The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Related articles in The JI
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kasprowicz, D. J.
Right arrow Articles by Ziegler, S. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kasprowicz, D. J.
Right arrow Articles by Ziegler, S. F.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Substance via MeSH
The Journal of Immunology, 2003, 171: 1216-1223.
Copyright © 2003 by The American Association of Immunologists

Scurfin (FoxP3) Controls T-Dependent Immune Responses In Vivo Through Regulation of CD4+ T Cell Effector Function 1

Deborah J. Kasprowicz, P. Scott Smallwood, Aaron J. Tyznik and Steven F. Ziegler2

Benaroya Research Institute at Virginia Mason, Seattle, WA 98101


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Scurfin, the protein product of the FoxP3 gene, is a forkhead-family transcription factor that negatively regulates T cell function. Mice carrying a loss-of-function mutation in FoxP3 (scurfy mice) present with fatal autoimmune-like disease caused by hyperresponsive CD4+ T cells. Mice that overexpress scurfin (FoxP3 Tg mice) possess fewer mature T cells with reduced functional capabilities compared with normal littermate control mice. We analyzed the ability of CD4+ T cells and B cells from FoxP3 Tg mice to respond to a T-dependent Ag and found that immunized FoxP3 Tg mice displayed reduced total and Ag-specific serum Ig and disorganized splenic architecture. However, when cultured in vitro, FoxP3 Tg B cells responded normally, suggesting that the poor Ab response was a result of defective T cell help in vivo. When challenged, CD4+ T cells from FoxP3 Tg mice display reduced up-regulation of CD40 ligand and fewer IFN-{gamma}-producing cells. Overall, these findings show that overexpression of scurfin reduces T cell responses in vivo such that CD4+ T cells cannot provide help to B cells during a T cell-dependent Ab response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mutations in the X-linked FoxP3 gene in scurfy mice leads to an autoimmune disease characterized by multiorgan lymphocytic infiltration that results in enlarged spleen, lymph nodes, and liver, as well as dermatitis and severe runting (1, 2, 3, 4, 5, 6, 7). In humans, mutations in FoxP3 have been linked to autoimmune diseases including X-linked autoimmunity allergic dysregulation syndrome (8), X-linked autoimmunity immunodeficiency syndrome (Refs. 9 and 10), and X-linked syndrome (immune dysfunction, polyendocrinopathy, enteropathy, X-linked; Ref. 11). In mice, disease is characterized by hyperresponsive CD4+ T cells that secrete large amounts of a broad range of cytokines and constitutively express activation markers including CD44, CD69, and CD25 (5, 12). Adoptive transfer of scurfy mutant CD4+ T cells to syngeneic recipients is sufficient to transfer disease (12), while depletion of CD4+ T cells prevents disease development (6). Mice typically succumb to disease within 3–4 wk of age, most likely due to the excess of inflammatory cytokines being produced. In this respect, scurfy mice resemble CTLA-4 null or TGF-{beta} null mice (13, 14, 15, 16, 17). Scurfin, the protein product of FoxP3, is a member of the forkhead/winged-helix family of transcriptional regulators (12). Recently, Schubert et al. (18) showed that scurfin acts as a transcriptional repressor, suggesting that scurfin is a negative regulator of T cell activation/function.

The scurfy mutation in mice results from a 2-bp insertion in the FoxP3 gene that generates a premature stop codon and a truncated protein product. Expression of a wild-type FoxP3 transgene prevents the development of disease in scurfy mutant animals (12). However, overexpression of wild-type scurfin in otherwise normal mice confers a dose-dependent decrease in CD4+ and CD8+ T cell numbers in peripheral lymphoid organs. In addition, CD4+ T cells display reduced proliferative capacity and cytokine production in response to stimulation (19). The severe suppression of T cell function in FoxP3 transgenic (Tg) 3 mice further supports a negative regulatory role for scurfin in normal T cells. Contrary to the decrease in T cells in FoxP3 Tg mice, B cell numbers are marginally increased. However, the ability of either T or B cells from these mice to respond to immunologic challenge has not been addressed in vivo.

A T cell-dependent (TD) Ab response requires the participation of both CD4+ T cells and B cells. Initially the B cell is stimulated through engagement of the B cell receptor with further signals received through cytokine receptors and CD40 (reviewed in Ref. 20, 21). These signals culminate in an Ab response that is classified as either Th1-dependent when Th1 cells secrete IFN-{gamma} and induce B cells to produce IgM and IgG2a or Th2-dependent when Th2 cells secrete IL-4 and induce B cells to produce IgM, IgG1, and IgE (21, 22, 23). In the present study, we show that FoxP3 Tg mice fail to mount an effective Th1- or Th2-dependent Ab response when immunized with the T-dependent Ag trinitrophenol-keyhole limpet hemocyanin (TNP-KLH). When examined more closely, B cells from the FoxP3 Tg mice displayed normal Ab responses to polyclonal stimulation in vitro. Likewise, they were indistinguishable from normal littermate control (NLC) B cells when cocultured with NLC T cells and rechallenged with Ag, in vitro. In contrast, neither FoxP3 nor NLC B cells responded when cultured under similar conditions with FoxP3 Tg T cells from immunized FoxP3 mice in vitro. Thus, overexpression of the transcriptional repressor scurfin results in the inability to mount a humoral immune response due to a failure to generate appropriate effector T cells.


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

FoxP3 Tg mice on the C57BL/6 background (19) were obtained from CellTech R&D (Bothell, WA) and maintained at the Benaroya Research Institute animal facility under specific pathogen-free conditions with autoclaved food and bedding. Mice were phenotyped for the presence of the transgene using DNA obtained from the tail and PCR with primers specific to the transgene. Mice were used at 7–8 wk of age. Mice were maintained and studies were conducted according to internal Animal Care and Usage Committee guidelines. Because no gender differences were noted in FoxP3 Tg mice, both male and female mice were used for experiments.

Reagents and Abs

The following reagents were purchased from Sigma-Aldrich (St. Louis, MO): 2,4,6-trinitrobenzenesulfonic acid and chicken OVA. KLH was obtained from Calbiochem (La Jolla, CA). The TNP derivatives of KLH (TNP25-KLH) and OVA (TNP20-OVA) were prepared as described previously (24). Recombinant mouse IL-2, IL-4, and IFN-{gamma} were purchased from BD PharMingen (San Diego, CA). We determined cell populations using the following surface markers: CD3-FITC (clone 145-2C11), CD4-PE or Tri-Color (clone L3T4), CD8-Tri-Color or allophycocyanin (clone CT-CD84), B220-PE (clone RA3-6B2), F4/80-Tri-Color (for macrophages and dendritic cells, clone CI:A3-1), CD154 (CD40 ligand (L))-biotin (clone MR-1), CD69-FITC (clone H1.2F3), CD25-FITC (clone 7D4), and PE-conjugated-streptavidin.

