Published online before print November 21, 2003
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,1
Departments of
* Medicine and
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
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B (NF-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
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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-B (NF-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.
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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.
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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.
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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.
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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.
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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.
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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.
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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)-
[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- 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.
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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- (IFN-) 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.
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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- 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.
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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- 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.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B kinase, and transcription factors, such as NF-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.
Received August 25, 2003; revised October 3, 2003; accepted October 21, 2003.
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B Mol. Cell. Biol. 18,558-565B and IB proteins: new discoveries and insights Annu. Rev. Immunol. 14,649-683[CrossRef][Medline]B and Rel proteins: evolutionarily conserved mediators of immune responses Annu. Rev. Immunol. 16,225-260[CrossRef][Medline]B activation prevents cell death Cell 87,565-576[CrossRef][Medline]B transcription factors and the control of apoptosis Semin. Cancer Biol. 8,113-119[CrossRef][Medline]B by the CD30 receptor is mediated by TRAF1 and TRAF2 Mol. Cell. Biol. 17,1535-1542[Abstract]B transcription factor Blood 87,2443-2449B activation J. Biol. Chem. 273,5808-5814This article has been cited by other articles:
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