Journal of Leukocyte Biology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.0803401 on November 21, 2003

Published online before print November 21, 2003
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0803401v1
75/2/350    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
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
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Toennies, H. M.
Right arrow Articles by Arch, R. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Toennies, H. M.
Right arrow Articles by Arch, R. H.
(Journal of Leukocyte Biology. 2004;75:350-357.)
© 2004 by Society for Leukocyte Biology

Expression of CD30 and Ox40 on T lymphocyte subsets is controlled by distinct regulatory mechanisms

Holly M. Toennies*, Jonathan M. Green*,{dagger} and Robert H. Arch*,{dagger},1

Departments of
* Medicine and
{dagger} Pathology and Immunology, Washington University, School of Medicine, St. Louis, Missouri

1 Correspondence: Washington University, School of Medicine, Campus Box 8052, 660 S. Euclid Ave., St. Louis, MO 63110. E-mail: arch{at}wustl.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Members of the TNF receptor (TNFR) superfamily are cell-surface proteins that can be found on most cell types including lymphocytes. Although some TNFR-related molecules are constitutively expressed, others, such as CD30 and Ox40, are induced upon activation of lymphocytes. CD30 and Ox40 are predominantly expressed on activated T helper (Th)2 cells. Both receptors can activate c-Jun N-terminal kinase (JNK) and nuclear factor-{kappa}B (NF-{kappa}B) and have been suggested to play costimulatory roles in lymphocyte activation. To gain further insight into events triggered by both TNFR-related molecules, a detailed analysis of their expression patterns has been performed. We found that CD30 and Ox40 were coexpressed on Th2 cells. However, in contrast to CD30, Ox40 was also expressed on Th1 cells. Although expression of both receptors is augmented by interleukin-4, only CD30 expression is dependent on signal transducer and activator of transcription (STAT)-6-mediated signaling. Differences in the regulatory pathways controlling expression of CD30 and Ox40 suggest distinct, functional effects triggered by the two TNFR-related molecules during lymphocyte activation.

Key Words: surface receptor • T cell differentiation • signal transduction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The TNF receptor (TNFR) superfamily contains a growing number of cell-surface proteins that are expressed on many cell types including cells of the immune system [1 ]. TNFR family members play pivotal roles in the differentiation, activation, and survival of cells [2 ]. Most members of the TNFR superfamily contain between two and four cysteine-rich domains in their extracellular domains but show little sequence homology in their cytoplasmic domains [3 ]. TNFR-related molecules do not have any known enzymatic activity and depend on the recruitment of cytoplasmic proteins for the activation of downstream signaling pathways.

Death domain-containing TNFR family members including TNFR-I and Fas recruit adaptor proteins with similar death domains, such as TNFR-associated death domain protein (TRADD) and Fas-associated death domain protein (FADD), triggering a cascade of proteases, termed caspases and subsequently, apoptosis (programmed cell death) [4 ]. TNFR-related proteins lacking a death domain including CD30 and Ox40 (CD134) interact with TNFR-associated factors to induce downstream signal-transduction events, such as activation of the c-Jun N-terminal kinase (JNK) and the transcription factor nuclear factor-{kappa}B (NF-{kappa}B) as well as the production of reactive oxygen species (ROS) [5 6 7 8 ]. All of these events have been shown to regulate the balance between cell survival and apoptosis [9 10 11 12 13 14 15 16 ]. It is interesting that signaling triggered by some TNFR-related proteins lacking a death domain, such as CD30 and TNFR-II, can also decrease the threshold to apoptosis triggered by TNFR-I [17 18 19 20 21 ]. Taken together, TNFR-related proteins are pivotal regulators of pro- and anti-apoptotic signal-transduction pathways.

The complex array of proteins including ligands, receptors, and intracellular adaptor proteins participating in TNFR signal transduction provides a large panel of regulatory mechanisms that modify the outcome of events induced by TNFR-related molecules. Expression levels of proteins, affinities of binding partners for each other, avidities within assembling multiprotein complexes, and competitive binding of intracellular proteins recruited to the cytoplasmic domains of the receptors are likely to play important roles in the outcome of signaling induced by TNFR-related molecules [22 ]. Therefore, depending on the type of the immune response and the cell types recruited to the site of an inflammation, signal-transduction pathways triggered by TNFR-related molecules will vary.

CD30 and Ox40 are not expressed on resting lymphocytes but are up-regulated in response to T cell activation. Both TNFR-related molecules have been described as costimulatory molecules during secondary immune responses and are thought to regulate cell survival and cytokine production [23 , 24 ]. To elucidate further the roles of CD30 and Ox40, their expression kinetics and expression patterns in response to lymphocyte activation triggered by a variety of stimuli were examined and directly compared. In this report, we demonstrate that CD30 and Ox40 are expressed on an overlapping subset of activated T helper (Th)2 cells but have subtle differences in their expression kinetics. In contrast to CD30, Ox40 was also expressed on lymphocytes cultured under Th1 conditions. It is interesting that although the expression levels of both TNFR-related proteins were increased in response to interleukin (IL)-4, only CD30 expression was dependent on signal transducer and activator of transcription (STAT)-6-mediated signal-transduction pathways. Differences in expression patterns and expression kinetics of CD30 and Ox40 as well as distinct regulatory mechanisms controlling their expression argue for distinct functions of the two TNFR-related molecules.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
C57/BL6 and BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME). T cell receptor (TCR) transgenic mice expressing a TCR specific for a peptide of ovalbumin (OVA) containing residues 323–336 (OVA323–336; DO11.10) and DO11.10 STAT-6-deficient mice were kind gifts from Kenneth M. Murphy (Washington University, School of Medicine, St. Louis, MO). All mice were kept at a specific, pathogen-free animal facility according to the guidelines of the Animal Studies Committee of Washington University.