Western blot analysis

Spleens and lymph nodes were isolated from NLC and FoxP3 Tg mice and single cell suspensions were generated. For analysis of total scurfin expression, cells were lysed directly in gel sample buffer. To analyze expression of scurfin in CD4+ T cells or B220+ B cells, splenocytes were labeled with Abs to CD4-Tri-Color and B220-PE and individual cell populations were isolated by cell sorting using a FACSVantage flow cytometer (BD Biosciences, San Jose, CA) and lysed in sample buffer. Lysates representing 1 x 106 cells were separated on SDS-PAGE gels, transferred to nylon filters, and probed with a rabbit polyclonal antisera raised against mouse scurfin. The filters were stripped and reprobed with antisera against extracellular signal-regulated kinase 1 and 2.

In vivo immunization strategy and serum isolation

Seven to 8-wk-old mice were bled via the retro-orbital vein before immunization to obtain steady state levels of serum Ig. Mice were immunized i.p. with TNP25-KLH (100 µg/mouse) in CFA at week 0 and with TNP25-KLH in IFA at weeks 6 and 12. Mice were bled every 7–10 days throughout the course of the experiment. After the final boost, mice were sacrificed and lymphoid organs were removed for cellular and histological characterization.

ELISA for the detection of in vitro-secreted or in vivo serum Ab

B cell supernatants were collected and immediately frozen at -80°C and serum was frozen at -20°C until analysis by ELISA as described below. Ninety-six-well, flexible round-bottom microtiter plates (secreted Ab; Dynatech Laboratories, Chantilly, VA) or flat-bottom microtiter plates (serum Ab; Fisher Scientific, Los Angeles, CA) were coated with 2 µg/ml of either goat anti-mouse Ig or IgG or 100 µg/ml TNP-KLH in PBS/azide overnight. Plates were washed with PBS and blocked with PBS/1% BSA at 37°C for 1 h. Serum samples were diluted in complete RPMI 1640 (cRPMI 1640) from 1/100 to 1/24,000 depending on the isotype assayed and six serial dilutions were assayed for each individual serum sample collected. Twenty microliters of Ab-containing supernatants or standards or 100 µl of serum were added to each well. Plates were incubated for 2 h (secreted Ab and serum IgM) at 37°C or overnight (serum IgG1 and IgG2a) in a humidified atmosphere and washed with PBS/0.05% Tween 20. A 1 µg/ml solution of alkaline phosphatase-conjugated goat anti-mouse IgM, IgG1, or IgG2a was added to each well. After 1 h at 37°C, plates were washed with PBS/0.05% Tween 20, and a developing solution consisting of 1 mg/ml p-nitrophenyl phosphate in 10 mM diethanolamine/0.5 mM MgCl2 buffer, pH 9.4, was added to each well. A standard curve for IgM, IgG1, and IgG2a Ab was prepared using known quantities of the myeloma proteins MOPC-104E, MOPC-21, and UPC-10, respectively, and the curve detected on a plate coated with 2 µg/ml goat anti-mouse Ig or IgG. The absorbance at 405 nm was determined on a UVmax kinetic microplate reader. The OD reading for each isotype that was within the linear portion of the standard curve was converted to the nanograms per milliliter amount of Ab and plotted. The lower limits of detection were as follows: IgM, <=2 ng/ml; IgG1, <=4 ng/ml; and IgG2a, <=1 ng/ml.

Isolation of small, dense resting B cells

Small, dense resting B cells were isolated from the spleens of unimmunized mice as previously described (25, 26). The small high-density resting B cells were collected from a Percoll density gradient at the interface of the 1.082 and 1.097 g/ml layers. This population contained ~90–95% B220+ B cells.

LPS culture conditions

Resting small, dense resting B cells were cultured at 5 x 104 cells/well in a flat-bottom 96-well plate in cRPMI 1640 ± LPS (5–50 µg/ml) in a final volume of 0.15 ml in a humidified atmosphere of 5% CO2 in air at 37°C. Some wells also received either mouse recombinant IL-4 (1 ng/ml) or IFN-{gamma} (1 ng/ml) at the initiation of culture. After 8 days, supernatants were collected and analyzed for the amount of IgM, IgG1, and IgG2a by ELISA.

T cell/B cell coculture conditions

B cells were isolated from TNP-KLH immunized mice as described above. T cells were enriched from TNP-KLH-immunized mouse lymph nodes using panning on anti-mouse Ig-coated plates (Southern Biotechnology Associates, Birmingham, AL) to remove B cells and complement-mediated lysis of CD8+ T cells. Cell populations were 85–95% pure. Each cell type (5 x 104) was plated in a flat-bottom 96-well plate with TNP-KLH (1.0 µg/ml) in cRPMI 1640 at a final volume of 0.15 ml. Cells were incubated for 8 days in a humidified atmosphere of 5% CO2 in air at 37°C. Supernatants were collected and analyzed for the amount of IgM, IgG1, and IgG2a by ELISA.

Reconstitution of RAG2-/- mice

Splenic CD4+ T cells from FoxP3 Tg and NLC mice were obtained by sort purification on a FACSVantage flow cytometer (BD Biosciences). B cells were isolated by complement-mediated lysis of T cells as described above. CD4+ T cells (1 x 106) and 2 x 106 B220+ B cells resuspended in PBS were adoptively transferred to 8-wk-old RAG2-/- mice via the tail vein. Mice were bled before and 1 wk following cell transfer for basal Ig levels. One week postcell transfer, mice were immunized i.p. with 100 µg of TNP25-KLH/CFA (150 µl/mouse) and after 6 wk with 100 µg of TNP25-KLH/IFA (150 µl/mouse). Mice were bled weekly following both primary and secondary immunizations.

Immunofluorescence staining and flow cytometric analysis of T and B cells

Each Ab was titrated to determine the optimal staining concentration for maximum signal. After 30 min, the cells were washed in balanced salt solution/FCS/azide. Cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences) gated on all viable cells. The FACSCalibur was calibrated daily using Rainbow Calibration Particles (Sherotech, Libertyville, IL). Data were analyzed using CellQuest software (BD Biosciences).

Intracellular cytokine staining

CD4+ T cells from FoxP3 Tg and NLC mice immunized as described above were isolated 48 h following secondary challenge. Cells were restimulated with PMA (5 µg/ml) and ionomycin (250 ng/ml) in cRPMI 1640 in the presence of brefeldin A (10 µg/ml). After 6 h, cells were stained for CD4 surface expression. Cells were washed in PBS, fixed in 2% paraformaldehyde for 20 min at room temperature, and then washed in PBS. Cells were permeabilized and stained in the presence of PBS/BSA (1%)/saponin (0.5%). Cells were incubated with each of the following Abs (1 µg/1 x 106 cells) for 30 min at room temperature: biotin-conjugated anti-mouse IFN-{gamma} (clone XMG1.2; BD PharMingen), allophycocyanin-conjugated streptavidin (Caltag Laboratories, Burlingame, CA), and PE-conjugated anti-mouse IL-4 (clone BVD4-1D11; BD PharMingen). Cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences) and data were analyzed using CellQuest software (BD Biosciences).

Statistics

Data were analyzed using the Statview statistics program (Cary, NC). A one-way ANOVA was initially performed to determine whether an overall statistically significant change existed before using the two-tailed unpaired Student’s t test. Statistically significant differences were reported when the p value was <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mice overexpressing FoxP3 have reduced serum Ig

As previously reported, the absence of scurfin in mice and humans results in chronic T cell activation and development of fatal autoimmune lymphoproliferative disease (1, 2, 3, 4, 5, 6, 7). In contrast, overexpression of scurfin in mice results in a reduced T lymphocyte population and diminished T cell function (19). FoxP3 Tg mice have a 25–50% and 50–75% reduction in CD4+ and CD8+ T cell numbers, respectively, and a concurrent increase in the B cell compartment when compared with NLC mice (Table I and Ref. 19). As shown in Fig. 1, scurfin protein overexpression in FoxP3 Tg mice is apparent in both total spleen and lymph node lysates. Scurfin protein is expressed at its highest amount in CD4+ T cells from both NLC and FoxP3 Tg mice with protein also detectable in B cells. In the present study, we first determined baseline serum Ig levels in FoxP3 Tg and NLC mice. Serum from 7- to 8-wk-old unimmunized FoxP3 Tg and NLC mice was obtained and analyzed for total and TNP-specific IgM, IgG1, and IgG2a by ELISA. Unimmunized FoxP3 Tg mice displayed a 2-fold reduction in serum IgG1 and 5-fold reduction in IgG2a, with no change in basal IgM, in comparison to NLC (Fig. 2). Neither FoxP3 Tg nor NLC mice displayed TNP-specific IgM, IgG1, or IgG2a (data not shown). Overall, these findings suggest that FoxP3 Tg mice were unable to maintain "wild-type" levels of steady state serum Ab of isotypes requiring T cell help. However, it was unclear whether the defect in serum Ab levels was due to a defect in the T cell or B cell compartment.


View this table:
[in this window]
[in a new window]
  		Table I. CD4+ T and B cell populations in FoxP3 Tg and NLC mice pre- and postimmunization with a T-dependent Ag

 


View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 1. Scurfin expression in NLC and FoxP3 Tg mice. Lysates were prepared from total spleen (A), total lymph node (B), CD4+ T cells from spleen (C), and B220+ B cells from spleen (D). Lysates were separated by SDS-PAGE (106 cells/lane), and scurfin levels were measured by immunoblotting using a rabbit polyclonal antisera raised against mouse scurfin. Representative of four FoxP3 Tg and four NLC mice examined.

 


View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 2. Reduced serum Ig in unimmunized FoxP3 Tg mice at 7 wk of age. Serum was obtained from 7-wk-old FoxP3 Tg and NLC mice and analyzed for total IgM, IgG1, and IgG2a by ELISA. Each symbol represents an individual mouse. At least 20 different serum samples were analyzed for each mouse strain with the mean value represented by a solid dash. Insets, Mean values are presented as a bar graph. *, p < 0.001.

 
Decreased T-dependent Ab response in FoxP3 Tg mice in vivo

We next determined whether serum Ab levels would be increased in FoxP3 Tg mice following antigenic challenge with a T-dependent Ag in vivo. Mice were immunized i.p. with TNP-KLH in CFA and then boosted at 6 wk with TNP-KLH in IFA. Serum samples were obtained every 10 days throughout the immunization period and were analyzed simultaneously for total and TNP-specific IgM, IgG1, and IgG2a by ELISA. As shown in Fig. 3, total serum IgM was similar between FoxP3 Tg and NLC mice, while TNP-specific IgM in FoxP3 Tg mice was significantly reduced up to 2-fold following both primary and secondary immunizations. Although the FoxP3 Tg mice showed a higher Ag-specific IgM response to secondary challenge than primary challenge, this response quickly returned to the preboosting level, suggesting a possible defect in the memory response. We observed a greater decrease in the IgG1 and IgG2a responses in FoxP3 Tg mice compared with IgM. Total IgG1 was modestly reduced, but TNP-specific IgG1 was significantly reduced by up to 4-fold in FoxP3 Tg mice when compared with the NLC. As with the IgM response, TNP-specific IgG1 levels were increased immediately following the second immunization, but the increase was not sustained and Ag-specific Ig levels rapidly decreased. Lastly, both total and TNP-specific IgG2a were severely reduced, up to 10-fold in both the primary and secondary immune response to TNP-KLH. FoxP3 Tg mice displayed a poor response to the primary immunization with almost undetectable IgG2a during the first 2 wk of the response and a weak response to the secondary challenge in comparison to the NLC.



View larger version (35K):
[in this window]
[in a new window]
  		FIGURE 3. Primary and secondary immune response of FoxP3 Tg and NLC mice immunized with the TD Ag TNP-KLH. FoxP3 Tg and NLC mice were bled on day 0 to obtain a baseline level of serum IgM, IgG1, and IgG2a. Mice were immunized i.p. with 100 µg of TNP-KLH/CFA (150 µl/mouse) and after 6 wk with 100 µg of TNP-KLH/IFA (150 µl/mouse). Serum was obtained by retro-orbital eye-bleed every 10 days following the primary and secondary immunizations. Arrows indicate TNP-KLH challenges. After the final serum isolation, all serum samples were analyzed for total (left panels) and TNP-specific (right panels) IgM, IgG1, and IgG2a by ELISA. Data are presented as the mean micrograms per milliliter (IgM and IgG1) or nanograms per milliliter (IgG2a) ± SE from four to five mice per group. *, p < 0.05.

 
Further analysis showed no difference in the avidity of the Ab produced by FoxP3 Tg mice vs NLC mice (data not shown), suggesting that B cells from FoxP3 Tg mice produce normal affinity Ab, but in reduced amounts. Finally, we found that splenic architecture in immunized FoxP3 Tg mice differed from NLC mice (data not shown). NLC spleens contained fairly uniform, evenly distributed B cell follicles. In contrast, FoxP3 Tg spleens contained a disorganized distribution of small B cell follicles with multiple follicles merging into one another (data not shown), suggesting that another consequence of scurfin overexpression in T cells from FoxP3 Tg mice is a disorganized splenic architecture. Lastly, when we examined T and B cell populations in FoxP3 Tg and NLC mice following immunization, we found that the number of splenic B cells in FoxP3 Tg was similar to NLC mice (Table I). CD4+ T cell numbers were reduced ~2-fold in both FoxP3 Tg and NLC mice compared with unimmunized mice with FoxP3 Tg CD4+ T cells expressing lower levels of CD62L and CD44 both pre- and postimmunization, but higher CD45RB postimmunization compared with NLC mice. In addition, there were minor changes in B cell populations, including IgM+, IgDhigh, and CD62Llow, that may suggest poor generation of memory B cells in FoxP3 Tg mice. In summary, these data show that FoxP3 Tg mice respond poorly to immunologic challenge failing to generate Ag-specific Abs efficiently in vivo.

Normal Ab response by FoxP3 Tg B cells in vitro

The results in the previous section could be explained by either defective T cells or B cells in the immunized FoxP3 Tg mice. To examine B cell function, we first determined whether B cells isolated from FoxP3 Tg mice were capable of responding to stimulus in vitro. Splenic B cells from FoxP3 Tg and NLC mice were purified and their ability to produce Ig following LPS stimulation was measured. Purified B cells were cultured with LPS for 8 days and then cell supernatants were analyzed for IgM, IgG1, and IgG2a. FoxP3 Tg B cells produced equivalent amounts of IgM, IgG1, and IgG2a compared with NLC cultures (Fig. 4A, left panels). We also determined the ability of IL-4 or IFN-{gamma} to enhance Ab production by B cells from FoxP3 Tg mice. Although IgG1 and IgG2a production by B cells was enhanced by the addition of IL-4 (Fig. 4A, middle panels) or IFN-{gamma} (Fig. 4A, right panels), respectively, there was no difference between the amount of Ab produced by B cells from FoxP3 Tg and NLC mice.



View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 4. Ab production by FoxP3 Tg B cells stimulated in vitro. Splenic B cells were enriched from either FoxP3 Tg or NLC mice by complement-mediated lysis of T cells. A, B cells (1 x 105) were plated in flat-bottom 96-well plated in cRPMI 1640 ± LPS (5 or 50 µg/ml) in a final volume of 150 µl. Some cultures also received either IL-4 (1 ng/ml, middle panels) or IFN-{gamma} (1 ng/ml, right panels). After 8 days of culture, supernatants were collected and analyzed by ELISA for total IgM, IgG1 and IgG2a. Data are presented as the mean nanograms per milliliter ± SE from triplicate wells and are representative of three separate experiments. There was no significant difference between FoxP3 Tg and NLC samples. B, Splenic B cells and lymph node CD4+ T cells from TNP-KLH immunized FoxP3 Tg and NLC mice were isolated as described in Materials and Methods. Four different conditions were assayed: 1) NLC B + CD4+ cells; 2) NLC B + FoxP3 Tg CD4+ cells; 3) FoxP3 Tg B + NLC CD4+ cells; and 4) FoxP3 Tg B + CD4+ cells. Cells (5 x 104) of each type were plated with 1 µg/ml TNP-KLH in cRPMI 1640 per well in flat-bottom 96-well plates to a final volume of 150 µl. After 8 days, supernatants were collected and analyzed for IgM, IgG1, and IgG2a by ELISA. Data are presented as the percent of the NLC/NLC response ± SE from a combination of three separate experiments. *, p < 0.05 or greater (as denoted) when compared with NLC B + CD4+ T cell cultures.

 
As a final test of the ability of Ab production by B cells from FoxP3 Tg mice, we isolated splenic CD4+ T cells and B cells from TNP-KLH-immunized FoxP3 Tg and NLC mice. We cultured FoxP3 Tg or NLC B cells with equivalent numbers of either FoxP3 Tg or NLC CD4+ T cells in the presence of TNP-KLH. If the defect in FoxP3 Tg mice resides in the inability of CD4+ T cells to provide adequate help, then we would expect both NLC and FoxP3 B cells to have lowered responses when cultured with FoxP3 Tg T cells. Similarly, if B cells from FoxP3 mice function normally, then coculture with NLC T cells should result in levels of Ag-specific Ab equivalent to NLC B cells. We found that NLC and FoxP3 Tg B cells, when cultured with TNP-KLH in the presence of NLC CD4+ T cells, produced similar levels of IgM, IgG1, and IgG2a (Fig. 4B). However, when either population of B cells was cultured with FoxP3 Tg CD4+ T cells, Ab levels were reduced 2- to 4-fold in comparison to the same cells cultured with NLC T cells, suggesting that the poor Ab response in immunized FoxP3 Tg mice is due to defective T cells, but not B cells. Lastly, IgM production was not significantly different whether B cells were cultured with either NLC or FoxP3 Tg T cells, suggesting that the IgM response was less dependent on the CD4+ T cells. Overall, these findings suggest that B cell Ab production is not intrinsically impaired in B cells that overexpress scurfin and that CD4+ T cells from FoxP3 Tg mice provide inadequate help to both NLC and FoxP3 Tg B cells during and immune response in vitro.

B cells from NLC mice generate a defective Ab response when adoptively transferred into RAG2-/- mice with FoxP3 Tg CD4+ T cells

As described above, these findings suggest that the inability of FoxP3 Tg mice to mount a humoral immune response is due to a defect in T cell function when scurfin is overexpressed. This interpretation is consistent with previous work showing that T cells from FoxP3 mice performed poorly when stimulated in vitro (7). There are at least two possible mechanisms by which FoxP3 Tg T cells could cause a poor Ig response in FoxP3 Tg mice. The first possibility is that T cell function is similar in FoxP3 Tg and NLC mice in vivo, and that the observed defect is caused by the severely reduced T cell numbers seen in FoxP3 Tg mice. The second possibility is that FoxP3 Tg T cells are defective in T cell help and, even if cell numbers were equivalent between mice, FoxP3 Tg T cells would not provide equivalent help to NLC B cells. To address these two possibilities, we purified CD4+ T cells from FoxP3 Tg and NLC mice and B220+ cells from NLC mice, adoptively transferred equal numbers of either FoxP3 Tg or NLC CD4+ T cells and B cells to syngeneic RAG2-/--deficient hosts, allowed these cells to home, and then challenged the mice with TNP-KLH/CFA. We found that RAG2-/- mice that received NLC CD4+ T and B cells responded to immunologic challenge by producing significant amounts of both total and Ag-specific IgM, IgG1, and IgG2a (Fig. 5). In contrast, mice that received FoxP3 Tg CD4+ T cells andNLC B cells produced significantly reduced amounts of all three isotypes, with the most dramatic effects on IgG1 and IgG2a isotypes. When cell populations were analyzed, we did not find significant differences between the number of either CD4+ or B220+ cells present in the different reconstitution conditions (data not shown), suggesting that the reduced amount of Ab was not due to fewer FoxP3 Tg CD4+ T cells present, but rather to defective help from FoxP3 Tg T cells.



View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 5. Primary and secondary immune response of RAG2-/- mice reconstituted with FoxP3 Tg and NLC B and CD4+ T cells and immunized with TNP-KLH. B220+ and CD4+ T cells were purified from FoxP3 Tg and NLC mice as described in Materials and Methods. B220+ (2 x 106) and 1 x 106 CD4+ T cells were adoptively transferred to RAG2-/- mice via the tail vein. One week postcell transfer, mice were immunized i.p. with 100 µg of TNP-KLH/CFA (150 µl/mouse) and after 6 wk with 100 µg of TNP-KLH/IFA (150 µl/mouse). Serum was obtained and analyzed as described in Fig. 3. Arrows indicate TNP-KLH challenges. Total (left panels) and TNP-specific (right panels) IgM, IgG1, and IgG2a were determined by ELISA. Data are presented as the mean micrograms per milliliter ± SE from four to five mice per strain. *, p < 0.05.

 
Defective CD40L expression and cytokine production by FoxP3 Tg T cells

The primary defect seen in FoxP3 mice in these experiments is the lack of a secondary response to antigenic challenge. Typically, secondary Ab responses involve switching of the Ig isotype, to either IgG1 and IgE in a Th2-like response or IgG2a in a Th1 response (21, 22, 23). This isotype switch is mediated through signals given to Ag-specific B cells by CD4+ T cells. These signals are mediated through both cytokines (IL-4 and IFN-{gamma}), as well as cell-cell contact (CD40-CD40L). To determine where the defect in FoxP3 Tg T cells resided, we first examined the ability of CD4+ T cells from these animals to up-regulate expression of activation markers, including CD40L, when stimulated through the TCR. When resting FoxP3 Tg CD4+ T cells were stimulated with anti-mouse CD3/anti-CD28 Abs in vitro, ~2-fold fewer cells expressed CD40L and CD69 and those that did up-regulate expression expressed a lower level than cells from NLC mice, while CD25 expression was normal (Fig. 6), suggesting that, in addition to reduced proliferation and cytokine production, FoxP3 Tg CD4+ T cells display inefficient up-regulation of activation markers including CD40L and CD69 that are required by B cells for isotype switching and germinal center formation.



View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 6. Activation marker expression on FoxP3 Tg or NLC CD4+ T cells stimulated with anti-CD3/anti-CD28 Ab in vitro. CD4+ splenic T cells from either FoxP3 Tg (bold profile) or NLC mice (filled profile) were stimulated with plate-bound anti-mouse CD3 Ab (clone 2C11, 5 µg/ml) and anti-mouse CD28 (clone 37.51, 5 µg/ml) for 18 h in cRPMI 1640. Cells were collected and stained with biotin-conjugated anti-mouse CD40L Ab and streptavidin-conjugated PE and either FITC-conjugated anti-mouse CD25 or CD69 Ab. Cells were analyzed on a FACSCalibur flow cytometer. Isotype control staining is indicated by dashed line. The percentage of cells is denoted in each histogram with FoxP3 Tg mice percentages in bold and are representative of four to eight individual experiments for each surface marker. The mean percent of CD40L+ cells was 36.4 ± 1.8 for NLC and 26.0 ± 2.2 for FoxP3 Tg mice (p < 0.003) and mean CD40L mean fluorescence intensity was 388 ± 20 for NLC and 267 ± 26 for FoxP3 Tg mice (p < 0.003).

 
In addition to CD40L expression, CD4 T cell-derived cytokines are critical for class switching and subsequent Ab production (20, 21). To determine the ability of T cells from immunized FoxP3 Tg mice to produce cytokines, we isolated CD4+ splenic T cells from FoxP3 Tg and NLC mice 48 h following secondary immunization and determined the number of cytokine-producing cells. Consistent with the inability of immunized FoxP3 mice to mount an effective IgG2a response, we found that 2-fold fewer T cells from these mice produced IFN-{gamma} following in vitro restimulation (Fig. 7). Although the percentage of IL-4-producing cells was similar between the two types of mice, total cell numbers were reduced in FoxP3 Tg mice due to reduced total numbers of CD4+ T cells. These data suggest that in addition to fewer T cells up-regulating CD40L and CD69, fewer IFN-{gamma}- and IL-4-producing cells are also generated in immunized FoxP3 Tg mice. Overall, these findings suggest CD4+ T cells from FoxP3 Tg mice possess multiple defects that are likely to be responsible for the poor response of intact FoxP3 Tg or reconstituted RAG2-/-mice to immunologic challenge in vivo.



View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 7. Cytokine-producing cells generated in FoxP3 Tg and NLC mice following immunization with TNP-KLH. CD4+ T cells from FoxP3 Tg and NLC mice immunized as in Fig. 3 were isolated 48 h following secondary challenge. Cells were restimulated with PMA (5 µg/ml) and ionomycin (250 ng/ml) in cRPMI 1640 in the presence of brefeldin A (10 µg/ml). After 6 h, cells were stained for CD4 surface expression, fixed and permeabilized, then stained with biotin-conjugated anti-mouse IFN-{gamma}, streptavidin-conjugated allophycocyanin, and PE-conjugated anti-mouse IL-4. Cells were analyzed on a FACSCalibur flow cytometer. Data are presented as (A) one representative mouse for each group and (B) the mean number of cytokine-secreting cells from six to eight mice per group. *, p < 0.05 when compared with NLC.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we show that overexpression of scurfin in FoxP3 Tg mice results in the inability to mount an effective humoral immune response. FoxP3 Tg mice displayed lower levels of circulating IgG when compared with their nontransgenic littermates, and had a dramatically reduced response when challenged with a specific Ag. However, B cells from FoxP3 Tg mice produced normal levels of Ig when stimulated in vitro, suggesting that a defect in T cell-derived help was responsible for the phenotype seen in these mice. Three additional lines of evidence support this hypothesis. First, T cells from FoxP3 Tg mice failed to provide help to B cells from NLC mice. Second, CD4+ T cells from FoxP3 Tg mice failed to up-regulate CD40L and CD69 expression when stimulated. Finally, significantly fewer IL-4- and IFN-{gamma}-producing splenic T cells were generated in immunized FoxP3 Tg mice as compared with NLC mice. Overall, these findings suggest that the poor Ag-specific Ab response generated by FoxP3 Tg mice immunized with a TD Ag in vivo is attributable to defective CD4+ T cells.

One novel finding from this study is that properly functioning T cells are required for maintaining normal levels of serum Ig following weaning. We found that by 3 wk postweaning, FoxP3 Tg mice displayed reduced levels of serum IgG1 and IgG2a levels compared with preweaning (Fig. 2 and data not shown), while NLC mice possessed higher levels of Ig at 7 wk compared with levels preweaning. Therefore, FoxP3 Tg mice could not maintain or increase serum Ig levels postweaning. This finding suggests that either soluble- (i.e., cytokine) or surface-mediated signals are required for the maintenance of basal serum Ig levels. Because IgG was more affected than IgM, it is likely that the same signals that are required for IgG switching during an immune response are involved in the maintenance of serum IgG.

Brunkow et al. (12) showed that FoxP3 mRNA expression in B cells from FoxP3 Tg mice was slightly increased compared with NLC mice, but at least 10-fold lower than in FoxP3 Tg CD4+ T cells. In addition herein, we report that scurfin protein expression is also higher in CD4+ T cells and B220+ cells from FoxP3 Tg mice (Fig. 1). However, because FoxP3 Tg B cells generate a normal response in vitro (Fig. 4), it appears that higher expression of scurfin in B cells does not detrimentally affect Ab production by these cells. In addition, because B cells from immunized FoxP3 Tg mice produced wild-type levels of Ab when cultured with NLC CD4+ T cells (Fig. 4B), exposure to FoxP3 Tg CD4+ T cells in vivo does not program FoxP3 Tg B cells with the inability to respond to T cell signals during an immune response. Finally, the IgM response of FoxP3 Tg mice to immunization with a T-independent Ag (TNP-Ficoll) was indistinguishable from NLC mice (data not shown). These data all suggest that the ability of B cells from FoxP3 Tg mice to produce Ab was unaffected. The question then arises as to the mechanism by which the scurfin regulates Ab production in vivo. The data presented herein show that scurfin overexpression affects CD4+ T cell helper function. Therefore, the failure of FoxP3 Tg mice to mount a TD response arises from a defect in the ability of CD4+ T cells in these mice to provide adequate help. Previous studies have reported that FoxP3 Tg CD4+ T cells produce low amounts of cytokine in comparison to NLC when stimulated in vitro (Refs. 12 and 19 , and D. J. Kasprowicz and S. F. Ziegler, unpublished observations) suggesting that these cells may also provide insufficient cytokine help in vivo. Consistent with this, immunized FoxP3 Tg mice possessed fewer IFN-{gamma} and IL-4-positive cells than NLC mice (Fig. 7). However, the percentage of IL-4-positive cells was similar between FoxP3 Tg and NLC mice, suggesting that the defect in IgG1 production may be a result of a defect elsewhere in CD4+ T cell effector function. For example, FoxP3 Tg CD4+ T cells proliferated poorly in response to in vitro stimulation (Ref. 19 and D. J. Kasprowicz and S. F. Ziegler, unpublished observations) suggesting that reduced expansion of CD4+ cells in vivo could also result in fewer cytokine-producing CD4+ T cells available to provide help to B cells. Lastly, it is likely that reduced expression of activation markers (e.g., CD40L and CD69) on CD4+ T cells from FoxP3 Tg mice also detrimentally affects Ab production by B cells in these mice. As shown in Fig. 6, FoxP3 Tg CD4+ T cells displayed fewer positive cells expressing a lower amount of surface CD40L and CD69 expression, as well as lower CD44 expression (Table I and Fig. 6) suggesting a possible defect in effector and memory T cell generation. Taken together, it appears that it is not the mere decrease in total CD4+ T cells in FoxP3 Tg mice that leads to a poor T-dependent Ab response, but rather a culmination of multiple defects in the ability of CD4+ T cells to become functionally activated that results in fewer effector T cells capable of providing sufficient help to the B cells during the immune response.

Many studies have reported that cytokines (e.g., IL-4, IFN-{gamma}) and surface signals (e.g., CD40L) from the CD4+ Th cell are required for B cell differentiation into Ab-secreting cells and for affinity maturation of Ab (20, 27, 28, 29, 30). Moreover, T cell participation via surface-mediated signals has been shown to be important for both the formation and maintenance of germinal centers. Therefore, it is not surprising that we also found poor splenic organization in TNP-KLH immunized FoxP3 Tg mice (data not shown). If FoxP3 Tg T cells cannot provide these signals, then B cells may not generate or maintain germinal centers and in the absence of germinal centers, B cells would not isotype switch and produce IgG isotypes (21). One possible role for scurfin in T cell function comes from recent work demonstrating that it acts as a transcriptional repressor, targeting the composite NF-AT/AP-1 sites found in the regulatory regions of cytokine genes (18). T cells that lack scurfin show constitutive NF-AT and AP-1 activity, while those that overexpress scurfin have reduced activity. Also, NF-AT and AP-1 have also been shown to be important for the expression of CD40L (31) and cytokines (e.g., IL-2, IL-4, IFN-{gamma}; Ref. 32, 33, 34), which may explain the poor induction of CD40L and cytokines in T cells from the FoxP3 Tg mice, respectively (Fig. 6 and 7). Taken as a whole, these data point to a lack of developed effector function in the FoxP3 Tg T cells due to an inability to properly express genes important for these functions. Experiments are currently under way to study the regulation of NF-AT and AP-1 activity in T cells from FoxP3 Tg mice.

