cells in the peripheral blood of cancer patients. Clin. Cancer Res. 9: 606-612. [Abstract/Free Full Text]
  8. Curiel, T. J., G. Coukos, L. Zou, X. Alvarez, P. Cheng, P. Mottram, M. Evdemon-Hogan, J. R. Conejo-Garcia, L. Zhang, M. Burow, et al 2004. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat. Med. 10: 942-949. [Medline]
  9. Sakaguchi, S., K. Fukuma, K. Kuribayashi, T. Masuda. 1985. Organ-specific autoimmune diseases induced in mice by elimination of T cell subset. I. Evidence for the active participation of T cells in natural self-tolerance; deficit of a T cell subset as a possible cause of autoimmune disease. J. Exp. Med. 161: 72-87. [Abstract/Free Full Text]
  10. Asano, M., M. Toda, N. Sakaguchi, S. Sakaguchi. 1996. Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation. J. Exp. Med. 184: 387-396. [Abstract/Free Full Text]
  11. Powrie, F., M. W. Leach, S. Mauze, L. B. Caddle, R. L. Coffman. 1993. Phenotypically distinct subsets of CD4+ T cells induce or protect from chronic intestinal inflammation in C.B-17 scid mice. Int. Immunol. 5: 1461-1471. [Abstract/Free Full Text]
  12. Jonuleit, H., E. Schmitt, M. Stassen, A. Tuettenberg, J. Knop, A. H. Enk. 2001. Identification and functional characterization of human CD4+CD25+ T cells with regulatory properties isolated from peripheral blood. J. Exp. Med. 193: 1285-1294. [Abstract/Free Full Text]
  13. Sakaguchi, S., N. Sakaguchi, M. Asano, M. Itoh, M. Toda. 1995. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor {alpha}-chains (CD25): breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155: 1151-1164. [Abstract]
  14. Shimizu, J., S. Yamazaki, T. Takahashi, Y. Ishida, S. Sakaguchi. 2002. Stimulation of CD25+CD4+ regulatory T cells through GITR breaks immunological self-tolerance. Nat. Immunol. 3: 135-142. [Medline]
  15. McHugh, R. S., M. J. Whitters, C. A. Piccirillo, D. A. Young, E. M. Shevach, M. Collins, M. C. Byrne. 2002. CD4+CD25+ immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor. Immunity 16: 311-323. [Medline]
  16. Herman, A. E., G. J. Freeman, D. Mathis, C. Benoist. 2004. CD4+CD25+ T regulatory cells dependent on ICOS promote regulation of effector cells in the prediabetic lesion. J. Exp. Med. 199: 1479-1489. [Abstract/Free Full Text]
  17. Hori, S., T. Nomura, S. Sakaguchi. 2003. Control of regulatory T cell development by the transcription factor Foxp3. Science 299: 1057-1061. [Abstract/Free Full Text]
  18. Fontenot, J. D., M. A. Gavin, A. Y. Rudensky. 2003. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol. 4: 330-336. [Medline]
  19. Khattri, R., T. Cox, S. A. Yasayko, F. Ramsdell. 2003. An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat. Immunol. 4: 337-342. [Medline]
  20. Fontenot, J. D., J. P. Rasmussen, L. M. Williams, J. L. Dooley, A. G. Farr, A. Y. Rudensky. 2005. Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity 22: 329-341. [Medline]
  21. Thornton, A. M., E. M. Shevach. 1998. CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J. Exp. Med. 188: 287-296. [Abstract/Free Full Text]
  22. Thornton, A. M., E. M. Shevach. 2000. Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific. J. Immunol. 164: 183-190. [Abstract/Free Full Text]
  23. Taams, L. S., J. Smith, M. H. Rustin, M. Salmon, L. M. Poulter, A. N. Akbar. 2001. Human anergic/suppressive CD4+CD25+ T cells: a highly differentiated and apoptosis-prone population. Eur. J. Immunol. 31: 1122-1131. [Medline]
  24. Asseman, C., S. Mauze, M. W. Leach, R. L. Coffman, F. Powrie. 1999. An essential role for interleukin 10 in the function of regulatory T cells that inhibit intestinal inflammation. J. Exp. Med. 190: 995-1004. [Abstract/Free Full Text]
  25. Nakamura, K., A. Kitani, I. Fuss, A. Pedersen, N. Harada, H. Nawata, W. Strober. 2004. TGF-beta1 plays an important role in the mechanism of CD4+CD25+ regulatory T cell activity in both humans and mice. J. Immunol. 172: 834-842. [Abstract/Free Full Text]
  26. Berhanu, A., J. Huang, S. M. Alber, S. C. Watkins, W. J. Storkus. 2006. Combinational rFlt3-ligand and rGM-CSF treatment promotes enhanced tumor infiltration by dendritic cells and anti-tumor CD8+ T cell cross-priming, but is ineffective as a therapy. Cancer Res. 66: 4895-4903. [Abstract/Free Full Text]
  27. Peng, Y., Y. Laouar, M. O. Li, E. A. Green, R. A. Flavell. 2004. TGF-beta regulates in vivo expansion of Foxp3-expressing CD4+CD25+ regulatory T cells responsible for protection against diabetes. Proc. Natl. Acad. Sci. USA 101: 4572-4577. [Abstract/Free Full Text]
  28. Ramos-Payan, R., M. Aguilar-Medina, S. Estrada-Parra, J. A. Gonzalez-y-Merchand, L. Favila-Castillo, A. Monroy-Ostria, I. C. Estrada-Garcia. 2003. Quantification of cytokine gene expression using an economical real-time polymerase chain reaction method based on SYBR Green I. Scand. J. Immunol. 57: 439-445. [Medline]
  29. Volman, T. J., R. J. Goris, J. W. van der Meer, T. Hendriks. 2004. Tissue- and time-dependent up-regulation of cytokine mRNA in a murine model for the multiple organ dysfunction syndrome. Ann. Surg. 240: 142-150. [Medline]
  30. Moon, J. A., H. T. Kim, I. Cho, Y. Y. Sheen, D. K. Kim. 2006. IN-1130, a novel transforming growth factor-beta type I receptor kinase (ALK5) inhibitor, suppresses renal fibrosis in obstructive nephropathy. Kidney Int. 70: 1234-1243. [Medline]
  31. Butcher, E. C., L. J. Picker. 1996. Lymphocyte homing and homeostasis. Science 272: 60-66. [Abstract]
  32. Stock, P., O. Akbari, G. Berry, G. J. Freeman, R. H. Dekruyff, D. T. Umetsu. 2004. Induction of T helper type 1-like regulatory cells that express Foxp3 and protect against airway hyperreactivity. Nat. Immunol. 5: 1149-1156. [Medline]
  33. Takahashi, T., T. Tagami, S. Yamazaki, T. Uede, J. Shimizu, N. Sakaguchi, T. W. Mak, S. Sakaguchi. 2000. Immunologic self-tolerance maintained by CD25+CD4+ regulatory T cells constitutively expressing CTLA-4. J. Exp. Med. 192: 303-310. [Abstract/Free Full Text]
  34. McKenna, H. J., K. L. Stocking, R. F. Miller, K. Brasel, T. De Smedt, E. Maraskovsky, C. R. Maliszewski, D. H. Lynch, J. Smith, B. Pulendran, et al 2000. Mice lacking flt3 ligand have deficient hematopoiesis affecting hematopoietic progenitor cells, dendritic cells and natural killer cells. Blood 95: 3489-3497. [Abstract/Free Full Text]
  35. Walzer, T., P. Brawand, D. Swart, J. Tocker, T. De Smedt. 2005. No defect in T-cell priming, secondary response, or tolerance in response to inhaled antigens in Fms-like tyrosine kinase 3 ligand-deficient mice. J. Allergy Clin. Immunol. 115: 192-199. [Medline]
  36. Wada, H., Y. Noguchi, M. W. Marino, A. R. Dunn, L. J. Old. 1997. T cell functions in granulocyte/macrophage colony-stimulating factor deficient mice. Proc. Natl. Acad. Sci. USA 94: 12557-12561. [Abstract/Free Full Text]
  37. Gangi, E., C. Vasu, D. Cheatem, B. S. Prabhakar. 2005. IL-10-producing CD4+CD25+ regulatory T cells play a critical role in granulocyte-macrophage colony-stimulating factor-induced suppression of experimental autoimmune thyroiditis. J. Immunol. 174: 7006-7013. [Abstract/Free Full Text]
  38. Akbari, O., R. H. DeKruyff, D. T. Umetsu. 2001. Pulmonary dendritic cells producing IL-10 mediate tolerance induced by respiratory exposure to antigen. Nat. Immunol. 2: 725-731. [Medline]
  39. McGuirk, P., C. McCann, K. H. G. Mills. 2002. Pathogen-specific T regulatory 1 cells induced in the respiratory tract by a bacterial molecule that stimulates interleukin 10 production by dendritic cells: a novel strategy for evasion of protective T helper type 1 responses by Bordetella pertussis. J. Exp. Med. 195: 221-231. [Abstract/Free Full Text]
  40. Bluestone, J. A., Q. Tang. 2005. How do CD4+CD25+ regulatory T cells control autoimmunity?. Curr. Opin. Immunol. 17: 638-642. [Medline]
  41. De la Barrera, S., M. Aleman, R. Musella, P. Schierloh, V. Pasquinelli, V. Garcia, E. Abbate, C. Sasiain Mdel. 2004. IL-10 down-regulates costimulatory molecules on Mycobacterium tuberculosis-pulsed macrophages and impairs the lytic activity of CD4 and CD8 CTL in tuberculosis patients. Clin. Exp. Immunol. 138: 128-138. [Medline]
  42. Thomas, D. A., J. Massague. 2005. TGF-beta directly targets cytotoxic T cell functions during tumor evasion of immune surveillance. Cancer Cells 8: 369-380. [Medline]
  43. Robertson, S. J., R. J. Messer, A. B. Carmody, K. J. Hasenkrug. 2006. In vitro suppression of CD8+ T cell function by friend virus-induced regulatory T cells. J. Immunol. 176: 3342-3349. [Abstract/Free Full Text]
  44. Jarnicki, A. G., J. Lysaght, S. Todryk, K. H. Mills. 2006. Suppression of antitumor immunity by IL-10 and TGF-beta-producing T cells infiltrating the growing tumor: influence of tumor environment on the induction of CD4+ and CD8+ regulatory T cells. J. Immunol. 177: 896-904. [Abstract/Free Full Text]
  45. Ramirez-Montagut, T., A. Chow, D. Hirschhorn-Cymerman, T. H. Terwey, A. A. Kochman, S. Lu, R. C. Miles, S. Sakaguchi, A. N. Houghton, M. R. van den Brink. 2006. Glucocorticoid-induced TNF receptor family related gene activation overcomes tolerance/ignorance to melanoma differentiation antigens and enhances antitumor immunity. J. Immunol. 176: 6434-6442. [Abstract/Free Full Text]
  46. Kanamaru, F., P. Youngnak, M. Hashiguchi, T. Nishioka, T. Takahashi, S. Sakaguchi, I. Ishikawa, M. Azuma. 2004. Costimulation via glucocorticoid-induced TNF receptor in both conventional and CD25+ regulatory CD4+ T cells. J. Immunol. 172: 7306-7314. [Abstract/Free Full Text]



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