In vitro activation of lymphocytes
Lymph nodes and spleens were removed and mechanically disrupted into single-cell suspensions in phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS). For in vitro stimulation, 5 x 106 lymphocytes per well were plated on six-well plates in RPMI 1640 supplemented with 10% FBS, 4 mM L-glutamine, 10 mM HEPES, 100 mM nonessential amino acids, 57 µM ß-mercaptoethanol, 100 U/ml penicillin, and 100 µg/ml streptomycin. As described in the figure legends, T cells were stimulated with phorbol 12-myristate 13-acetate (PMA; 25 ng/ml) and ionomycin (1 µM), plate-bound anti-CD3 antibodies (Ab; 2C11), alone or in the presence of soluble anti-CD28 Ab (PV-1), or an antigenic peptide of OVA (OVA323–336). Coating plates with 2C11 was performed by incubating plates with the indicated concentrations of Ab in PBS at 4°C overnight. For costimulation, soluble PV-1 (1 µg/ml) was added to the cultures in the presence of antigen (Ag)-presenting cells (APCs). T cells were skewed toward the Th1 and the Th2 lineages, respectively, by adding recombinant mouse (rm)IL-12 (25 µg/ml) and anti-IL-4 Ab (10 µg/ml) or rmIL-4 (50 ng/ml) and anti-IL-12 Ab (10 µg/ml).

Ab and cytokines
Anti-CD4 (H129.1), anti-CD8 (53-6.7), anti-CD25 (PC61), anti-CD30 (mCD30.1), anti-IL-4 (11B11), and anti-IL-12 (C15.1) Ab were purchased from PharMingen (San Diego, CA). Anti-Ox40 Ab was purchased from Serotech (Oxford, UK). PV-1 was a kind gift from Jeffrey Bluestone (University of California-San Francisco). For fluorescein-activated cell sorter (FACS) analysis, all Abs were used directly conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE), as indicated or in combination with FITC- or PE-conjugated secondary Ab specific for the immunoglobulin isotype of the primary Ab. For in vitro stimulation of lymphocytes, purified Ab and when available, Ab preparations tested for their low endotoxin concentrations were used.

IL-4 was purchased from R&D Systems (Minneapolis, MN). rmIL-12 was purchased from Sigma Aldrich (St. Louis, MO).

FACS of surface expression of lymphocyte markers
Resting and in vitro-activated lymphocytes, harvested on the indicated days, were washed, counted, and resuspended in PBS containing 2% FBS. A total of 2 x 105 cells were stained for 30 min at 4°C in a volume of 50 µl with the previously described Ab diluted to a concentration that had been tested for optimal staining. The stained cells were washed twice and resuspended in PBS/FBS. For all experiments, Ab of irrelevant specificities were used as appropriate isotype controls to determine background levels of unspecific staining and set dot-blot quadrants. For all samples, analysis was performed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA) and CellQuest software, collecting at least 104 live cells. For purposes of clarity, only 2500 events are depicted in the presented dot blots.

Enzyme-linked immunosorbent assay (ELISA) of cytokines
ELISA determined cytokines in the culture supernatants, according to the manufacturer’s (PharMingen) protocol. In brief, wells of microtiter plates were coated with capture Ab diluted in coating buffer. The wells were washed and blocked before appropriate dilutions of culture supernatants, and standards were added. After 2 h of incubation at room temperature (RT), the wells were aspirated and washed. Detection Ab and avidin horseradish peroxidase were added and incubated for 1 h at RT. After aspirating and washing the wells, substrate was added to the wells and incubated for 30 min at RT. After stopping the reaction with stop solution, absorbance was read at 450 nm. The cytokine concentrations were calculated using dilutions of recombinant cytokines as standards.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The TNFR-related molecules CD30 and Ox40 are not expressed on resting lymphocytes but are up-regulated during lymphocyte activation
Lymph node cells (LNC) and splenocytes (SC) isolated from naïve C57/BL6 mice were analyzed by flow cytometry (FACS) using Ab specific for mouse CD4, CD8, CD25, CD30, CD69, Ox40, and Ab with unrelated specificity as negative controls. Consistent with previously published findings, expression of CD30 and Ox40 could not be detected on CD69-negative, CD25-negative resting lymphocytes (data not shown) [24 25 26 ].

Both TNFR-related molecules are up-regulated in response to T cell activation [24 25 26 ]. However, regulation and kinetics of their expression are not fully understood. To compare the effects of signaling triggered by the TCR and the costimulatory receptor CD28 on the expression of CD30 and Ox40, LNC and SC from naïve C57/BL6 mice were stimulated in vitro with plate-bound Ab specific for CD3 (2C11) to mimic TCR stimulation in the presence or absence of a costimulatory signal provided by cross-linking with anti-CD28 Ab (PV-1). Neither CD30 nor Ox40 expression was detected after stimulation of T cells with low concentrations (0.1 µg/ml) of plate-bound anti-CD3 alone; however, costimulation triggered by 1 µg/ml soluble anti-CD28 Ab caused expression of Ox40 but not of CD30 (Fig. 1A ). Higher concentrations of anti-CD3 Ab (1 µg/ml) coated to plastic alone or in combination with soluble anti-CD28 Ab resulted in higher percentages of cells expressing Ox40 and in increased expression levels of Ox40 (Fig. 1B) . In contrast, CD30 expression was not triggered by high concentrations of anti-CD3 Ab alone and was only slightly increased by CD28 costimulation. These results demonstrated differences in the regulation of expression of CD30 and Ox40 during T cell activation and suggested that CD30 expression requires signaling events in addition to pathways triggered by the TCR and CD28.



View larger version (39K):
[in this window]
[in a new window]
  		Figure 1. Lymphocyte activation results in expression of CD30 and Ox40. LNC and SC were stimulated with plate-bound anti-CD3 Ab with or without soluble anti-CD28 as described in Materials and Methods. Three days post-stimulation, cells were stained with the indicated Ab and analyzed by FACS. (A) Costimulation of LNC and SC with a low concentration (0.1 µg/ml) of coated anti-CD3 and soluble anti-CD28 (1 µg/ml) Ab. (B) Costimulation of SC with a high concentration (1.0 µg/ml) of coated anti-CD3 and soluble anti-CD28 (1 µg ml) Ab. The data shown represent one experiment out of five using three to five mice per experimental group.

 
To test if proximal events of TCR or CD28 signaling play a role in inducing expression of CD30 and Ox40, LNC and SC were stimulated with the phorbol ester PMA and the calcium ionophore ionomycin. Ox40 but no detectable CD30 was found on CD4+ and CD8+ T cells in cultures of LNC and SC stimulated with PMA and ionomycin (Fig. 2 and data not shown). Taken together, these findings indicate that expression of CD30 and Ox40 is regulated by distinct mechanisms and argue that signaling events in addition to TCR/CD28 stimulation are involved in regulating CD30 expression during T cell activation.



View larger version (41K):
[in this window]
[in a new window]
  		Figure 2. Lymphocyte stimulation with PMA and ionomycin results in expression of Ox40 but not CD30. LNC and SC were cultured with PMA (25 ng/ml) and ionomycin (1 µM) for 3 days, stained with the indicated Ab, and analyzed by FACS. Shown is one representative experiment out of five using three to five animals per experimental group.

 
CD30 and Ox40 expression is regulated by the Th2 cytokine IL-4
CD30 and Ox40 can be expressed on activated Th2 cells and have been suggested to play a costimulatory role in this T cell subset [23 , 24 , 26 ]. To characterize further the effects of Th2 differentiation on the expression of CD30 and Ox40, T cells were stimulated with anti-CD3 and anti-CD28 Ab in the presence or absence of exogenous IL-4 (Fig. 3 ). As expected, the percentage of cells expressing CD30 and Ox40 increased when rmIL-4 was added to the culture to drive Th2 differentiation. It is interesting that expression of CD30 and Ox40 was significantly increased on CD4- cells in the cultures, and expression levels and percentages of both receptors on CD4+ cells did not change (data not shown). These results suggest a role of CD30 and Ox40 on Th cells as well as other lymphocytes.



View larger version (47K):
[in this window]
[in a new window]
  		Figure 3. CD30 and Ox40 expression levels increase under Th2 culture conditions. Resting SC were stimulated with plate-bound anti-CD3 (1 µg/ml) and soluble anti-CD28 Ab (1 µg/ml) in the presence or absence of 50 ng/ml rmIL-4. After 3 days of stimulation, the cells were stained with the indicated Ab and analyzed by FACS.

 
CD30 and Ox40 expression levels are elevated in response to T cell activation by Ag
To address how expression of CD30 and Ox40 is regulated under physiologic conditions, SC from naïve TCR transgenic DO11.10 mice were stimulated with an antigenic peptide of OVA323–336. In vitro stimulation of DO11.10 SC with OVA323–336 results in accumulation of CD4+ Th2 cells in the culture [27 , 28 ]. Three days post-stimulation, the majority of CD4+ T cells showed significantly elevated levels of CD30 and Ox40 (data not shown). To test if CD30 and Ox40 are coexpressed on the same subset of T cells and to gain further insight into the expression kinetics of both receptors, double-stainings with Ab specific for the two TNFR-related molecules were performed over a period of 4 days post-Ag stimulation. CD30 and Ox40 were coexpressed on the same T cell subset, but differences in their expression kinetics were found, as indicated by the changed ratio of Ox40+ and CD30+ T cells (Fig. 4A and 4B ). Ox40 expression was detected 1 day post-stimulation, and CD30 expression was observed 12–24 h after Ox40 expression. Surface levels of both receptors peaked 48–72 h post-stimulation. Analysis of cells at later time points revealed decreasing expression of Ox40 and CD30. Seven days post-stimulation, expression of both TNFR-related molecules reached baseline levels (Fig. 4A and data not shown). The slightly delayed increase of expression of CD30 compared with Ox40 suggests a role of CD30 in the late phase of an immune response and/or Th cell differentiation (Fig. 4B) .



View larger version (43K):
[in this window]
[in a new window]
  		Figure 4. Ag stimulation of T cells results in increased expression of CD30 and Ox40. Resting SC from TCR transgenic DO11.10 mice were cultured with 0.3 µM OVA323–336 peptide for up to 4 days. (A) Each day, aliquots of cells were stained with the indicated Ab and analyzed by FACS. (B) To analyze differences in the expression kinetics of CD30 and Ox40, the ratios of Ox40-positive (upper and lower right quadrants) and CD30-positive (upper right quadrants) were calculated. Shown is one experiment representative of two using cells pooled from five mice per experiment. n.d., not determined. (C) Resting SC from TCR transgenic DO11.10 mice were stimulated for 3 days with plate-bound anti-CD3 Ab in the presence or absence of soluble anti-CD28 Ab and IL-4. Subsequently, the cells were harvested and stained with Ab specific for CD30 and Ox40 and analyzed by FACS. Shown is one experiment representative of two with similar results using cells pooled from two mice per experiment.

 
To test if the discrepancy between transgenic lymphocytes stimulated with Ag and nontransgenic lymphocytes stimulated with mitogens is a result of the different genetic backgrounds, SC from DO11.10 TCR transgenic as well as from nontransgenic BALB/c mice were activated with plate-bound Ab specific for CD3 in the presence or absence of soluble anti-CD28 Ab and rmIL-4 (Fig. 4C and data not shown). These experiments confirmed the results obtained with lymphocytes from mice on the C57/BL6 background. Although cross-linking CD3 alone triggered expression of Ox40 predominantly, costimulation by cross-linking CD28 as well as addition of exogenous IL-4 promoted expression of both receptors. Taken together, these findings argue that the observed differences in response to distinct stimuli are not a result of the differences in the genetic background.

CD30 and Ox40 expression levels show differences in response to T cell differentiation
DO11.10 cells differentiate predominantly toward the Th2 phenotype even in the absence of exogenous cytokines such as IL-4 or blocking Ab such as anti-IL-12 or anti-interferon (IFN)-{gamma} [28 ]. To test if differences in CD30 and Ox40 expression are a result of differences in the responsiveness to Th1 or Th2 cytokines, SC were stimulated in vitro with Ag in the presence of exogenous IL-12 and anti-IL-4 or exogenous IL-4 and anti-IL-12 to drive Th cell differentiation. Skewing toward the Th1 and Th2 lineages, respectively, was determined by analyzing IFN-{gamma} and IL-4 levels in the culture supernatants (Fig. 5A ). CD30 and Ox40 were coexpressed on T cells skewed toward the Th2 lineage (Fig. 5B) . However, although more than 40% of the cells in cultures skewed toward the Th1 lineage expressed Ox40, less than 8% of the cells also expressed CD30 on their surface. Consistent with our previous results (Fig. 4) , CD30 expression was significantly delayed when compared with Ox40 expression under these conditions (Fig. 5C) . These results suggest that CD30 expression is regulated by Th2-specific signaling events, and pathways common to Th1 and Th2 cells trigger Ox40 expression.



View larger version (47K):
[in this window]
[in a new window]
  		Figure 5. T cell differentiation results in differences in the expression patterns of CD30 and Ox40. Resting SC from DO11.10 mice were differentiated into Th1 or Th2 cells as described in Materials and Methods. Three and 4 days post-activation, (A) interferon-{gamma} (IFN-{gamma}) and IL-4 levels in the culture supernatants were determined by ELISA, and (B) cells were stained with the indicated Ab and analyzed by FACS. (C) Percentages of CD30+ (upper left and right quadrants) and Ox40+ (upper and lower right quadrants) cells accumulating over time in cultures skewed toward the Th1 or Th2 lineages. Shown is one representative experiment of three using cells pooled from at least five mice per experiment.

 
STAT-6-mediated signaling is essential for regulating CD30 but not Ox40 expression
STAT-6 is a critical regulator of IL-4-mediated events. To test if the expression of CD30 and/or Ox40 is regulated by STAT-6, SC from STAT-6-/- and STAT-6+/+ animals on the DO11.10 background were stimulated with OVA323–336 under Th2 culture conditions for 4 days. Consistent with a role of IL-4 in regulating Ox40 expression, the percentage of CD4+ T cells expressing Ox40 was found to be slightly decreased on STAT-6-/- CD4+ T cells (38.9%) when compared with STAT-6+/+ CD4+ T cells (47.9%). Although CD30 and Ox40 expression could be detected on a similar percentage of STAT-6+/+ CD4+ T cells (49.0% vs. 47.9%), only 11.8% of STAT-6-/- CD4+ T cells expressed CD30 (Fig. 6A ). To test if the lack of CD30 expression was a result of an artificial Th1 phenotype of the STAT-6-/- lymphocytes in these experiments, IFN-{gamma} and IL-4 levels were determined in the cultures (Fig. 6B) . As controls, parallel cultures were skewed toward the Th1 lineage. Although STAT-6+/+ and STAT-6-/- cultures had comparable levels of IL-4 in the supernatants when skewed toward the Th2 lineage, IFN-{gamma} was significantly reduced in comparison with Th1 cultures, indicating that the STAT-6-/- cells were not artificially skewed toward the Th1 lineage. Therefore, although expression of Ox40 and CD30 increased in response to IL-4, only CD30 expression is dependent on STAT-6-mediated events. Increased expression of CD30 and Ox40 in response to distinct signaling events suggests that the two TNFR-related molecules play roles during distinct phases of the immune response and/or have distinct effector functions in Th2 cells.



View larger version (28K):
[in this window]
[in a new window]
  		Figure 6. STAT-6 is essential for regulating CD30 but not Ox40 expression. SC from STAT-6+/+ and STAT-6-/- DO11.10 mice were stimulated with 0.3 µM OVA323–336 and skewed toward the Th2 lineage as described in Materials and Methods. (A) Four days post-activation of the indicated molecules was determined surface expression by FACS. (B) In parallel, IFN-{gamma} and IL-4 levels in culture supernatants were analyzed by ELISA. Th1 and Th2 culture conditions were used as positive and negative controls, respectively. The data shown are representative for the results of three independent experiments performed with cells of two mice per genotype.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The TNFR-related proteins CD30 and Ox40 are transiently expressed during activation of primary T cells and are also expressed on a number of T cell tumors. CD30 and Ox40 have been linked to a number of immunological disorders and diseases including graft-versus-host disease, Crohn’s disease, experimental allergic encephalomyelitis, and diabetes [29 30 31 32 33 34 35 36 ]. Downstream signaling events triggered by CD30 and Ox40 include activation of kinases, such as JNK and inhibitor of {kappa}B kinase, and transcription factors, such as NF-{kappa}B, as well as ROS production [5 , 8 , 37 ]. CD30 and Ox40 can play costimulatory roles during secondary activation of lymphocytes, in particular, Th2 cells, promoting cytokine production as well as proliferation, differentiation, and survival of T cells [23 , 26 , 38 , 39 ]. Furthermore, Ox40 is thought to play a role in cell-mediated cytotoxicity and adhesion of T cells [40 41 42 ]. In contrast, CD30 has also been suggested to be involved in negative selection in the thymus and to regulate cell survival and apoptosis [17 , 18 , 43 , 44 ].

Expression of CD30 and Ox40 on overlapping subsets of activated lymphocytes and similar signaling events triggered by the receptors suggest additional functional events triggered by the receptors. To gain further insight into the roles of these TNFR-related molecules in T cell activation and differentiation, detailed analysis of their expression patterns was performed. A variety of stimuli were used to activate T cells in vitro, including Ag presented in the context of the major histocompatibility complex on APCs, cross-linking the TCR and the costimulatory molecule CD28 with Ab, and pharmacologic agents, such as PMA and ionomycin. These studies revealed significant differences in T cell responses as measured by cell proliferation and cytokine production (data not shown) and in the regulation of expression of CD30 and Ox40. Although Ox40 expression could be triggered by CD3 signaling alone, CD30 expression was only detected on T cells after activation with anti-CD3 Ab in combination with a costimulatory signal provided by CD28 cross-linking. These findings indicated differences in regulatory pathways controlling expression of the two TNFR-related molecules.

To characterize molecular mechanisms regulating the expression of CD30 and Ox40, T cells were activated with pharmacologic agents. The phorbol ester PMA in combination with the calcium ionophore ionomycin induces T cell proliferation but bypasses early events of TCR signaling. Stimulation of lymphocytes with PMA and ionomycin resulted in expression of Ox40 but not CD30, confirming distinct, regulatory mechanisms for the expression of the two receptors. Further experiments are required to determine if lack of proximal signaling events triggered by the TCR or costimulatory receptors, such as CD28, or the requirement of additional signals, e.g., events induced by cytokine receptors, resulted in inefficient CD30 expression.

CD30 and Ox40 are predominantly expressed on Th2 cells [23 , 24 ]. To compare the expression levels and kinetics of the two receptors under Th2 conditions, T cells were stimulated by cross-linking CD3 and CD28 in the presence of exogenous IL-4. Consistent with previously published findings, increased percentages of cells expressing the receptors were detected in response to IL-4-induced signaling. It is interesting that CD4- cells in the cultures showed significantly increased expression levels of CD30 and Ox40, and their expression levels on CD4+ were much less affected by IL-4 in the media. These findings suggest that both TNFR-related molecules play a functional role on cell types other than CD4+ Th2 cells during the late phases of an immune response.

To study the expression of CD30 and Ox40 under physiologic conditions, we chose a TCR transgenic mouse model (DO11.10), which in response to stimulation with a peptide of OVA (OVA323–336), results predominantly in the generation of CD4+ Th2 cells [28 ]. Three days of in vitro stimulation of DO11.10 SC with Ag resulted in the accumulation of activated, i.e., CD25-expressing, CD4+ T cells, which expressed detectable surface levels of CD30 and Ox40. A closely related TNFR family member, 4-1BB, could not be detected under these culture conditions (data not shown), consistent with its proposed role on Th1 cells and CD8+ T cells [45 46 47 48 49 50 ]. Kinetics analysis confirmed that the receptors were coexpressed on the majority of activated CD4+ T cells but demonstrated slightly delayed expression of CD30 in comparison to Ox40. Expression of both receptors peaked three days post-stimulation and decreased at later time points (data not shown). The observed differences between TCR transgenic and nontransgenic lymphocytes were not a result of differences in the genetic background. Mitogenic stimulation of DO11.10 SC with Ab specific for CD3 and CD28 resulted in an expression pattern of CD30 and Ox40 similar to the one defined for nontransgenic lymphocytes. Furthermore, in the presence of IL-4, expression of CD30 and Ox40 was enhanced, suggesting a role of both TNFR-related molecules in Th2 cells.

To define further the expression patterns of CD30 and Ox40 during T cell differentiation, DO11.10 SC were activated in vitro and skewed toward the Th1 or Th2 lineage. These studies indicated that CD30 and Ox40 were coexpressed on Th2 cells activated by Ag. However, Ox40, but not CD30, could also be detected on activated Th1 cells, confirming differences in the regulation of the two TNFR-related molecules seen after T cell stimulation with PMA and ionomycin or Ab specific for CD3 and CD28. In contrast to T cells activated by cross-linking of CD3 and CD28, Ag stimulation of lymphocytes triggered expression of CD30 and Ox40 exclusively on CD4+ T cells. These findings emphasize that culture conditions during lymphocyte activation are critical in regulating the expression of TNFR-related molecules and probably the downstream signaling events induced by the receptors.

Expression of CD30 on Th2 but not Th1 cells and expression of Ox40 on both types of Th cells suggested a role of STAT-6 in regulating the expression of CD30 but not Ox40. In vitro stimulation of STAT-6-/- SC confirmed the role of STAT-6 in regulating CD30 expression. Although lack of STAT-6 resulted in significantly decreased CD30 expression on Th2 cells, Ox40 expression was only slightly impaired. Analysis of cytokine levels in cultures of STAT-6+/+ and STAT-6-/- lymphocytes revealed that STAT-6 deficiency did not result in artificial skewing that could be responsible for the differences in CD30 and Ox40 expression. Although these experiments do not rule out a role of STAT-6 in regulating Ox40 expression, they indicate that Ox40 expression is regulated, at least in part, by other mechanisms. This is consistent with the finding that Ox40 expression can be triggered by CD3 stimulation alone and is found on activated Th1 cells. Further studies are necessary to determine the mechanisms downstream of the TCR and CD28 that regulate expression of TNFR-related molecules. In addition, more detailed analysis of the promoter regions of CD30 and Ox40 is required to define transcription factors regulating their expression.

Our studies indicate that distinct signaling mechanisms regulate expression of CD30 and Ox40 on overlapping T cell subsets, suggesting functional differences between the receptors. CD30 and Ox40 function as costimulatory molecules that induce activation of kinases and transcription factors [5 , 37 , 51 ]. Ox40 has been shown to promote differentiation of IL-4-producing Th2 cells as well as T cell survival and generation of memory T cells in the late phase of an immune response [42 , 52 ]. It is interesting that Ox40-mediated signaling pathways seem to be required for regulating CD30 expression (Jane Yen, Susan Kuechenberg, and R. H. Arch, unpublished results). In contrast, CD30 can sensitize cells to apoptosis and thus, could be responsible for terminating the immune response [17 , 18 ]. Taken together, our findings argue that CD30 and Ox40 play discrete, functional roles in T cell activation and/or differentiation and are crucial in modulating Th1/Th2 cell ratios in the late phase of T cell activation.


   ACKNOWLEDGEMENTS
 
This research was supported in part by grants of the National Institutes of Health (5 RO1 HL67312-02), the Diabetes Training Center (DRTC) of Washington University, School of Medicine (NIH 5 P60 DK20579), and an Investigator Award of the Cancer Research Institute (R. H. A.)

Received August 25, 2003; revised October 3, 2003; accepted October 21, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Locksley, R. M., Killeen, N., Lenardo, M. J. (2001) The TNF and TNF receptor superfamilies: integrating mammalian biology Cell 104,487-501[CrossRef][Medline]
  2. Arch, R. H., Thompson, C. B. (1999) Lymphocyte survival–the struggle against death Annu. Rev. Cell Dev. Biol. 15,113-140[CrossRef][Medline]
  3. Bodmer, J. L., Schneider, P., Tschopp, J. (2002) The molecular architecture of the TNF superfamily Trends Biochem. Sci. 27,19-26[CrossRef][Medline]
  4. Hofmann, K. (1999) The modular nature of apoptotic signaling proteins Cell. Mol. Life Sci. 55,1113-1128[CrossRef][Medline]
  5. Arch, R. H., Thompson, C. B. (1998) 4-1BB and Ox40 are members of a tumor necrosis factor (TNF)-nerve growth factor receptor subfamily that bind TNF receptor-associated factors and activate nuclear factor {kappa}B Mol. Cell. Biol. 18,558-565[Abstract/Free Full Text]
  6. Gedrich, R. W., Gilfillan, M. C., Duckett, C. S., Van Dongen, J. L., Thompson, C. B. (1996) CD30 contains two binding sites with different specificities for members of the tumor necrosis factor receptor-associated factor family of signal transducing proteins J. Biol. Chem. 271,12852-12858[Abstract/Free Full Text]
  7. Arch, R. H., Gedrich, R. W., Thompson, C. B. (1998) Tumor necrosis factor receptor-associated factors (TRAFs)–a family of adapter proteins that regulates life and death Genes Dev 12,2821-2830[Free Full Text]
  8. Chandel, N. S., Schumacker, P. T., Arch, R. H. (2001) Reactive oxygen species are downstream products of TRAF-mediated signal transduction J. Biol. Chem. 276,42728-42736[Abstract/Free Full Text]
  9. Baldwin, A. S., Jr (1996) The NF-{kappa}B and I{kappa}B proteins: new discoveries and insights Annu. Rev. Immunol. 14,649-683[CrossRef][Medline]
  10. Ghosh, S., May, M. J., Kopp, E. B. (1998) NF-{kappa}B and Rel proteins: evolutionarily conserved mediators of immune responses Annu. Rev. Immunol. 16,225-260[CrossRef][Medline]
  11. Karin, M., Liu, Z., Zandi, E. (1997) AP-1 function and regulation Curr. Opin. Cell Biol. 9,240-246[CrossRef][Medline]
  12. Liebermann, D. A., Gregory, B., Hoffman, B. (1998) AP-1 (Fos/Jun) transcription factors in hematopoietic differentiation and apoptosis Int. J. Oncol. 12,685-700[Medline]
  13. Liu, Z. G., Hsu, H., Goeddel, D. V., Karin, M. (1996) Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-{kappa}B activation prevents cell death Cell 87,565-576[CrossRef][Medline]
  14. Natoli, G., Costanzo, A., Ianni, A., Templeton, D. J., Woodgett, J. R., Balsano, C., Levrero, M. (1997) Activation of SAPK/JNK by TNF receptor 1 through a noncytotoxic TRAF2-dependent pathway Science 275,200-203[Abstract/Free Full Text]
  15. Sonenshein, G. E. (1997) Rel/NF-{kappa}B transcription factors and the control of apoptosis Semin. Cancer Biol. 8,113-119[CrossRef][Medline]
  16. Finkel, T., Holbrook, N. J. (2000) Oxidants, oxidative stress and the biology of ageing Nature 408,239-247[CrossRef][Medline]
  17. Arch, R. H., Gedrich, R. W., Thompson, C. B. (2000) Translocation of TRAF proteins regulates apoptotic threshold of cells Biochem. Biophys. Res. Commun. 272,936-945[CrossRef][Medline]
  18. Chiarle, R., Podda, A., Prolla, G., Podack, E. R., Thorbecke, G. J., Inghirami, G. (1999) CD30 overexpression enhances negative selection in the thymus and mediates programmed cell death via a Bcl-2-sensitive pathway J. Immunol. 163,194-205[Abstract/Free Full Text]
  19. Weiss, T., Grell, M., Hessabi, B., Bourteele, S., Müller, G., Scheurich, P., Wajant, H. (1997) Enhancement of TNF receptor p60-mediated cytotoxicity by TNF receptor p80: requirement of the TNF receptor-associated factor-2 binding site J. Immunol. 158,2398-2404[Abstract]
  20. Weiss, T., Grell, M., Siemienski, K., Mühlenbeck, F., Dürkop, H., Pfizenmaier, K., Scheurich, P., Wajant, H. (1998) TNFR80-dependent enhancement of TNFR60-induced cell death is mediated by TNFR-associated factor 2 and is specific for TNFR60 J. Immunol. 161,3136-3142[Abstract/Free Full Text]
  21. Fotin-Mleczek, M., Henkler, F., Samel, D., Reichwein, M., Hausser, A., Parmryd, I., Scheurich, P., Schmid, J. A., Wajant, H. (2002) Apoptotic crosstalk of TNF receptors: TNF-R2 induces depletion of TRAF2 and IAP proteins and accelerates TNF-R1-dependent activation of caspase-8 J. Cell Sci. 115,2757-2770[Abstract/Free Full Text]
  22. Kuhné, M. R., Robbins, M., Hambor, J. E., Mackey, M. F., Kosaka, Y., Nishimura, T., Gigley, J. P., Noelle, R. J., Calderhead, D. M. (1997) Assembly and regulation of the CD40 receptor complex in human B cells J. Exp. Med. 186,337-342[Abstract/Free Full Text]
  23. Del Prete, G., De Carli, M., D’Elios, M. M., Daniel, K. C., Almerigogna, F., Alderson, M., Smith, C. A., Thomas, E., Romagnani, S. (1995) CD30-mediated signaling promotes the development of human T helper type 2-like T cells J. Exp. Med. 182,1655-1661[Abstract/Free Full Text]
  24. Ohshima, Y., Yang, L. P., Uchiyama, T., Tanaka, Y., Baum, P., Sergerie, M., Hermann, P., Delespesse, G. (1998) OX40 costimulation enhances interleukin-4 (IL-4) expression at priming and promotes the differentiation of naive human CD4+ T cells into high IL-4-producing effectors Blood 92,3338-3345[Abstract/Free Full Text]
  25. Calderhead, D. M., Buhlmann, J. E., van den Eertwegh, A. J., Claassen, E., Noelle, R. J., Fell, H. P. (1993) Cloning of mouse Ox40: a T cell activation marker that may mediate T–B cell interactions J. Immunol. 151,5261-5271[Abstract]
  26. Gilfillan, M. C., Noel, P. J., Podack, E. R., Reiner, S. L., Thompson, C. B. (1998) Expression of the costimulatory receptor CD30 is regulated by both CD28 and cytokines J. Immunol. 160,2180-2187[Abstract/Free Full Text]
  27. Murphy, K. M., Heimberger, A. B., Loh, D. Y. (1990) Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivo Science 250,1720-1723[Abstract/Free Full Text]
  28. Hsieh, C. S., Macatonia, S. E., O’Garra, A., Murphy, K. M. (1995) T cell genetic background determines default T helper phenotype development in vitro. J. Exp. Med. 181,713-721[Abstract/Free Full Text]
  29. Schwab, U., Stein, H., Gerdes, J., Lemke, H., Kirchner, H., Schaadt, M., Diehl, V. (1982) Production of a monoclonal antibody specific for Hodgkin and Sternberg-Reed cells of Hodgkin’s disease and a subset of normal lymphoid cells Nature 299,65-67[CrossRef][Medline]
  30. Tsukada, N., Kobata, T., Aizawa, Y., Yagita, H., Okumura, K. (1999) Graft-versus-leukemia effect and graft-versus-host disease can be differentiated by cytotoxic mechanisms in a murine model of allogeneic bone marrow transplantation Blood 93,2738-2747[Abstract/Free Full Text]
  31. Dürkop, H., Latza, U., Hummel, M., Eitelbach, F., Seed, B., Stein, H. (1992) Molecular cloning and expression of a new member of the nerve growth factor receptor family that is characteristic for Hodgkin’s disease Cell 68,421-427[CrossRef][Medline]
  32. Weinberg, A. D., Wegmann, K. W., Funatake, C., Whitham, R. H. (1999) Blocking OX-40/OX-40 ligand interaction in vitro and in vivo leads to decreased T cell function and amelioration of experimental allergic encephalomyelitis J. Immunol. 162,1818-1826[Abstract/Free Full Text]
  33. Chiarle, R., Podda, A., Prolla, G., Gong, J., Thorbecke, G. J., Inghirami, G. (1999) CD30 in normal and neoplastic cells Clin. Immunol. 90,157-164[CrossRef][Medline]
  34. Latza, U., Foss, H. D., Dürkop, H., Eitelbach, F., Dieckmann, K. P., Loy, V., Unger, M., Pizzolo, G., Stein, H. (1995) CD30 antigen in embryonal carcinoma and embryogenesis and release of the soluble molecule Am. J. Pathol. 146,463-471[Abstract]
  35. Gerli, R., Lunardi, C., Vinante, F., Bistoni, O., Pizzolo, G., Pitzalis, C. (2001) Role of CD30+ T cells in rheumatoid arthritis: a counter-regulatory paradigm for Th1-driven diseases Trends Immunol 22,72-77[CrossRef][Medline]
  36. Lyons, P. A., Hancock, W. W., Denny, P., Lord, C. J., Hill, N. J., Armitage, N., Siegmund, T., Todd, J. A., Phillips, M. S., Hess, J. F., Chen, S. L., Fischer, P. A., Peterson, L. B., Wicker, L. S. (2000) The NOD Idd9 genetic interval influences the pathogenicity of insulitis and contains molecular variants of Cd30, Tnfr2, and Cd137 Immunity 13,107-115[CrossRef][Medline]
  37. Duckett, C. S., Gedrich, R. W., Gilfillan, M. C., Thompson, C. B. (1997) Induction of nuclear factor {kappa}B by the CD30 receptor is mediated by TRAF1 and TRAF2 Mol. Cell. Biol. 17,1535-1542[Abstract]
  38. Gruss, H. J., Ulrich, D., Dower, S. K., Herrmann, F., Brach, M. A. (1996) Activation of Hodgkin cells via the CD30 receptor induces autocrine secretion of interleukin-6 engaging the NF-{kappa}B transcription factor Blood 87,2443-2449[Abstract/Free Full Text]
  39. Bowen, M. A., Lee, R. K., Miragliotta, G., Nam, S. Y., Podack, E. R. (1996) Structure and expression of murine CD30 and its role in cytokine production J. Immunol. 156,442-449[Abstract]
  40. Imura, A., Hori, T., Imada, K., Kawamata, S., Tanaka, Y., Imamura, S., Uchiyama, T. (1997) OX40 expressed on fresh leukemic cells from adult T-cell leukemia patients mediates cell adhesion to vascular endothelial cells: implication for the possible involvement of OX40 in leukemic cell infiltration Blood 89,2951-2958[Abstract/Free Full Text]
  41. Rogers, P. R., Croft, M. (2000) CD28, Ox-40, LFA-1, and CD4 modulation of Th1/Th2 differentiation is directly dependent on the dose of antigen J. Immunol. 164,2955-2963[Abstract/Free Full Text]
  42. Rogers, P. R., Song, J., Gramaglia, I., Killeen, N., Croft, M. (2001) Ox40 promotes Bcl-xL and Bcl-2 expression and is essential for long-term survival of CD4 T cells Immunity 15,445-455[CrossRef][Medline]
  43. Amakawa, R., Hakem, A., Kundig, T. M., Matsuyama, T., Simard, J. J., Timms, E., Wakeham, A., Mittruecker, H. W., Griesser, H., Takimoto, H., Schmits, R., Shahinian, A., Ohashi, P., Penninger, J. M., Mak, T. W. (1996) Impaired negative selection of T cells in Hodgkin’s disease antigen CD30-deficient mice Cell 84,551-562[CrossRef][Medline]
  44. Grell, M., Zimmermann, G., Gottfried, E., Chen, C. M., Grünwald, U., Huang, D. C., Wu Lee, Y. H., Dürkop, H., Engelmann, H., Scheurich, P., Wajant, H., Strasser, A. (1999) Induction of cell death by tumour necrosis factor (TNF) receptor 2, CD40 and CD30: a role for TNF-R1 activation by endogenous membrane-anchored TNF EMBO J 18,3034-3043[CrossRef][Medline]
  45. Hurtado, J. C., Kim, S. H., Pollok, K. E., Lee, Z. H., Kwon, B. S. (1995) Potential role of 4-1BB in T cell activation. Comparison with the costimulatory molecule CD28 J. Immunol. 155,3360-3367[Abstract]
  46. Hurtado, J. C., Kim, Y. J., Kwon, B. S. (1997) Signals through 4-1BB are costimulatory to previously activated splenic T cells and inhibit activation-induced cell death J. Immunol. 158,2600-2609[Abstract]
  47. Miller, R. E., Jones, J., Le, T., Whitmore, J., Boiani, N., Gliniak, B., Lynch, D. H. (2002) 4-1BB-specific monoclonal antibody promotes the generation of tumor-specific immune responses by direct activation of CD8 T cells in a CD40-dependent manner J. Immunol. 169,1792-1800[Abstract/Free Full Text]
  48. Pollok, K. E., Kim, Y. J., Zhou, Z., Hurtado, J., Kim, K. K., Pickard, R. T., Kwon, B. S. (1993) Inducible T cell antigen 4-1BB.Analysis of expression and function J. Immunol. 150,771-781[Abstract]
  49. Shuford, W. W., Klussman, K., Tritchler, D. D., Loo, D. T., Chalupny, J., Siadak, A. W., Brown, T. J., Emswiler, J., Raecho, H., Larsen, C. P., Pearson, T. C., Ledbetter, J. A., Aruffo, A., Mittler, R. S. (1997) 4-1BB costimulatory signals preferentially induce CD8+ T cell proliferation and lead to the amplification in vivo of cytotoxic T cell responses J. Exp. Med. 186,47-55[Abstract/Free Full Text]
  50. Takahashi, C., Mittler, R. S., Vella, A. T. (1999) Cutting edge: 4-1BB is a bona fide CD8 T cell survival signal J. Immunol. 162,5037-5040[Abstract/Free Full Text]
  51. Kawamata, S., Hori, T., Imura, A., Takaori-Kondo, A., Uchiyama, T. (1998) Activation of OX40 signal transduction pathways leads to tumor necrosis factor receptor-associated factor (TRAF)2- and TRAF5-mediated NF-{kappa}B activation J. Biol. Chem. 273,5808-5814[Abstract/Free Full Text]
  52. Ohshima, Y., Tanaka, Y., Tozawa, H., Takahashi, Y., Maliszewski, C., Delespesse, G. (1997) Expression and function of OX40 ligand on human dendritic cells J. Immunol. 159,3838-3848[Abstract]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
Y. Tone, Y. Kojima, K. Furuuchi, M. Brady, Y. Yashiro-Ohtani, M. L. Tykocinski, and M. Tone
OX40 Gene Expression Is Up-Regulated by Chromatin Remodeling in Its Promoter Region Containing Sp1/Sp3, YY1, and NF-{kappa}B Binding Sites
J. Immunol., August 1, 2007; 179(3): 1760 - 1767.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
E. M. Esparza and R. H. Arch
Glucocorticoid-Induced TNF Receptor Functions as a Costimulatory Receptor That Promotes Survival in Early Phases of T Cell Activation
J. Immunol., June 15, 2005; 174(12): 7869 - 7874.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0803401v1
75/2/350    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
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
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Toennies, H. M.
Right arrow Articles by Arch, R. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Toennies, H. M.
Right arrow Articles by Arch, R. H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS