Skip Navigation


International Immunology Advance Access originally published online on August 16, 2006
International Immunology 2006 18(10):1461-1471; doi:10.1093/intimm/dxl079
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
18/10/1461    most recent
dxl079v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (2)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Melencio, L.
Right arrow Articles by Nagarkatti, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Melencio, L.
Right arrow Articles by Nagarkatti, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?


© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Role of CD4+CD25+ T regulatory cells in IL-2-induced vascular leak

Louise Melencio1, Robert J. McKallip1, Hongbing Guan1, Rupal Ramakrishnan1, Reena Jain2, Prakash S. Nagarkatti1 and Mitzi Nagarkatti1

1 Department of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, 6439 Garner's Ferry Road, Columbia, SC 29209, USA
2 Department of Pathology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298, USA

Correspondence to: M. Nagarkatti; E-mail: mnagark{at}med.sc.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
T regulatory cells (CD4+CD25+) play an important role in the regulation of the immune response. However, little is known about the ability of T regulatory cells to regulate endothelial cell (EC) damage following activation of lymphocytes with IL-2. Therefore, in the current study, we examined the role of T regulatory cells and the subsequent Th1/Th2 bias in IL-2-mediated EC injury using the well-characterized C57BL/6 (Th1-biased) and BALB/c (Th2-biased) models. Following IL-2 treatment, BALB/c mice were less susceptible to IL-2-induced vascular leak syndrome (VLS) compared with C57BL/6 mice. Splenocytes from BALB/c mice displayed less cytotoxicity against ECs compared with those from C57BL/6 mice. Interestingly, BALB/c mice had significantly higher numbers of CD4+CD25+ T regulatory cells, which proliferated more profoundly following IL-2 treatment, compared with CD4+CD25+ T regulatory cells from C57BL/6 mice. In addition, T regulatory cells from naive BALB/c mice were more potent suppressors of anti-CD3 mAb-stimulated proliferation of T cells than similar cells from C57BL/6 mice. Depletion of T regulatory cells in both BALB/c and C57BL/6 mice led to a significant increase in IL-2-induced VLS. Together, the results from this study suggest that CD4+CD25+ T regulatory cells play an important role in the regulation of IL-2-induced EC injury.

Keywords: IL-2, Th1/Th2 cytokine, T regulatory cells, vascular leak


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Endothelial cell (EC) injury is a widely occurring pathological condition seen during a variety of infections, autoimmunity, transplantation, graft-versus-host disease and following immunotherapy with cytokines and immunotoxins (1–6). IL-2 therapy has been shown to be effective against human melanomas and renal cell carcinomas (7–9). It is also being tested to treat AIDS and other viral infections (10). However, IL-2 therapy is also accompanied by severe life-threatening toxicity characterized by EC injury leading to capillary or vascular leak syndrome (VLS) (8, 11, 12). It is becoming increasingly clear that EC injury can be mediated by effector lymphocytes (13–16). For example, following IL-2 treatment of mice, we observed a significant increase in the number and percentage of CD8+ T cells, NK cells and NKT cells. These cells possessed significant ability to bind to and lyse EC targets (17).

In addition to activating effector cells, IL-2 plays an important role in the development and maintenance of T regulatory cells (18). Recent studies suggest that T regulatory cells play an important role in the regulation of the immune response. Defects or depletion of T regulatory cell populations have been shown to result in autoreactive disease, implicated in type 1 diabetes development, myasthenia gravis, and increased immunity in HIV patients (19–21). The exact mechanism by which T regulatory cells control the immune response remains unclear. However, there is evidence to suggest that inhibition of IL-2 transcription by T regulatory cells may play a central role in their ability to suppress proliferation (22). T regulatory cells can influence the response of a number of cells including CD8+ T cells and CD4+ T cells (23, 24). However, few studies have examined the role of T regulatory cells in EC damage, and in particular, the injury caused by lymphokine-activated killer (LAK) cells in IL-2-induced immunotherapy. In addition, there is evidence to suggest that T regulatory cells are preferentially suppressive toward Th1 T cell clones rather than Th2 clones (25). Th1 cytokines, such as IL-2, IL-12, IFN-{gamma}, are often associated with inflammation, control of intracellular pathogens, delayed-type hypersensitivity reaction and anti-cancer activity (26). Uncontrolled Th1 responses are associated with organ-specific autoimmune disease, such as arthritis, multiple sclerosis and type 1 diabetes (26). In comparison, Th2 cytokines such as IL-4 and IL-5 are involved in controlling extracellular pathogens. Dysfunctional Th2 immunity is associated with allergy-type reactions, IgE-based disease and systemic autoimmune disease (26). To date, little is known about the role of Th1/Th2 skewing in the development and control of VLS.

In the current study, we examined the role of CD4+CD25+ T regulatory cells and the effect of Th1/Th2 bias in the development and control of IL-2-induced VLS. Using the C57BL/6 (Th1-biased) and BALB/c (Th2-biased) mouse models (27–29), we observed that C57BL/6 mice are significantly more susceptible than BALB/c mice to IL-2-induced VLS and that the resistance to IL-2-induced VLS was associated with Th2-biased cytokine production along with increased T regulatory cell activity.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
C57BL/6 and BALB/c female mice, 5–7 weeks of age, were obtained from the National Cancer Institute [(NCI), Bethesda, MD, USA]. MHC class II-deficient (Abb B6.129-H2-Ab1tm1GruN12) homozygous mice were obtained from Taconic (Germantown, NY, USA). Mice were given food and water ad libitum.

Cell lines
TME, an EC line, P815, an NK-resistant cell line, and Yac-1, an NK-sensitive cell line, were maintained on RPMI containing 10% FCS at 37°C, 5% CO2.

Antibodies
Antibodies—FITC-conjugated anti-CD4, FITC-conjugated anti-CD8, PE-conjugated anti-CD25, PE-conjugated anti-CD3—were obtained from BD PharMingen (San Diego, CA, USA). FITC-conjugated anti-DX5 was obtained from eBiosciences (San Diego, CA, USA).

Flow cytometric analysis of cell sorting
Splenocytes from mice treated with high-dose IL-2, as described below, or splenocytes stimulated with IL-2 in vitro were analyzed for the expression of various cell-surface markers. Nonspecific staining was blocked by incubation of the cells with Fc block (PharMingen) for 15 min. The expression of various cell-surface markers was determined by staining the cells with FITC- or PE-conjugated mAbs for 30 min on ice, followed by washing three times. The cells (5 x 104) were analyzed by flow cytometry. Intracellular levels of Foxp3 were determined by fixing the cells for 30 min with 4% PFA. Next, the cells were permeabilized by suspending the cells in the BD Perm/Wash solution for 15 min. The cells were stained with fluorescently labeled mAbs specific for Foxp3 (eBioscience) followed by flow cytometric analysis. In experiments in which CD4+CD25+ and CD4+CD25– cells were isolated, nonspecific staining was blocked by incubation of the cells with Fc block (PharMingen) for 15 min. Next, the cells were stained with FITC-conjugated anti-CD4 and PE-conjugated anti-CD25 (7D4) for 30 min. CD4+CD25+ and CD4+CD25– cells were sorted using MoFlo cell sorter (Cytomation, Fort Collins, CO, USA).

IL-2
Recombinant IL-2 was provided by the NCI Biological Resources Branch (Rockville, MD, USA).

Quantification of VLS
Vascular leak was studied by measuring the extravasation of Evan's blue, which when given intravenously (i.v.) binds to plasma proteins, particularly albumin, and following extravasation can be detected in various organs as described previously (15, 30, 31). Vascular leak was induced by injection of IL-2 as previously described (14, 15). Groups of five mice were injected intra-peritoneally with 75 000 U of rIL-2 or PBS as a control, three times a day for 3 days. On day 4, the mice received one injection and 2 h later were injected i.v. with 0.1 ml of 1% Evan's blue in PBS. After 2 h, the mice were exsanguinated under anesthesia, and the heart was perfused with heparin in PBS, as described previously (32). The lungs were harvested and placed in formamide at 37°C overnight. The Evan's blue in the organs was quantified by measuring the absorbance of the supernatant at 650 nm with a spectrophotometer. In experiments examining the effect of CD4+CD25+ T regulatory cell depletion on IL-2-induced VLS, mice were treated with 1 mg per mouse of anti-CD25 (PC61.5.3) ascites antibody (Cedarlane Laboratories, Westbury, NY, USA) 24 h prior to the first IL-2 injection.

Histological analysis
Lung tissue samples were removed from mice treated with IL-2 or vehicle. Samples were fixed using 10% formaldehyde and embedded in paraffin. Sections, 3 µm thick, were stained with hematoxylin and eosin and analyzed for perivascular infiltrate.

Generation of LAK cells
Spleens were harvested from C57BL/6 and BALB/c mice and were prepared into a single-cell suspension using a laboratory homogenizer (Stomacher, Tekmar, Cincinnati, OH, USA). Contaminating erythrocytes were removed by re-suspending the cells in 3 ml RBC lysing buffer (Sigma-Aldrich, St Louis, MO, USA) for 5 min and then washing three times in RPMI containing 10% FBS. After the third wash, the viable spleen cells were quantified by trypan blue dye exclusion and counting using a hemocytometer. The splenocytes were adjusted to 5 x 106 ml–1 and were cultured in vitro for 48 h with 1 x 103 U ml–1 of IL-2 in RPMI containing 10% FBS. The cells were harvested, and viable cells were purified by density gradient centrifugation using Ficoll–Hypaque (Sigma-Aldrich). Such cells will be referred to as LAK cells. LAK cells were tested for cytotoxicity against P815 tumor, YAC-1 tumor or TME EC targets using the 51Cr release assay. Briefly, 1 x 106 target cells were labeled with 51NaCrO4 at 37°C for 1 h. The LAK cells were plated in triplicate into 96-well round-bottomed plates at varying effector to target ratios and incubated for 4 h at 37°C. In these experiments, the LAK cells are defined as the effector cells that mediate lysis of the 51NaCrO4-labeled EC or tumor cells, which are defined as the target cells.

Quantification of cytokine and chemokine production
To measure cytokines and chemokines secreted in vivo, serum samples were taken from the blood of IL-2 or PBS-treated mice (n = 3) and collected into an Eppendorf tube containing 25 µl of 50 U ml–1 heparin. Blood samples were centrifuged at 5000 r.p.m. for 10 min. Serum was separated from RBCs. To measure cytokines and chemokines secreted in vitro, cells (2.5 x 106 ml–1) were cultured with 1 x 103 U ml–1 IL-2 overnight. The supernatant was collected. Cytokine levels were quantified using the Bio-Rad 18-plex assay (Bio-Rad Laboratories, Hercules, CA, USA).

Quantification of CD4+CD25+ T regulatory cell activity
CD4+CD25– T cells (5 x 104 per well) were cultured along with irradiated (200 rads) syngeneic adherent T cell-depleted splenocytes (5 x 104 per well) and soluble anti-CD3 antibody (5 µg ml–1) in the presence or absence of syngeneic CD4+CD25+ (2.5 x 104 per well) cells. The proliferative response was measured on day 4, following an 8-h pulse with [3H]-thymidine (0.5 µCi per well) and scintillation counting.

Statistical analysis
Analysis of variance and Student's t-test were used to determine statistical significance and P <0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
C57BL/6 mice show increased sensitivity to IL-2-induced vascular leak when compared with BALB/c mice
In initial experiments, we compared the susceptibility of C57BL/6 and BALB/c mice with IL-2-induced VLS. To this end, groups of C57BL/6 and BALB/c mice were injected with 75 000 U of IL-2 three times daily for 3 days and once on day 4. On the last day, the mice were injected with 1% Evan's blue and VLS was studied by determining the extravasation of the dye in the lungs. As depicted in Fig. 1(A), following IL-2 treatment, BALB/c mice were significantly less susceptible to IL-2-induced VLS compared with C57BL/6 mice. Interestingly, the decreased susceptibility of the BALB/c mice to IL-2-induced VLS was not due to unresponsive lymphocytes since a similar increase in splenic cellularity was observed in both strains following IL-2 treatment (Fig. 1B).


Figure 1
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 C57BL/6 mice show increased sensitivity to IL-2-induced vascular leak when compared with BALB/c mice. C57BL/6 and BALB/c mice were injected intra-peritoneally with 7.5 x 104 U of IL-2 alone three times daily for 3 days and once on day 4 or with PBS. Two hours after the last injection with IL-2, the mice were injected with 1% Evans blue, and VLS was studied by determining the extravasation of Evans blue dye in the lungs (A). The vertical bars represent the percent increase in VLS ± SEM following IL-2 treatment compared with that in the PBS-treated controls as described in Methods. Spleens were removed from C57BL/6 and BALB/c mice treated with PBS or IL-2 and the vertical bars represent cellularity (x106 per mouse) which was determined by trypan blue dye exclusion (B). Asterisks indicate statistically significant difference when compared with the PBS-treated controls, P < 0.05.

 
The effect of IL-2 treatment on the expansion of effector cell populations in BALB/c and C57BL/6 mice in vivo
In order to determine the mechanism leading to BALB/c resistance to IL-2-induced VLS, we compared the relative percentages of the various splenic cell populations following PBS or IL-2 treatment in BALB/c and C57BL/6 mice. Following IL-2 administration (Fig. 2), we found that there was a decrease in the percentage of CD4+ T cells and an increase in CD8+, NK (DX5+CD3–) and NKT (DX5+CD3+) cell populations in both C57BL/6 and BALB/c mice. This was also reflected by an increase in the total number of CD4+, CD8+, NK and NKT cells. FACS analysis also indicated that following IL-2 treatment, there was an increase in the expression of the activation marker CD69 for both strains. These data suggested that the observed resistance of BALB/c mice to IL-2-induced VLS was not due to reduced activation or expansion of cells reportedly involved in mediating EC damage following IL-2 treatment.


Figure 2
View larger version (58K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 The effect of IL-2 treatment on the expansion of effector cell populations in BALB/c and C57BL/6 mice in vivo. Splenocytes from C57BL/6 and BALB/c mice treated with PBS or IL-2, as described in Methods, were stained for the following cell-surface markers: CD4, CD8, CD3, DX5 and CD69. Cells were then analyzed by flow cytometry, as described in Methods. Histograms are displayed as the percentage of CD4/8+ T cells and mean fluorescence intensity before and following IL-2 treatment (A). Dot plots were used to depict NK (CD3–DX5+) and NKT (CD3+DX5+) cells. Cellularity was calculated from the percentage of positively labeled cells and total cellularity and depicted as average cellularity (x106 per mouse) ± SEM, n = 3 (B).

 
BALB/c mice show reduced perivascular infiltration following IL-2 treatment compared with C57BL/6 mice
Next, we examined whether the observed resistance of BALB/c mice to IL-2-induced VLS was due to reduced lymphocytic infiltration into the lungs. To this end, groups of C57BL/6 and BALB/c mice were injected with 7.5 x 104 U of IL-2 three times daily for 3 days and once on day 4. On day 4, the lungs were harvested and stained with hematoxylin and eosin (Fig. 3). The lungs from the PBS-treated mice did not show any perivascular infiltration. In contrast, the IL-2-treated C57BL/6 mice exhibited significant perivascular infiltration consisting mainly of lymphocytes around the pulmonary vein, whereas IL-2-treated BALB/c mice presented with minimal perivascular mononuclear infiltration, suggesting that BALB/c resistance to IL-2-induced VLS may be due, at least in part, to a reduction in perivascular infiltration.


Figure 3
View larger version (97K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 BALB/c mice show reduced perivascular infiltration following IL-2 treatment compared with C57BL/6 mice. C57BL/6 and BALB/c mice were treated with vehicle or IL-2 as described previously. Lungs were removed, fixed in 10% formaldehyde and embedded in paraffin and stained with H&E, and perivascular infiltration was studied.

 
IL-2-activated splenocytes from BALB/c mice are less effective at lysing target cells compared with IL-2-activated splenocytes from C57BL/6 mice
In addition to infiltration into the lung, the ability of LAK cells to lyse ECs plays an important role in the development of VLS. Therefore, we compared the cytotoxic activity of LAK cells from BALB/c mice and C57BL/6 mice. More specifically, splenocytes from BALB/c and C57BL/6 mice treated for 4 days with high-dose IL-2 were directed against various target cells, including TME (Fig. 4A), an EC line, P815 (Fig. 4B), an NK-resistant cell line, and Yac-1 (Fig. 4C), an NK-sensitive cell line. The results showed that following in vivo treatment with high-dose IL-2, splenocytes from BALB/c mice had less cytotoxic activity against all target cell lines when compared with the cytotoxic activity of the C57BL/6 splenocytes. This indicated that reduced IL-2-induced VLS in BALB/c may be attributed to reduced IL-2-stimulated cytotoxic activity. Furthermore, the reduced cytotoxic activity was not specific for EC cells but included both NK-sensitive and NK-insensitive targets.


Figure 4
View larger version (10K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Splenocytes from IL-2-treated BALB/c mice are less effective at lysing target cells compared with splenocytes from IL-2-treated C57BL/6 mice. Splenocytes from BALB/c and C57BL/6 mice treated for 4 days with high-dose IL-2, as described in Methods, were tested for cytolytic activity against 51Cr-labeled TME EC (A), as well as YAC-1 (B) and P815 (C) tumor cells. LAK activity was determined using the 4-h 51Cr release assay. The data indicate the mean percent cytotoxicity of triplicate cultures ± SEM. Asterisk indicates statistically significant difference when C57BL/6 is compared with BALB/c results, P < 0.05.

 
Differences in cytokine and chemokine production by splenocytes from BALB/c versus C57BL/6 mice following IL-2 exposure in vitro
The profile of cytokines and chemokines produced following stimulation of the immune system can greatly influence the resulting response. Therefore, we examined whether exposure to IL-2 had a differential effect on the levels and/or types of cytokines and chemokines produced by BALB/c versus C57BL/6 mice. To this end, splenocytes from BALB/c and C57BL/6 mice were cultured for 48 h with or without IL-2 (1 x 103 U ml–1) and the supernatants were collected. The cytokine and chemokine levels were measured using the Bio-Rad 18-plex assay. The results showed that there were marked differences in cytokine and chemokine production (Fig. 5A). More specifically, we noted that higher levels of the pro-inflammatory cytokines, such as IFN-{gamma}, IL-12 and IL-6, as well as chemokines, such as MIP-1 ß and RANTES, were produced by splenocytes from C57BL/6 following IL-2 exposure, whereas BALB/c splenocytes not only produced lower amounts but the levels were further decreased following IL-2 exposure. In addition, experiments were conducted to examine differences in cytokine and chemokine production between C57BL/6 mice and BALB/c mice following treatment with IL-2 in vivo. To this end, C57BL/6 and BALB/c mice were treated for 4 days with high-dose IL-2. Serum samples were collected on days 1 and 4 and were subsequently tested for cytokine and chemokine production using the Bio-Rad 18-plex assay. The results confirmed the in vitro data demonstrating increased levels of the Th1 cytokines IL-6 and IL-12 as well as an increase in the levels of RANTES in the C57BL/6 mice when compared with the levels in the BALB/c mice (Fig. 5B). A number of reports suggest a possible role for IL-10 in T regulatory cell activity in vivo (33). Therefore, we examined the effect of IL-2 treatment on the level of IL-10 in the serum of BALB/c and C57BL/6 mice compared with vehicle-treated mice (Fig. 5B). The results showed that there was a significant increase in the level of IL-10 produced by the BALB/c mice following IL-2 treatment compared with the level of IL-10 produced by the C57BL/6 mice. Together, these results suggest that a reduced capacity to produce pro-inflammatory cytokines and chemokines as well as an increased production in IL-10 following exposure to IL-2 may contribute to the reduced sensitivity of BALB/c mice to IL-2-induced VLS.


Figure 5
View larger version (19K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Differences in cytokine production by splenocytes from BALB/c versus C57BL/6 mice following IL-2 exposure in vitro. Splenocytes from naive BALB/c and C57BL/6 mice were removed and cultured overnight with media alone or IL-2 and media. Supernatants were collected and cytokine and chemokine levels were measured using the Bio-Rad 18-plex assay (A). C57BL/6 and BALB/c mice were treated with vehicle or IL-2 as described previously. Serum samples were collected and cytokine and chemokine levels were measured in the Bio-Rad 18-plex assay (B). Asterisk indicates statistically significant difference when compared with the PBS-treated groups, P < 0.05.

 
Increased levels of CD4+CD25+ T regulatory cells in BALB/c mice before and after IL-2 treatment in vivo compared with C57BL/6 mice
Recent studies suggest that T regulatory cells can play an important role in the outcome of an immune response. Therefore, we examined whether there were differences in the levels of CD4+CD25+ T regulatory cells in BALB/c compared with C57BL/6 mice before and/or after exposure to IL-2. Prior to IL-2 treatment, the percentage as well as the absolute number of T regulatory cells were slightly higher in the BALB/c mice compared with the C57BL/6 mice. Following IL-2 treatment, we observed a significant increase in the percentage and the absolute number of CD4+CD25+ cells in both C57BL/6 and BALB/c (Fig. 6A and B). However, the increase in CD4+CD25+ T regulatory cells was more pronounced in the BALB/c mice. These results were confirmed using intracellular staining of CD4+ cells with antibodies specific for Foxp3 which demonstrated that the absolute number of CD4+Foxp3+ before and after stimulation with IL-2 was significantly higher in the BALB/c mice when compared with C57BL/6 mice (Fig. 6C).


Figure 6
View larger version (41K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Increased levels of CD4+CD25+ T regulatory cells in BALB/c mice before and after IL-2 treatment in vivo compared with C57BL/6 mice. Splenocytes from PBS- or IL-2-treated C57BL/6 and BALB/c mice treated with IL-2 or PBS were stained for CD4 and CD25 expression and analyzed using flow cytometric analysis (A). Absolute cell number was determined by using total cellularity, determined by trypan blue dye exclusion and percentage of CD4+CD25+ cells (B). CD4+ splenocytes from PBS- and IL-2-treated BALB/c and C57BL/6 mice were analyzed for Foxp3 and ß-actin mRNA expression was determined by RT–PCR (C). The absolute number of CD4+CD25+ cells in the spleen (C) and lymph nodes (D) was determined by multiplying the total cellularity of the spleen or thymus by the percentage of CD4+FoxP3+ cells in the spleen and thymus, respectively.

 
CD4+CD25+ T regulatory cell from BALB/c show elevated suppressive activity compared with CD4+CD25+ T regulatory cells from C57BL/6 mice
In addition to differences in T regulatory cell number, it is possible that alterations in T regulatory activity may contribute to the relative resistance of BALB/c mice to IL-2-induced VLS. Therefore, we compared the suppressive activity of the T regulatory cells from BALB/c and C57BL/6 mice. More specifically, CD4+CD25– cells from BALB/c and C57BL/6 mice were cultured with irradiated syngeneic T cell-depleted splenocytes and soluble anti-CD3 mAbs in the presence or absence of syngeneic CD4+CD25+ cells. T regulatory cells from both PBS- and IL-2-treated mice were examined. Results depicted in Fig. 7 show that although CD4+CD25+ cells from both groups of PBS-treated mice had suppressive activity, the T regulatory cells from the BALB/c mice were significantly more suppressive than those from the C57BL/6 mice. In addition, CD4+CD25+ cells from IL-2-treated mice were significantly more suppressive than CD4+CD25+ cells from PBS-treated mice. However, the CD4+CD25+ from the IL-2-treated BALB/c mice were significantly more potent in suppressing CD4+CD25– T cell proliferation compared with their C57BL/6 counterparts. Together, these results suggest that CD4+CD25+ cells from BALB/c mice are significantly more active at suppressing the proliferative response following stimulation with anti-CD3 mAbs.


Figure 7
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 CD4+CD25+ T regulatory cells from BALB/c show elevated suppressive activity compared with CD4+CD25+ T regulatory cells from C57BL/6 mice. CD4+CD25+ T regulatory cells from PBS- and IL-2-treated C57BL/6 and BALB/c mice were cultured for 48 h with CD4+CD25– T cells along with 5 µg ml–1 of anti-CD3 antibody and irradiated adherent T cell-depleted syngeneic splenic cells. During the final 8 h of culture, the cells were pulsed with 2 µCi [3H]-thymidine. Thymidine incorporation was determined by ß-scintillation counting. The data represent mean ± SEM from triplicate wells.

 
The role of CD4+CD25+ T regulatory cells in IL-2-induced VLS
Next we examined the potential role of CD4+CD25+ T regulatory cells in the development and control of IL-2-induced VLS. More specifically, we determined whether depletion of T regulatory cells would have any effect on the severity of vascular leak following IL-2 treatment. To this end, C57BL/6 and BALB/c mice were injected with anti-CD25 mAbs (1 mg per mouse) 24 h prior to injection with IL-2. To determine the effectiveness of the anti-CD25 mAb treatment, splenocytes from anti-CD25 mAb and isotype antibody-treated control mice were stained for CD4 and CD25. Data depicted in Fig. 8(A) indicate that the percentage of CD4+CD25+ cells in IL-2-injected mice treated with anti-CD25 mAbs was <0.1%, compared with the 3–5% in the control-treated mice. Depicted by Fig. 8(B), following depletion of T regulatory cells and IL-2 treatment, we observed an increase in the levels of vascular leak for both C57BL/6 and BALB/c mice. To further confirm the role of T regulatory cells in IL-2-induced VLS, we examined the levels of vascular leak in MHC class II knockout (KO) mice, which lack functional CD4+CD25+ T regulatory cells (Fig. 8C). The results showed that the MHC class II KO displayed significantly higher levels of IL-2-induced VLS when compared with wild-type (WT) mice. Taken together, the results from these experiments suggest that T regulatory cells play an important role in the control of IL-2-induced VLS.


Figure 8
View larger version (26K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 8 The role of CD4+CD25+ T regulatory cells in IL-2-induced VLS. Groups of mice were injected with anti-CD25 antibody 24 h prior to receiving PBS or IL-2 treatment. The effect of anti-CD25 mAb treatment on the level of CD4+CD25+ T regulatory cells was determined by flow cytometric analysis (A). The effect of anti-CD25 mAbs on IL-2-induced VLS in vivo was determined by injecting the mice with isotype or anti-CD25 ascites antibody (1 mg per mouse) 24 h prior to the 4-day treatment with IL-2, as described in Methods (B). Vascular leak was determined as described above. IL-2-induced VLS in MHC class II KO mice compared with WT mice was performed by treating the mice with PBS or IL-2, as described in Methods and determining the levels of vascular leak as described above (C).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Work from our laboratory as well as others has shown that IL-2-mediated VLS can be induced by effector cells, such as CD8+ T cell, NK cells, NKT cells as well as polymorphonuclear neutrophils (17, 34). The involvement of these cells is further supported by results from the current study in which we demonstrated that following IL-2 exposure there was a significant increase in the numbers of CD8+ T cells, NK and NKT cells. However, the increase in cell number was not reflective of sensitivity to IL-2-induced VLS, suggesting another mechanism of control. More specifically, we noted that in spite of comparable increases in these populations, BALB/c mice were less sensitive to IL-2-induced VLS.

Another possible mechanism of regulation of the IL-2-induced VLS may be through control of cytokine production. Numerous studies demonstrate that cytokine profiles can have a significant impact on the development of various immunological disorders. More specifically, a shift toward a Th1 response has been associated with increased susceptibility to autoimmune diseases such as multiple sclerosis and rheumatoid arthritis, while a Th2-dominant response has been associated with allergic disease and asthma (35). In the current study, we demonstrated in the IL-2-induced VLS-sensitive C57BL/6 mice that IL-2 treatment led to a significant increase in IFN-{gamma}. In addition, following IL-2 exposure, the levels of the chemokines, RANTES and MIP-1 alpha were elevated in the C57BL/6 mice. Interestingly, MIP-1 alpha, RANTES and IFN-{gamma} have been linked as a group of ‘type 1 cytokines’ used by NK cells in the innate phase of the immune response and also used by CTL in the antigen-specific phase (36). However, a comparable up-regulation of these cytokines was not seen in the IL-2-induced VLS-resistant BALB/c mice, suggesting that control of these cytokines may play an important role in the development of vascular leak following IL-2 exposure. Additionally, the altered regulation of chemokines, MIP-1 ß and RANTES seen in the BALB/c mice may at least in part explain the observed decrease in lymphocytic infiltration in the lungs. Furthermore, the reduced cytolytic activity of IL-2-activated splenocytes in BALB/c mice may be related to the decreased production of IFN-{gamma}.

One possible explanation for the observed differences in the cytokine profiles observed in the BALB/c versus C57BL/6 mice could be related to the presence and activity of T regulatory cells. In experiments, examining the levels and activity of CD4+CD25+ T regulatory cells in the BALB/c mice revealed increased levels and increased activity of these cells when compared with CD4+CD25+ T regulatory cells from similarly treated C57BL/6 mice and that depletion of T regulatory cells led to an increase in the level of vascular leak following IL-2 treatment. It has been reported that CD4+CD25+ T regulatory cells tend to play more significant role in the regulation of the Th1 response. For example, CD4+CD25+ from human thymus completely suppressed the proliferative response of Th1 clones but showed significantly less suppressive activity against Th2 clones (25). The ability of T regulatory cells to inhibit the Th1 response has also been demonstrated in a number of other models including schistosomiasis, in which T regulatory cells directly suppress the Th1 response and not the Th2 response (33). Therefore, the observed differences in cytokine profiles of BALB/c versus C57BL/6 mice reported in this as well as other studies may be dependent on the differential presence and/or activity of the T regulatory cells, where we hypothesize that the presence of higher numbers or more active T regulatory cells leads to a Th2-biased cytokine pattern and lower levels of T regulatory cells with reduced activity shifts the cytokine production toward a Th1 response. More importantly, these data may suggest an important role for T regulatory cells in human disease. Cytokine profiles have been implicated in a number of immunological diseases. For example, it has been shown that individuals with a high risk of developing type 1 diabetes produced less Th1 cytokines, such as IFN-{gamma}, following stimulation with auto-antigen (37). Furthermore, Th1 and Th2 cytokine profiles have been implicated in the development of rheumatoid arthritis, multiple sclerosis, as well as cancer (38–40). Taken together with these reports, the results from this study suggest that, in the future, it may be possible to predict whether or not a patient will respond to specific immunologically based therapy, such as IL-2 treatment of metastatic melanoma, or to predict an individual's susceptibility to specific immunologically relevant diseases by screening patients for the presence and/or activity of T regulatory cells.

In this study, we showed a significant increase in the number of CD4+CD25+ T regulatory cells following in vivo treatment with IL-2. This observation may have significant clinical implication in that it suggests that IL-2 may be useful for expansion of T regulatory cells for use in the treatment of number of immunologically based diseases. The development and expansion of CD4+CD25+ T regulatory cells in vivo remain unclear. Although most investigators believe that the thymus plays a central role in the development of T regulatory cells, the origins of T regulatory cells expanded in the periphery remain controversial. Two of the most recognized models for T regulatory cell expansion in the periphery are the ‘dedicated T reg cell lineage’ and the ‘adaptive T reg cell effectors’ models (41). In the dedicated T reg cell lineage model, Foxp3-expressing T regulatory cells are developed primarily in the thymus and expansion in the periphery is due to direct stimulation of thymus-derived T regulatory cells by IL-2 produced by activated effector cells. In contrast to this model, the adaptive T reg cell effectors model suggests that Foxp3-expressing T regulatory cells are generated both in the thymus as well as de nevo from nonregulatory effector cells following activation. Although we cannot discount the adaptive model, the results from this study support a critical aspect of the dedicated T reg lineage model in that stimulation with IL-2 was sufficient to increase the number of Foxp3-expressing T regulatory cell in the periphery.

IL-2-induced VLS is believed to be mediated primarily by components of the innate immune system. CD4+CD25+ T regulatory cells play an important role in the regulation of the immune response. However, most reports demonstrate the ability of T regulatory cells to directly affect the activity of cells involved in the acquired immune response. For example, a number of reports suggest a role of T regulatory cells in the controlling of autoimmune diseases, including inflammatory bowel disease, type 1 diabetes and experimental autoimmune encephalomyelitis (42). To date, very little has been reported about the ability of CD4+CD25+ T regulatory cells to control the response of the innate immune system. In the current study, we provide novel evidence that suggests a role of T regulatory cells in directly controlling innate immunity. However, the mechanism by which CD4+CD25+ T regulatory cells control EC damage following IL-2 damage remains unclear. Initial reports examining the mechanism of T regulatory cell suppressor functions in vitro suggested that the main mechanism of suppression was due to inhibition of IL-2 transcription and that cell contact was required (22). Interestingly, it was shown that CD4+CD25+ T cell suppression of the immune response occurred in an antigen nonspecific manner (43). To date, the mechanism of T regulatory action in vivo remains unclear. A number of reports suggest that cell contact may be less important in vivo and that T regulatory cell production of IL-10 and/or transforming growth factor (TGF)-ß may play a more important role. For example, IL-10-deficient mice develop spontaneous colitis which can be attenuated by adoptive transfer of T regulatory cells from WT but not IL-10- or TGF-ß-deficient mice (44–46). In the current study, we were unable to detect significant changes in the levels of IL-10 following in vitro stimulation of BALB/c or C57BL/6 splenocytes with IL-2. However, examination of IL-10 production in vivo revealed a significant increase in the level of IL-10 in the serum of BALB/c mice, whereas no significant increase was seen in the C57BL/6 mice, suggesting a possible role of IL-10 in the control of IL-2-induced VLS.

The current study suggests that enrichment of CD4+CD25+ cells may be a novel way to control IL-2-induced VLS. However, a number of reports also suggest that T regulatory cells can play a significant role in suppressing the immune response to a number of cancers including melanoma. For example, depletion of CD4+CD25+ cells leads to enhanced anti-tumor immunity and tumor rejection in a number of mouse tumor models (47–50). This enhanced tumor immunity was believed to be primarily related to an enhanced activity of tumor-specific CD8+ CTLs. However, a possible role of T regulatory cells on the anti-tumor activity of CD4+ and NK cells also exists. In addition, a number of reports suggest that elevated numbers of CD4+CD25+ T regulatory cells are present in patients with melanoma and that the T regulatory cells possess the ability to suppress the responses of tumor infiltrating lymphocytes (51, 52). Furthermore, it has been shown that there may be a correlation between the response against metastatic melanoma and the development of pulmonary edema following IL-2 therapy. More specifically, it was shown that patients who responded better to the anti-tumor effects of IL-2 also were more susceptible to IL-2-induced pulmonary edema (53), suggesting the possibility that reducing VLS may reduce the anti-tumor response. Therefore, studies directed at dissecting the differences in the roles and/or mechanisms of T regulatory cell control of the anti-tumor response and the development of VLS may prove useful in further enhancing the usefulness of IL-2 therapy.

In conclusion, in the current study, we demonstrated a novel role of CD4+CD25+ T regulatory cells in the development and control of IL-2-induced vascular leak. More specifically, we showed that BALB/c mice were relatively resistant to IL-2-induced VLS when compared with C57BL/6 mice. This resistance was found to be directly related to reduced perivascular infiltration in the lungs as well as a reduced capacity of IL-2-stimulated splenocytes to lyse EC targets. In addition, the resistance of BALB/c mice to IL-2-induced VLS was associated with decreased production of several pro-inflammatory cytokines. Upon examination of the presence and activity of CD4+CD25+ T regulatory cells in these two strains, we noted that there were more CD4+CD25+ T regulatory cells present in the BALB/c when compared with the C57BL/6 mice and that the T regulatory cells isolated from the BALB/c were better able to suppress the proliferative response of CD4+CD25– cells following stimulation with anti-CD3 mAbs. Furthermore, we directly established the role of CD4+CD25+ T regulatory cells in IL-2-induced VLS by demonstrating that depletion of T regulatory cells led to an increase in the severity of IL-2-induced VLS. Taken together, the results from this study suggest that novel approaches to enhance the ability of CD4+CD25+ T regulatory cells to suppress IL-2-induced VLS without suppressing the anti-tumor immune response, possibly by site-specific depletion or enhancement of T regulatory cells, may be one approach to significantly improve the efficacy of IL-2 therapy to treat cancer.


   Acknowledgements
 
This work was supported in part by grants from National Institutes of Health (RO1 AI 053703, RO1 DA 016545, RO1 AI 058300, RO1 HL 058641 and RO1 ES 09098).


   Abbreviations
 
EC, Endothelial cell
i.v., intravenous
KO, knockout
LAK, lymphokine-activated killer
NCI, National Cancer Institute
TGF, transforming growth factor
VLS, vascular leak syndrome
WT, wild type

   Notes
 
Transmitting editor: L. Glimcher

Received 9 February 2006, accepted 18 July 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Baluna R and Vitetta ES. (1997) Vascular leak syndrome: a side effect of immunotherapy. Immunopharmacology 37:117.[CrossRef][Web of Science][Medline]
  2. Doherty PC, Allan JE, Lynch F, Ceredig R. (1990) Dissection of an inflammatory process induced by CD8+ T cells. Immunol. Today 11:55.[CrossRef][Web of Science][Medline]
  3. Hewicker M and Trautwein G. (1987) Sequential study of vasculitis in MRL mice. Lab. Anim. 21:335.[Abstract/Free Full Text]
  4. McCluskey RT and Fienberg R. (1983) Vasculitis in primary vasculitides, granulomatoses, and connective tissue diseases. Hum. Pathol. 14:305.[Web of Science][Medline]
  5. Moyer CF and Reinisch CL. (1984) The role of vascular smooth muscle cells in experimental autoimmune vasculitis. I. The initiation of delayed type hypersensitivity angiitis. Am. J. Pathol. 117:380.[Abstract]
  6. Tsukada N, Matsuda M, Miyagi K, Yanagisawa N. (1993) Cytotoxicity of T cells for cerebral endothelium in multiple sclerosis. J. Neurol. Sci. 117:140.[CrossRef][Web of Science][Medline]
  7. Fyfe G, Fisher RI, Rosenberg SA, Sznol M, Parkinson DR, Louie AC. (1995) Results of treatment of 255 patients with metastatic renal cell carcinoma who received high-dose recombinant interleukin-2 therapy. J. Clin. Oncol. 13:688.[Abstract/Free Full Text]
  8. Rosenberg SA, Lotze MT, Muul LM, et al. (1985) Observations on the systemic administration of autologous lymphokine-activated killer cells and recombinant interleukin-2 to patients with metastatic cancer. N. Engl. J. Med. 313:1485.[Abstract]
  9. Rosenberg SA, Mule JJ, Spiess PJ, Reichert CM, Schwarz SL. (1985) Regression of established pulmonary metastases and subcutaneous tumor mediated by the systemic administration of high-dose recombinant interleukin 2. J. Exp. Med. 161:1169.[Abstract/Free Full Text]
  10. Jacobson EL, Pilaro F, Smith KA. (1996) Rational interleukin 2 therapy for HIV positive individuals: daily low doses enhance immune function without toxicity. Proc. Natl Acad. Sci. USA 93:10405.[Abstract/Free Full Text]
  11. Bechard DE, Gudas SA, Sholley MM, et al. (1989) Nonspecific cytotoxicity of recombinant interleukin-2 activated lymphocytes. Am. J. Med. Sci. 298:28.[Web of Science][Medline]
  12. Vial T and Descotes J. (1995) Clinical toxicity of cytokines used as haemopoietic growth factors. Drug Saf. 13:371.[Web of Science][Medline]
  13. Nakajima T, Schulte S, Warrington KJ, et al. (2002) T-cell-mediated lysis of endothelial cells in acute coronary syndromes. Circulation 105:570.[Abstract/Free Full Text]
  14. Rafi AQ, Zeytun A, Bradley MJ, et al. (1998) Evidence for the involvement of Fas ligand and perforin in the induction of vascular leak syndrome. J. Immunol. 161:3077.[Abstract/Free Full Text]
  15. Rafi-Janajreh AQ, Chen D, Schmits R, et al. (1999) Evidence for the involvement of CD44 in endothelial cell injury and induction of vascular leak syndrome by IL-2. J. Immunol. 163:1619.[Abstract/Free Full Text]
  16. Yoneda O, Imai T, Goda S, et al. (2000) Fractalkine-mediated endothelial cell injury by NK cells. J. Immunol. 164:4055.[Abstract/Free Full Text]
  17. McKallip RJ, Fisher M, Do Y, et al. (2003) Targeted deletion of CD44v7 exon leads to decreased endothelial cell injury but not tumor cell killing mediated by IL-2 activated cytolytic lymphocytes. J. Biol. Chem. 44:43818.
  18. Scheffold A, Huhn J, Hofer T. (2005) Regulation of CD4(+)CD25(+) regulatory T cell activity: it takes (IL-)two to tango. Eur. J. Immunol. 35:1336.[CrossRef][Web of Science][Medline]
  19. Lindley S, Dayan CM, Bishop A, Roep BO, Peakman M, Tree TI. (2005) Defective suppressor function in CD4(+)CD25(+) T-cells from patients with type 1 diabetes. Diabetes 54:92.[Abstract/Free Full Text]
  20. Eggena MP, Barugahare B, Jones N, et al. (2005) Depletion of regulatory T cells in HIV infection is associated with immune activation. J. Immunol. 174:4407.[Abstract/Free Full Text]
  21. Balandina A, Lecart S, Dartevelle P, Saoudi A, Berrih-Aknin S. (2005) Functional defect of regulatory CD4(+)CD25+ T cells in the thymus of patients with autoimmune myasthenia gravis. Blood 105:735.[Abstract/Free Full Text]
  22. Thornton AM and Shevach EM. (1998) CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J. Exp. Med. 188:287.[Abstract/Free Full Text]
  23. Shevach EM, McHugh RS, Piccirillo CA, Thornton AM. (2001) Control of T-cell activation by CD4+ CD25+ suppressor T cells. Immunol. Rev. 182:58.[CrossRef][Web of Science][Medline]
  24. Earle KE, Tang Q, Zhou X, et al. (2005) In vitro expanded human CD4+CD25+ regulatory T cells suppress effector T cell proliferation. Clin. Immunol. 115:3.[Web of Science][Medline]
  25. Cosmi L, Liotta F, Angeli R, et al. (2004) Th2 cells are less susceptible than Th1 cells to the suppressive activity of CD25+ regulatory thymocytes because of their responsiveness to different cytokines. Blood 103:3117.[Abstract/Free Full Text]
  26. Kidd P. (2003) Th1/Th2 balance: the hypothesis, its limitations, and implications for health and disease. Altern. Med. Rev. 8:223.[Medline]
  27. Leipner C, Grun K, Schneider I, Gluck B, Sigusch HH, Stelzner A. (2004) Coxsackievirus B3-induced myocarditis: differences in the immune response of C57BL/6 and Balb/c mice. Med. Microbiol. Immunol. (Berl) 193:141.[CrossRef][Medline]
  28. Ulett GC, Ketheesan N, Hirst RG. (2000) Cytokine gene expression in innately susceptible BALB/c mice and relatively resistant C57BL/6 mice during infection with virulent. Burkholderia pseudomallei. Infect. Immun. 68:2034.
  29. Biedermann T, Zimmermann S, Himmelrich H, et al. (2001) IL-4 instructs TH1 responses and resistance to Leishmania major in susceptible BALB/c mice. Nat. Immunol. 2:1054.[CrossRef][Web of Science][Medline]
  30. Udaka K. (1970) Simple method for quantification of enhanced vascular permeability. Proc. Soc. Exp. Biol. Med. 133:1384.[CrossRef][Medline]
  31. Mustafa A, McKallip RJ, Fisher M, Duncan R, Nagarkatti PS, Nagarkatti M. (2002) Regulation of interleukin-2-induced vascular leak syndrome by targeting CD44 using hyaluronic acid and anti-CD44 antibodies. J. Immunother. 25:476.[CrossRef][Medline]
  32. Li L, Elliott JF, Mosmann TR. (1994) IL-10 inhibits cytokine production, vascular leakage, and swelling during T helper 1 cell-induced delayed-type hypersensitivity. J. Immunol. 153:3967.[Abstract]
  33. McKee AS and Pearce EJ. (2004) CD25+CD4+ cells contribute to Th2 polarization during helminth infection by suppressing Th1 response development. J. Immunol. 173:1224.[Abstract/Free Full Text]
  34. Assier E, Jullien V, Lefort J, et al. (2004) NK cells and polymorphonuclear neutrophils are both critical for IL-2-induced pulmonary vascular leak syndrome. J. Immunol. 172:7661.[Abstract/Free Full Text]
  35. Wahl SM, Vazquez N, Chen W. (2004) Regulatory T cells and transcription factors: gatekeepers in allergic inflammation. Curr. Opin. Immunol. 16:768.[CrossRef][Web of Science][Medline]
  36. Dorner BG, Scheffold A, Rolph MS, et al. (2002) MIP-1alpha, MIP-1beta, RANTES, and ATAC/lymphotactin function together with IFN-gamma as type 1 cytokines. Proc. Natl Acad. Sci. USA 99:6181.[Abstract/Free Full Text]
  37. Karlsson Faresjo MG and Ludvigsson J. (2005) Diminished Th1-like response to autoantigens in children with a high risk of developing type 1 diabetes. Scand. J. Immunol. 61:173.[CrossRef][Web of Science][Medline]
  38. Ohmura K, Nguyen LT, Locksley RM, Mathis D, Benoist C. (2005) Interleukin-4 can be a key positive regulator of inflammatory arthritis. Arthritis Rheum. 52:1866.[CrossRef][Web of Science][Medline]
  39. Sospedra M and Martin R. (2005) Immunology of multiple sclerosis. Annu. Rev. Immunol. 23:683.[CrossRef][Web of Science][Medline]
  40. Kusuda T, Shigemasa K, Arihiro K, Fujii T, Nagai N, Ohama K. (2005) Relative expression levels of Th1 and Th2 cytokine mRNA are independent prognostic factors in patients with ovarian cancer. Oncol. Rep. 13:1153.[Web of Science][Medline]
  41. Fontenot JD and Rudensky AY. (2005) A well adapted regulatory contrivance: regulatory T cell development and the forkhead family transcription factor Foxp3. Nat. Immunol. 6:331.[CrossRef][Web of Science][Medline]
  42. Paust S and Cantor H. (2005) Regulatory T cells and autoimmune disease. Immunol. Rev. 204:195.[CrossRef][Web of Science][Medline]
  43. Thornton AM and Shevach EM. (2000) Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific. J. Immunol. 164:183.[Abstract/Free Full Text]
  44. Kuhn R, Lohler J, Rennick D, Rajewsky K, Muller W. (1993) Interleukin-10-deficient mice develop chronic enterocolitis. Cell 75:263.[CrossRef][Web of Science][Medline]
  45. Maloy KJ, Salaun L, Cahill R, Dougan G, Saunders NJ, Powrie F. (2003) CD4+CD25+ T(R) cells suppress innate immune pathology through cytokine-dependent mechanisms. J. Exp. Med. 197:111.[Abstract/Free Full Text]
  46. Nakamura K, Kitani A, Fuss I, et al. (2004) TGF-beta 1 plays an important role in the mechanism of CD4+CD25+ regulatory T cell activity in both humans and mice. J. Immunol. 172:834.[Abstract/Free Full Text]
  47. Golgher D, Jones E, Powrie F, Elliott T, Gallimore A. (2002) Depletion of CD25+ regulatory cells uncovers immune responses to shared murine tumor rejection antigens. Eur. J. Immunol. 32:3267.[CrossRef][Web of Science][Medline]
  48. Onizuka S, Tawara I, Shimizu J, Sakaguchi S, Fujita T, Nakayama E. (1999) Tumor rejection by in vivo administration of anti-CD25 (interleukin-2 receptor alpha) monoclonal antibody. Cancer Res. 59:3128.[Abstract/Free Full Text]
  49. Shimizu J, Yamazaki S, Sakaguchi S. (1999) Induction of tumor immunity by removing CD25+CD4+ T cells: a common basis between tumor immunity and autoimmunity. J. Immunol. 163:5211.[Abstract/Free Full Text]
  50. Nagai H, Horikawa T, Hara I, et al. (2004) In vivo elimination of CD25+ regulatory T cells leads to tumor rejection of B16F10 melanoma, when combined with interleukin-12 gene transfer. Exp. Dermatol. 13:613.[CrossRef][Web of Science][Medline]
  51. Javia LR and Rosenberg SA. (2003) CD4+CD25+ suppressor lymphocytes in the circulation of patients immunized against melanoma antigens. J. Immunother. 26:85.[CrossRef][Web of Science][Medline]
  52. Viguier M, Lemaitre F, Verola O, et al. (2004) Foxp3 expressing CD4+CD25(high) regulatory T cells are overrepresented in human metastatic melanoma lymph nodes and inhibit the function of infiltrating T cells. J. Immunol. 173:1444.[Abstract/Free Full Text]
  53. Quan WD Jr,, Khan N, Ramirez M, et al. (2005) Correlation between development of pulmonary edema and response of pulmonary metastases of metastatic melanoma and kidney cancer to high-dose continuous-infusion interleukin-2. Cancer Biother. Radiopharm. 20:151.[CrossRef][Web of Science][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Clin. Cancer Res.Home page
T. Kottke, J. Thompson, R. M. Diaz, J. Pulido, C. Willmon, M. Coffey, P. Selby, A. Melcher, K. Harrington, and R. G. Vile
Improved Systemic Delivery of Oncolytic Reovirus to Established Tumors Using Preconditioning with Cyclophosphamide-Mediated Treg Modulation and Interleukin-2
Clin. Cancer Res., January 15, 2009; 15(2): 561 - 569.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
H. Guan, P. S. Nagarkatti, and M. Nagarkatti
Blockade of Hyaluronan Inhibits IL-2-Induced Vascular Leak Syndrome and Maintains Effectiveness of IL-2 Treatment for Metastatic Melanoma
J. Immunol., September 15, 2007; 179(6): 3715 - 3723.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
18/10/1461    most recent
dxl079v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (2)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Melencio, L.
Right arrow Articles by Nagarkatti, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Melencio, L.
Right arrow Articles by Nagarkatti, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?