In summary, we have shown that mice that overexpress the FoxP3 gene have dramatically reduced TD Ab responses, which correlates with defective CD4+ T cell function. These animals have severely reduced T cell numbers, and the T cells present have a diminished capacity to proliferate, produce cytokines, and up-regulate activation markers when stimulated, all factors that could lead to diminished B cell Ab production. These data show that the transcriptional repressor scurfin is critical for normal CD4+ T cell function and for the successful coordination of a normal response to immunological challenge in vivo.


   Acknowledgments
 
We thank Dr. Fred Ramsdell and Sue Anne Yasako for the rabbit antisera against mouse scurfin. We thank Drs. Andy Farr, Steve Levin, and Christopher Wilson for critical reading of the manuscript. We also thank Dr. Farr for assistance with histological techniques, Theingi Aye and Angele Benard for technical assistance, and Karen Aiken for maintenance of the animal facility, and K. "Aru" Arumuganathan for cell sorting.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Grant AI48779 (to S.F.Z.). Back

2 Address correspondence and reprint requests to Dr. Steven F. Ziegler, Benaroya Research Institute at Virginia Mason, Seattle, WA 98101. E-mail address: sziegler{at}vmresearch.org Back

3 Abbreviations used in this paper: Tg, transgenic; TD, T cell-dependent; NLC, normal littermate control; TNP-KLH, trinitrophenol-keyhole limpet hemocyanin; L, ligand; cRPMI 1640, complete RPMI 1640. Back

Received for publication November 5, 2002. Accepted for publication May 22, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Lyons, M. F., J. Peters, P. H. Glenister, S. Ball, E. Wright. 1990. The scurfy mouse mutant has previously unrecognized hematological abnormalities and resembles Wiskott-Aldrich syndrome. Proc. Natl. Acad. Sci. USA 87:2433.[Abstract/Free Full Text]
  2. Godfrey, V. L., J. E. Wilkinson, L. B. Russell. 1991. X-linked lymphoproliferative disease in scurfy (sf) mutant mouse. Am. J. Pathol. 138:1379.[Abstract]
  3. Godfrey, V. L., J. E. Wilkinson, E. M. Rinchik, L. B. Russell. 1991. Fatal lymphoproliferative disease in scurfy (sf) mice requires T cells that mature in a sf thymic environment: potential model for thymic education. Proc. Natl. Acad. Sci. USA 88:5528.[Abstract/Free Full Text]
  4. Godfrey, V. L., B. T. Rouse, J. E. Wilkinson. 1994. Transplantation of T cell-mediated, lymphoreticular disease from the scurfy (sf) mouse. Am. J. Pathol. 145:281.[Abstract]
  5. Kanangat, S., P. Blair, R. Reddy, M. Deheshia, V. Godfrey, B. T. Rouse, J. E. Wilkinson. 1996. Disease in the scurfy (sf) mouse is associated with over-expression of cytokine genes. Eur. J. Immunol. 26:161.[Medline]
  6. Blair, P. J., S. J. Bultman, J. C. Haas, B. T. Rouse, J. E. Wilkinson, V. L. Godfrey. 1994. CD4+CD8- T cells are the effector cells in disease pathogenesis in the scurfy (sf) mouse. J. Immunol. 153:3764.[Abstract]
  7. Clark, L. B., M. W. Appleby, M. E. Brunkow, J. E. Wilkinson, S. F. Ziegler, F. Ramsdell. 1999. Cellular and molecular characterization of the scurfy mouse mutant. J. Immunol. 162:2546.[Abstract/Free Full Text]
  8. Chatila, T. A., F. Blaeser, N. Ho, H. M. Lederman, C. Voulgaropoulos, C. Helms, A. M. Bowcock. 2000. JM2, encoding a forkhead-related protein, is mutated in X-linked autoimmunity–allergic dysregulation syndrome. J. Clin. Invest. 106:R75.
  9. Powell, B. R., N. R. Buist, P. Stenzel. 1982. An X-linked syndrome of diarrhea, polyendocrinopathy, and fatal infection in infancy. J. Pediatr. 100:731.[Medline]
  10. Satake, N., M. Nakanishi, M. Okano, K. Tomizawa, A. Ishizaka, K. Kojima, M. Onodera, T. Ariga, A. Satake, Y. Sakiyama. 1993. A Japanese family of X-linked auto-immune enteropathy with haemolytic aneamia and polyendocrinopathy. Eur. J. Pediatr. 152:313.[Medline]
  11. Bennett, C. L., J. Christie, F. Ramsdell, M. E. Brunkow, P. J. Ferguson, L. Whitesell, T. E. Kelly, F. T. Saulsbury, P. F. Chance, H. D. Ochs. 2001. The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat. Genet. 27:20.[Medline]
  12. Brunkow, M. E., E. W. Jeffery, K. A. Hjerrild, B. Paeper, L. B. Clark, S. A. Yasayko, J. E. Wilkinson, D. Galas, S. F. Ziegler, F. Ramsdell. 2001. Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat. Genet. 27:68.[Medline]
  13. Tivol, E. A., F. Borriello, A. N. Schweitzer, W. P. Lynch, J. A. Bluestone, A. H. Sharpe. 1995. Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity 3:541.[Medline]
  14. Waterhouse, P., J. M. Penninger, E. Timms, A. Wakeham, A. Shahinian, K. P. Lee, C. B. Thompson, H. Griesser, T. W. Mak. 1995. Lymphoproliferative disorders with early lethality in mice deficient in CTLA-4. Science 270:985.[Abstract/Free Full Text]
  15. Shull, M. M., I. Ormsby, A. B. Kier, S. Pawlowski, R. J. Diebold, M. Yin, R. Allen, C. Sidman, G. Proetzel, D. Calvin. 1992. Targeted disruption of the mouse transforming growth factor-{beta}1 gene results in multifocal inflammatory disease. Nature 359:693.[Medline]
  16. Kulkarni, A. B., C. G. Huh, D. Becker, A. Geiser, M. Lyght, K. C. Flanders, A. B. Roberts, M. B. Sporn, J. M. Ward, S. Karlsson. 1993. Transforming growth factor {beta}1 null mutation in mice causes excessive inflammatory response and early death. Proc. Natl. Acad. Sci. USA 90:770.[Abstract/Free Full Text]
  17. Christ, M., N. L. McCartney-Francis, A. B. Kulkarni, J. M. Ward, D. E. Mizel, C. L. Mackall, R. E. Gress, K. L. Hines, H. Tian, S. Karlsson. 1994. Immune dysregulation in TGF-{beta}1-deficient mice. J. Immunol. 153:1936.[Abstract]
  18. Schubert, L. A., E. J. Jeffery, Y. Zhang, F. Ramsdell, S. F. Ziegler. 2001. Scurfin (FOXP3) acts as a repressor of transcription and regulates T cell activation. J. Biol. Chem. 276:37672.[Abstract/Free Full Text]
  19. Khattri, R., D. J. Kasprowicz, T. Cox, M. Mortrud, M. Appleby, M. Brunkow, S. F. Ziegler, F. Ramsdell. 2001. The amount of scurfin protein determines peripheral T cell number and responsiveness. J. Immunol. 167:6312.[Abstract/Free Full Text]
  20. Foy, T. M., A. Aruffo, J. Bajorath, J. E. Buhlman, R. J. Noelle. 1996. Immune regulation by CD40 and its ligand gp39. Annu. Rev. Immunol. 14:591.[Medline]
  21. Han, S., B. Zheng, Y. Takahashi, G. Kelsoe. 1997. Distinctive characteristics of germinal center B cells. Semin. Immunol. 9:255.[Medline]
  22. Stevens, T. L., A. Bossie, V. M. Sanders, R. Fernandez-Botran, R. L. Coffman, T. R. Mosmann, E. S. Vitetta. 1988. Regulation of antibody isotype secretion by subsets of antigen-specific helper T cells. Nature 334:255.[Medline]
  23. Mosmann, T. R., R. L. Coffman. 1989. TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu. Rev. Immunol. 7:145.[Medline]
  24. Myers, C. D., V. M. Sanders, E. S. Vitetta. 1986. Isolation of antigen-binding virgin and memory B cells. J. Immunol. Methods 92:45.[Medline]
  25. Snow, E. C., E. S. Vitetta, J. W. Uhr. 1983. Activation of antigen-enriched B cells. I. Purification and response to thymus-independent antigens. J. Immunol. 130:607.[Abstract]
  26. Vitetta, E. S., A. Bossie, R. Fernandez-Botran, C. D. Myers, K. G. Oliver, V. M. Sanders, T. L. Stevens. 1987. Interaction and activation of antigen-specific T and B cells. Immunol. Rev. 99:193.[Medline]
  27. Finkelman, F. D., J. Holmes, I. M. Katona, J. F. Urban, J. P. Beckmann, L. S. Park, K. A. Schooley, R. L. Coffman, T. R. Mosmann, W. E. Paul. 1990. Lymphokine control of in vivo immunoglobulin isotype selection. Annu. Rev. Immunol. 8:303.[Medline]
  28. Warren, W. D., M. T. Berton. 1995. Induction of germline {gamma}-1 and epsilon Ig gene expression in murine B cells: interleukin 4 and the CD40 ligand-CD40 interaction provide distinct but synergistic signals. J. Immunol. 155:5637.[Abstract]
  29. Greenfield, E. A., K. A. Nguyen, V. K. Kuchroo. 1998. CD28/B7 costimulation: a review. Crit. Rev. Immunol. 18:389.[Medline]
  30. Han, S., K. Hathcock, B. Zheng, T. B. Kepler, R. Hodes, G. Kelsoe. 1995. Cellular interaction in germinal centers: roles of CD40 ligand and B7-2 in established germinal centers. J. Immunol. 155:556.[Abstract]
  31. Tsysykova, A. V., E. N. Tsitsikov, R. S. Geha. 1996. The CD40L promoter contains nuclear factor of activated T cells-binding motifs which require AP-1 binding for activation of transcription. J. Biol. Chem. 271:3763.[Abstract/Free Full Text]
  32. Rao, A.. 1994. NF-ATp: a transcription factor required for co-ordinate induction of several cytokine genes. Immunol. Today 15:274.[Medline]
  33. Rooney, J. W., T. Hoey, L. H. Glimcher. 1995. Coordinate and cooperative roles for NF-AT and AP-1 in the regulation of the murine IL-4 gene. Immunity 2:473.[Medline]
  34. Sica, A., L. Dorman, V. Viggiano, M. Cippitelli, P. Ghosh, N. Rice, H. A. Young. 1997. Interaction of NF-{kappa}B and NFAT with the interferon-{gamma} promoter. J. Biol. Chem. 272:30412.[Abstract/Free Full Text]

Related articles in The JI:

IN THIS ISSUE

The JI 2003 171: 1121-1122. [Full Text]  



This article has been cited by other articles:


Home page
 DiabetesHome page
M. Tritt, E. Sgouroudis, E. d'Hennezel, A. Albanese, and C. A. Piccirillo
Functional Waning of Naturally Occurring CD4+ Regulatory T-Cells Contributes to the Onset of Autoimmune Diabetes
Diabetes, January 1, 2008; 57(1): 113 - 123.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
K. A. Smith, S. Efstathiou, and A. Cooke
Murine Gammaherpesvirus-68 Infection Alters Self-Antigen Presentation and Type 1 Diabetes Onset in NOD Mice
J. Immunol., December 1, 2007; 179(11): 7325 - 7333.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
C. Chen, E. A. Rowell, R. M. Thomas, W. W. Hancock, and A. D. Wells
Transcriptional Regulation by Foxp3 Is Associated with Direct Promoter Occupancy and Modulation of Histone Acetylation
J. Biol. Chem., December 1, 2006; 281(48): 36828 - 36834.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
R. Mallone, S. A. Kochik, H. Reijonen, B. Carson, S. F. Ziegler, W. W. Kwok, and G. T. Nepom
Functional avidity directs T-cell fate in autoreactive CD4+ T cells
Blood, October 15, 2005; 106(8): 2798 - 2805.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
K. S. Lee, G. Sen, and C. M. Snapper
Endogenous CD4+ CD25+ Regulatory T Cells Play No Apparent Role in the Acute Humoral Response to Intact Streptococcus pneumoniae
Infect. Immun., July 1, 2005; 73(7): 4427 - 4431.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
N. Carpino, W. E. Thierfelder, M.-s. Chang, C. Saris, S. J. Turner, S. F. Ziegler, and J. N. Ihle
Absence of an Essential Role for Thymic Stromal Lymphopoietin Receptor in Murine B-Cell Development
Mol. Cell. Biol., March 15, 2004; 24(6): 2584 - 2592.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Related articles in The JI
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kasprowicz, D. J.
Right arrow Articles by Ziegler, S. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kasprowicz, D. J.
Right arrow Articles by Ziegler, S. F.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS