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The Journal of Immunology, 2005, 175: 5629-5636.
Copyright © 2005 by The American Association of Immunologists

CD4-Dependent Signaling Is Required for a Late Checkpoint during Th2 Development Associated with Resistance to Activation-Induced Cell Death1

Zohreh Tatari-Calderone*, Jennifer L. Brogdon{dagger}, Kevin W. Tinsley*, Anahita Ramezani* and David Leitenberg2,*,{ddagger}

* Department of Immunology, The George Washington University, Washington, DC 20037; {dagger} Department of Immunobiology, Yale University, New Haven, CT 06520; and {ddagger} Department of Pediatrics, The George Washington University and Childrens Research Institute, Childrens National Medical Center, Washington, DC 20037


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Previous studies have found that class II-restricted T cells from CD4-deficient mice reconstituted with a tail-less CD4 transgene have a specific defect in the development of Th2 effector cells; however, the reason for this defect was not clear. Following stimulation with a high potency peptide and exogenous IL-4, CD4-dependent signaling is required for optimal generation of a Th2 effector population. However, initial IL-4 and GATA-3 transcription is appropriately induced, suggesting that the initial stages of Th2 development are intact and independent of CD4 after priming with a strong agonist peptide. In addition to the defect in Th2 development, CD4 mutant T cells are also relatively resistant to activation-induced cell death (AICD). Furthermore, inhibition of AICD in wild-type T cells causes a defect in Th2 development similar to that seen in the CD4 mutant T cells. These data support the hypothesis that CD4-dependent signaling pathways regulate a distinct checkpoint in the expansion and commitment phase of Th2 development, which is related to dysregulation of AICD.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The development of Th lymphocyte effector function is a multistep process that occurs over several days following initial recognition of peptide ligand presented by APCs. Over the last several years it has become clear that Th subset development is regulated by a complex interplay between TCR, costimulatory and cytokine receptor signaling events, which in turn regulate the expression and activity of a variety of transcription factors that control the distinct patterns of gene expression necessary for Th2 effector cell function (1, 2, 3). Despite extensive study, many issues regarding the relative roles, timing, and potential cross-talk between TCR, costimulatory and cytokine receptor signals involved in controlling Th development remain unclear.

CD4 plays an important role in regulating T cell activation, by serving as a coreceptor for the T cell Ag receptor during initial Ag recognition and promoting the activation and recruitment of the src family kinase lck to the CD3/TCR complex. In addition, CD4 has been shown to play a less well-understood role in regulating the function and development of effector and memory T lymphocytes (4, 5, 6). Several years ago, we and other groups independently found that CD4-dependent signaling was required for the optimal development of IL-4-secreting Th2 effector cells in vitro and in vivo but not for the development of Th1 effector cells (7, 8, 9). The specific failure in Th2 development was observed in class II MHC-restricted, CD4-deficient T cells (or CD4-deficient T cells reconstituted with a tail-less CD4 transgene), as well as in situations where wild-type CD4+ T cells were primed with peptide presented by mutant class II MHC molecules unable to interact with CD4. Surprisingly, the requirement for CD4 expression was not overcome by the addition of large amounts of exogenous IL-4 and/or following stimulation with anti-CD3 or strong agonist peptides which do not require CD4 coreceptor function (8, 9). These data suggest that the role of CD4 in promoting Th2 development is a relatively late event in the differentiation pathway.

In support of this hypothesis, we have found that CD4 mutant and wild-type T cells produce similar levels of IL-4 and GATA-3 messenger RNA early after priming with a strong agonist peptide and exogenous IL-4, despite exhibiting a defect in Th2 cytokine production upon restimulation. These data further indicate that initial Th2 priming events are intact in the absence of CD4 signaling and that CD4-dependent signaling pathways define a previously unrecognized late checkpoint in Th2 development, which is independent of a coreceptor requirement for initial TCR activation. Coincident with the defect in Th2 development there is also a significant defect in Fas signaling and activation-induced cell death (AICD)3 in CD4 mutant T cells. Our data suggest that the defect in AICD is linked to Th2 development because inhibition of AICD after T cell priming in wild-type T cells inhibits Th2 development and IL-4 production similar to that seen in CD4 mutant T cells. We propose a model in which the differential susceptibility to AICD during Th development is a critical selection step, which enriches for committed Th2 effector cells from a diverse pool of Th2 precursor cells.


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

AND TCR transgenic mice in which CD4+ T cells express a TCR specific for carboxyl terminus of moth cytochrome c (pMCC) peptide in the context of I-Ek or I-Eb have been previously described (10), and are maintained in our breeding colony as heterozygotes on a B10.BR background. The CD4–/– {delta}cyt mice were originally provided by D. Littman (New York University, New York, NY) (11) and were backcrossed seven to nine times onto a B10.BR background with the AND TCR transgenic mice as previously described (9).

Purification of APC and CD4+ T cells

T cell-depleted APC were prepared by Ab-mediated complement lysis from total splenocytes as described (9). The APC were treated with 50 µg/ml mitomycin C (Sigma-Aldrich) before use. CD4+CD8– T cells from lymph nodes and spleens of transgenic mice were isolated by immunomagnetic negative selection, using Abs against CD8, CD32/CD16, B220, and MHC class II, followed by incubation with anti-mouse and anti-rat Ig-coated magnetic beads (Polysciences). Purity of the recovered V{alpha}11+/CD4+ T cells as determined by staining with anti-CD4 and anti-V{alpha}11 mAb is usually 85–95%.

T cell stimulation and detection of IL-4 and IFN-{gamma} production

Induction of naive T cell differentiation was performed as previously described with slight modifications. Briefly, purified T cells (0.5 x 106/ml) from AND TCR transgenic wild-type or {delta}cyt mice and mitomycin C- treated, T cell-depleted splenocytes (1 x 106 cell/ml) were incubated with 5 µg/ml pMCC (agonist peptide of moth cytochrome c (81–103), pMCC = VFAGLKKANERADLIAYLKQATK) under neutral conditions in the presence of rmIL-2 (25 U/ml) or under Th2 skewing conditions (with IL-2, rmIL-4 (10–20 ng/ml) (Endogen), and anti-IFN-{gamma} (XMG1.2) (2.5 µg/ml) (Harlan Bioproducts), or Th1 skewing conditions (with IL-2, rmIL-12 (10 ng/ml), anti-IL-4 (11B11)). After 4 days of priming, T cells were harvested, and dead cells were removed by using gradient centrifugation. Viable cells were then incubated for a rest period of 2 days with fresh APCs only. For the secondary culture, rested T cells (0.5 x 106/ml) were restimulated with pMCC (5 µg/ml) and fresh APC (1 x 106/ml) for an additional period of 2 days. Cytokine concentrations in the supernatants were determined by ELISA kit (Pierce-Endogen). Supernatants were diluted serially in duplicate, and the concentration of cytokine determined in relation to a reference standard supplied by the manufacturer. In some experiments, we added Jo-2 anti-mouse Fas mAb or anti-Fas ligand (FasL) mAb (BD Pharmingen) at varying times after initial stimulation.

Intracellular cytokine analysis was performed following 12–16 h of stimulation with agonist peptide and the addition of monensin (Golgistop; BD Biosciences) for the final 4–6 h of culture. Cells were harvested and surface labeled with anti-CD4 before fixation and permeabilization (Fix and Perm; Caltag Laboratories), and labeling with Abs to IL-4 and IFN-{gamma} (BD Pharmingen).

RNase protection assay

Two or 3 days after stimulation of CD4+ T cells with APC and peptide, the cells were harvested and dead cells removed by gradient centrifugation. In some experiments, CD4+ T cells were further isolated by immunomagnetic negative selection as described above. The purity of CD4+ T cells was then determined by FACS analysis and was >98% by CD4 and V{alpha}11 labeling. Total RNA was isolated using TRIzol (Invitrogen Life Technologies). RNase protection assays were conducted according to the manufacturer’s protocol (BD Biosciences), using multiprobe template sets from the Riboquant multiprobe kit (BD Biosciences).

Semiquantitative RT-PCR

For semiquantitative RT-PCR, 2 µg of total RNA were used to prepare first strand cDNA using SuperScript first strand RT-PCR kit (Invitrogen Life Technologies) following manufacturer’s protocol. The specific primers for FLIP were (from 5' to 3') GTC ACA TGA CAT AAC CCA GAT TGT and antisense (from 5' to 3') GTA CAG ACT GCT CTC CCA AGC ACT. The primers for FasL were (sense 5'to 3') ATC CCT CTG GAA TGG GAA GA and (antisense 5'to 3') CCA TAT CTG TCC AGT AGT CG. Primers for {beta}2-microglobulin were (sense 5' to 3') TGA CCG GCT TGT ATG CTA TC and (antisense 5' to 3') CAG TGT GAG CCA GGA TAT AG. Concentrations of input cDNAs were equalized by comparing different dilutions with the band intensities of {beta}2-microglobulin amplification products. The integrity of the PCR was controlled by parallel amplification of plasmid pMus3 (gift of Dr. D. Shire, Sanofi Recherche, Labège, France, and N. Noben-Trauth, George Washington University, Washington, D.C.).

Northern blot analysis

After 2 days in the primary culture, cells were harvested and dead cells were moved by gradient centrifugation. Total cellular RNA was isolated using TRIzol reagent (Invitrogen Life Technologies), and 10 µg of total RNA from each sample were fractionated on 1.2% agarose/formaldehyde gels, transferred to {zeta}-Probe GT membranes (Bio-Rad), and hybridized with the indicated cDNA probes in QuickHyb buffer (Stratagene). The GATA-3 construct was kindly provided by R. Flavell (Yale University, New Haven, CT). UV shadowing of the membrane to visualize 28S and 18S rRNA was performed to ensure equivalent sample loading.

Immunoprecipitation and Western blotting

CD4+ T cells (106/ml) have been primed with agonist peptide and APC (2 x 106) for 3 days. Cells were harvested and starved for 2 h in a medium culture without cytokines. They are then stimulated with different concentrations of IL-4 for 10 min. Cells were then lysed in ice-cold lysis buffer (20 mM Tris (pH 7.2), 1% Nonidet P-40, 150 mM NaCl, 1 mM MgCl2, 1 mM EGTA) containing protease and phosphatase inhibitors (10 mM Na4P2O7, 10 mM H20, 1 mM Na3VO4, 50 mM NaF, 1 mM PMSF, 10 µg/ml aprotinin). Cell lysates are pretreated with anti-STAT-6 and JAK-3 Abs (Santa Cruz Biotechnology and Upstate Biotechnology, respectively). Immunoprecipitates were washed four times and analyzed for tyrosine phosphorylation by Western blot. Phosphotyrosine-containing proteins were detected by blotting with anti-phosphotyrosine mAb (4G10; Upstate Biotechnology) followed by goat anti-mouse IgG HRP conjugate (Bio-Rad) and detected by ECL as described by the manufacturer (Amersham Biosciences).

AICD analysis

T cell from TCR transgenic mice, wild-type, or {delta}cyt were stimulated with mitomycin C-treated T cell-depleted splenocytes (106/ml) and 5 µg/ml pMCC under neutral or Th2 skewing conditions as described above for 3 days. T cells were harvested and dead cells were removed by using gradient centrifugation. Viable cells were then incubated for an additional 24 h with 10 U/ml rmIL-2. For the secondary culture, T cells (5 x 105/ml) were restimulated with fresh APC (106/ml) and pMCC (5 µg/ml) for 16 h. Cells are then harvested and stained with annexin-FITC and propidium iodide-PE using annexin V-FITC apoptosis detection kit II (BD Biosciences) and CD4-allophycocyanin (BD Biosciences) according to the manufacturer’s instructions. In some experiments, 150 ng/ml rhsFasL and 1 µg/ml cross-linker (Alexis Biochemicals Axxora) were added at the time of restimulation. Analysis was based on a CD4+ cell gate. Data were collected on a FACSCalibur (BD Biosciences) and analyzed with CellQuest software.

Caspase 8 activity analysis

To evaluate caspase 8 activation after restimulation with agonist peptide, CD4 wild-type and {delta}cyt blasts were generated and restimulated with agonist peptide as described above in the AICD assay. After 16 h of restimulation, the cells were washed and stained for caspase 8 activity using carboxyfluorescein FLICA, apoptosis detection kit, caspase assay (Immunochemistry Technologies). The apoptotic cells containing cleaved caspase substrate were detected in the FL1 channel. Cells are analyzed by gating on CD4+ T cells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD4 regulation of Th subset differentiation

To investigate the role of CD4 in regulating peripheral T cell differentiation, we have used T cells from CD4-deficient mice, reconstituted with a mutant CD4 transgene, which contains a deletion of the cytoplasmic tail of CD4 (CD4 {delta}cyt) and inter-crossed with AND TCR transgenic mice. T cells from AND TCR transgenic mice recognize an agonist peptide from moth cytochrome c (pMCC) in the context of I-Ek MHC molecules and are activated in the absence of CD4 coreceptor function (9, 12). Importantly, thymic development of functional "CD4 lineage" AND TCR transgenic T cells also occurs relatively efficiently in the absence of CD4 signaling when selected on a homozygous I-Ek background (9, 13).

To evaluate the developmental potential of these cells, purified CD4+CD8– T cells from the spleen and lymph nodes were primed in vitro with agonist peptide (pMCC) under neutral or Th2 skewing conditions. After 4 days of stimulation, and a 2 day "rest" period, the cells were then harvested and normalized for cell number and restimulated with pMCC for an additional 48 h. In cell cultures stimulated under Th2 skewing conditions, there is a significant decrease in IL-4, IL-5, and IL-10 production in CD4 {delta}cyt T cells compared with the wild-type control cells, whereas IFN-{gamma} production is suppressed similarly in both groups (Fig. 1). In contrast, when the cells were primed under neutral or Th1 skewing conditions, both wild-type CD4 and CD4 {delta}cyt cells produce a similar amount of IFN-{gamma} (Fig. 1). Th2 cytokines are not significantly induced in both wild-type and CD4 mutant T cells following stimulation with agonist peptide under neutral conditions.



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  		FIGURE 1. Specific defect in Th2 development in the absence of CD4 signaling. Purified CD4 T cells from wild-type (WT) or {delta}cyt mice expressing AND TCR transgene were primed with 5 µg/ml agonist peptide (pMCC) and 25 U/ml IL-2 under Th2, Th1, or neutral skewing conditions for 4 days as described in Materials and Methods. Cells were then washed and rested for an additional 2 days before challenging with agonist peptide and APC. A, After 48 h, culture supernatants were collected and analyzed for IL-4, IFN-{gamma}, IL-5, and IL-10 by ELISA. This experiment is representative of more than three independent experiments. B, Flow cytometric intracellular cytokine analysis of cells primed under Th2 or Th1 conditions after overnight stimulation with agonist peptide and APC. These data are representative of three independent experiments.

 
Consistent with previous results, this data suggests that CD4-dependent signaling is not required for the development of Th1 effector cells, yet is critical for the optimal development of Th2 effector cells (9). Although the production of Th2 cytokines is decreased in the CD4 mutant T cells, it is important to note that Th2 development is not completely abrogated. As seen in the ELISA data and following intracellular single cell analysis of cytokine production when the CD4 mutant T cells are primed under Th2 conditions, IFN-{gamma} production is suppressed and there is continued production of Th2-associated cytokines, although at a much lower level than in wild-type CD4 T cells (Fig. 1, A and B). Thus, it appears that in the absence of CD4-dependent signaling, cells are not converted into Th1 or Th0 cells but simply fail to develop as efficiently into a population of Th2 effector cells capable of high rate cytokine synthesis.

Defect in Th2 development is independent of initial TCR signal transduction events

The defect in Th2 differentiation after priming with a strong agonist peptide does not appear related to a defect in initial TCR-mediated signals, because the agonist peptide used in these studies is able to stimulate early biochemical signaling events in the absence of CD4 expression (9). Consistent with this finding, we also found no defect in cytokine gene transcription early after priming. As shown by RNase protection assays, wild-type and CD4 {delta}cyt cells produced equivalent levels of IL-4 mRNA 48 h after initial priming with agonist peptide plus exogenous IL-4 (Fig. 2A).



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  		FIGURE 2. Induction of early IL-4 and GATA-3 transcription is not dependent upon CD4 signaling. Purified WT or {delta}cyt CD4 T cells were stimulated with pMCC and the indicated IL-4 concentrations. A, After 48 h, the CD4 T cells were harvested, and total RNA was isolated. Cytokine RNA levels were analyzed by RNase protection assay (RPA); 10 µg of total RNA were used in each reaction. Equal loading of RNA was assessed using the housekeeping genes L32. B, Purified CD4 WT or {delta}cyt T cells were stimulated as above, and total RNA was extracted at day 3 or day 4 and assessed for GATA-3 expression by Northern blot; 10 µg of RNA were loaded per lane, and equal sample loading was confirmed by UV light shadowing of 28S and 18S rRNA. These data are representative of two independent experiments.

 
As GATA-3 is both a critical regulator as well as a hallmark of Th2 development, we also examined GATA-3 expression in the CD4 mutant T cells. Similar to the RNase protection assay data, induction of GATA-3 transcription following agonist peptide and IL-4 stimulation is also unchanged in wild-type and CD4 {delta}cyt cells (Fig. 2B). Thus, these results suggest that the initial signaling events induced by TCR recognition of peptide by CD4 {delta}cyt T cells in conjunction with exogenous IL-4 are sufficient for the development of a Th2 precursor population. It also suggests that the defect in Th2 differentiation, seen in the absence of CD4 signaling, occurs during the maturation and/or clonal expansion phase of Th2 development. In total, this data lead to a model in which there are at least two checkpoints that regulate Th2 development. One is during the initial development of a Th2 precursor population, which is CD4-signaling independent if T cells are activated using a high potency stimulus. The other is at a later checkpoint that promotes the selection of a mature Th2 effector cell population and is CD4 dependent.

IL-4R signaling

To examine factors important in Th2 development that may be influenced by CD4 signaling, we initially focused on IL-4R signaling. As indicated in Figs. 1 and 2, several aspects of IL-4R signaling appear grossly intact as shown by efficient suppression of IFN-{gamma} production and induction of GATA-3 transcription. A more direct analysis of early IL-4R signaling events also reveals that STAT-6 and JAK-3 phosphorylation are induced similarly in both CD4 wild-type and CD4 {delta}cyt T cells (Fig. 3). This data indicates that proximal IL-4R signaling events are not affected in the absence of CD4 signaling.



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  		FIGURE 3. Tyrosine phosphorylation of STAT-6 and JAK-3 following IL-4 signaling is independent of CD4 expression. Purified CD4 {delta}cyt or WT controls were stimulated with agonist peptide in the presence or absence of IL-4. Three days after stimulation, the viable T cells were isolated and rested for 3 h and then 5 x 106 cells group were restimulated with the indicated dose of IL-4 for 10 min and immunoprecipitated with antisera to STAT-6 or JAK-3. Immunoprecipitated proteins were separated by 8% SDS-PAGE and assessed for tyrosine phosphorylation by Western blot. The same membranes were reprobed for total STAT-6 or JAK-3. These data are representative of three independent experiments.

 
CD4 signaling regulates AICD

Consistent with our previous observations that CD4 mutant T cells are efficiently activated by the agonist peptide used in these studies, CFSE labeling before stimulation with peptide in the presence of exogenous IL-2 reveals that CD4 {delta}cyt T cells exhibit a significantly greater rate of cell division in cells primed under both neutral and Th2 skewing conditions (Fig. 4). A possible explanation for this finding is that the CD4 mutant T cells were more resistant to AICD than wild-type cells resulting in prolonged survival and enhanced cell division. Indeed, when wild-type or CD4 {delta}cyt T cell blasts were restimulated with agonist peptide, the wild-type cells underwent apoptotic cell death to a much greater extent than the CD4 mutant T cells (Fig. 5A). Importantly, although the overall level of cell death is lower in cells primed for 3 days under Th2 conditions there remains a significant decrease in death in the absence of CD4 signaling compared with wild-type CD4 T cells (Fig. 5B). This defect in AICD is also seen when the CD4 mutant blasts are stimulated with recombinant soluble FasL followed by cross-linking (Fig. 5C). These data indicate that there is a defect in AICD in the CD4 mutant T cells, which is at least due in part to a defect in the Fas signaling pathway.



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  		FIGURE 4. Increased cell division in CD4 mutant T cells. Purified CD4+ or CD4 {delta}cyt T cells were labeled with 0.5 µM CFSE and stimulated with agonist peptide (pMCC) plus IL-2 under neutral or Th2 skewing conditions as described in Fig. 1. Dilution of CFSE labeling is dependent on cell division and was assessed by flow cytometry at 3 days after initial stimulation. The solid line denotes wild-type cells and the dotted line CD4 {delta}cyt T cells. These data are representative of three independent experiments.

 


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  		FIGURE 5. CD4 signaling is required for AICD. Purified CD4 {delta}cyt and wild-type (WT) T cells were primed with agonist peptide (pMCC) for 3 days under neutral Th2 skewing conditions as described in Fig. 1, then harvested and recultured with 10 U/ml IL-2 in the absence of peptide stimulation of 24 h. AICD was then induced by restimulation of CD4 T cells with 5 µg/ml agonist peptide (pMCC) (A and B) or following exposure to soluble FasL plus cross-linking (C). After 16 h, cells were harvested and stained with FITC-annexin V, PE-propidium iodide, and allophycocyanin-CD4. CD4+ cells were gated for analysis. Apoptotic cells were defined by annexin V and propidium iodide labeling.

 
One possible explanation for the decreased AICD in the CD4 mutant T cells is failure to express components of the death receptor signaling pathways (e.g., FasL), and/or overexpress negative regulators of AICD (e.g., FLIP). In semiquantitative RT-PCR assays, and/or in RNase protection assays we have not found any evidence for decreased expression of Fas, FasL, Fas-associated death domain protein, caspase 8, TRAIL, TNFR, or increased expression of FLIP (Fig. 6). Consistent with this data, we have found that FasL expression as well as expression of other death receptor signaling components is more sustained over time in CD4 mutant cells compared with wild-type T cells. Typically, expression of FasL is transient on T cells because membrane expression allows cis-interactions with Fas resulting in cell death of the FasL expressing cells. When FasL membrane expression was examined in wild-type and CD4 mutant T cells, we found that expression peaked at day 1 after restimulation of wild-type T cells but was not detectable at day 2. In contrast, the CD4 mutant T cells exhibited sustained detectable levels of FasL expression through day 2 (Fig. 7). Flow cytometric analysis of Fas expression was also equivalent in CD4 wild-type and CD4 mutant T cells (data not shown). In total, this data suggests that in the absence of CD4-dependent signaling, there is a defect in the signaling events mediating AICD independent of Fas and FasL expression.



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  		FIGURE 6. Similar expression levels of AICD signaling components and regulatory proteins in wild-type and CD4 mutant T cells. A, Purified wild-type and mutant CD4 T cells were stimulated with agonist peptide for 3 days. Total RNA was extracted and subjected to an RNase protection assay using commercially available reagents (mAPO-3 kit; BD Pharmingen). B, RNA from the same cell populations was assessed for FasL and FLIP mRNA using a semiquantitative RT-PCR technique using the pMus3 plasmid-containing {beta}2-microglobulin as an internal competitor.

 


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  		FIGURE 7. Sustained increase in FasL expression in CD4 mutant T cells. Purified wild-type and dcyt CD4 T cells were stimulated as in Fig. 5 and then restimulated with agonist peptide. Flow cytometric analysis of FasL expression in wild-type (dark line) and {delta}cyt (pale line) CD4 T cells was done 1 and 2 days after restimulation. Data shown are FasL expression using a "gate" on CD4+ T cells. The shadowed histogram represents labeling with an isotype control Ab.

 
The defect in Fas signaling in the CD4 mutant T cells appears to occur before activation of caspase 8 in the cell death pathway, because caspase 8 activation was also significantly decreased in the CD4 mutant cells (Fig. 8). Activation of caspase 8 occurs early on in the death receptor signal transduction cascade upon assembly of the death-inducing signaling complex (DISC). This process requires appropriate oligomerization of death effector domains within Fas-associated death domain protein following association with Fas. Thus failure to activate caspase 8, in conjunction with appropriate Fas and FasL expression, suggests that the defect in AICD in the CD4 mutant T cells may be related to dysregulation of the assembly or composition of the DISC.



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  		FIGURE 8. Purified wild-type or dcyt CD4 T cells were primed under neutral conditions as described in Fig. 5 and then left unstimulated (–) or restimulated with agonist peptide (+) for 14 h. Caspase 8 activation was detected using a fluorescent reagent specific for active caspase 8 (FAM-LETD-FMK) obtained from Immunochemistry Technologies. This reagent is cell permeable and binds to active caspase 8. In the absence of caspase 8 activity unbound reagent is washed away. The percentage of cells containing fluorochrome-bound active caspase 8 was determined by flow cytometric analysis after gating on CD4+ T cells.

 
CD4 mutant T cell defect in AICD is linked to the defect in Th2 development

In experiments designed to link the defect in AICD described above to the defect in Th2 development described earlier, we have inhibited AICD using anti-Fas or anti-FasL Abs in wild-type CD4 cells and assessed the subsequent impact on Th2 development. Initial experiments were done to confirm that anti-Fas and anti-FasL Abs would block AICD in wild-type CD4 T cells to the low levels seen in the {delta}cyt T cells following Ag restimulation (Fig. 9, A and C). Subsequent experiments went on to assess the effect of blocking AICD with Fas or FasL Ab during Th2 priming. In these experiments, wild-type CD4+ T cells were first stimulated under Th2 cytokine skewing conditions as described in Fig. 1. One or 2 days after the initial stimulation, anti-Fas (Jo-2) or anti FasL (MFL3) was added. When cells were analyzed for IL-4 production after being treated with Ab during priming only and then restimulated with peptide, there was a defect in IL-4 production similar to that seen in the CD4 {delta}cyt T cells after challenge (Fig. 9, B and D). There was little or no effect on Th1 development as shown by IFN-{gamma} production in cells primed under neutral or Th1 skewing conditions following treatment with anti-Fas or anti-FasL (Fig. 9, B and D). No significant IFN-{gamma} production was detected in cells primed under Th2 conditions upon treatment with anti-Fas or anti-FasL as shown by ELISA or intracellular cytokine analysis (data not shown).



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  		FIGURE 9. Inhibition of AICD causes decreased Th2 development. A and C, WT and {delta}cyt CD4 T cells were assayed for AICD as described in Fig. 5 in the presence or absence of anti-Fas (2 µg/ml Jo-2) or anti-FasL (5 µg/ml) added during restimulation with agonist peptide. B, Purified CD4 T cells from AND TCR transgenic mice were stimulated under neutral or Th2 skewing conditions as in Fig. 1. Two days after initial stimulation, 2 µg/ml anti-Fas or isotype control Ab were added to the cultures. The cells were maintained in culture for an additional 2 days, then harvested, washed, and rested for 2 days before restimulation with agonist peptide and APC. After 48 h, culture supernatants were collected and analyzed for the detection of IL-4 or IFN-{gamma} production by ELISA. IFN-{gamma} production was below detectable limits in cells primed under Th2 skewing conditions under all conditions. D, Cells were stimulated under Th2 or Th1 skewing conditions as described in Fig. 1, and 1 day after priming 5 µg/ml anti-FasL or isotype control Ab were added to the cultures. Following 4 days of stimulation, the cells were harvested, washed, and restimulated for 2 additional days with agonist peptide and APC only. Supernatants were collected and analyzed for IL-4 and IFN-{gamma} by ELISA. These data are representative of at least two independent experiments.

 
In total, these data suggest that inhibition of AICD after initial priming of naive CD4 T cells specifically inhibits the development of Th2 effector cells. Furthermore, this data suggests that the defects we have described in the regulation of AICD in the absence of CD4 signaling are related to the defect in Th2 development.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The role of CD4 as a coreceptor that promotes initial T cell Ag receptor-mediated activation events is well established. However, the role of CD4 in regulating previously activated T cells and ongoing immune responses is less well understood. In the present study, we have found that CD4 signaling is required for a distinct checkpoint during Th2 effector cell development, which is independent of initial T cell stimulation. Although production of Th2-associated cytokines is significantly diminished in T cells primed under Th2 conditions in the absence of CD4 signaling, initial Th2 development appeared intact as defined by the appropriate induction of IL-4 and GATA-3 transcription. These findings are quite distinct from other studies in itk, SAP, and fyn-deficient mice in which Th2 development was compromised in cells primed under neutral conditions, but was recovered upon the addition of exogenous IL-4 in conjunction with a high potency TCR stimulus (14, 15, 16). In these cases, the defect in Th2 development appears related to initial T cell signal transduction events that are required for IL-4 production. In contrast, CD4 mutant T cells appear to be initially primed to undergo Th2 differentiation after stimulation with a strong agonist peptide and exogenous IL-4, but do not become a fully developed Th2 effector population when compared with wild-type CD4 T cells.

In addition to the defect in Th2 development, T cells unable to signal through CD4 are relatively insensitive to AICD and Fas-mediated death receptor signaling. The defect in Th2 development and AICD appear linked as shown in experiments in which inhibition of Fas-mediated cell death in wild-type T cells also results in a defect in Th2 development. These data suggest that the appropriate regulation of AICD during Th development is an important factor in controlling the selection of different effector cell subset populations. It has been recognized for some time that mature Th2 effector populations are relatively resistant to AICD (17, 18). We propose that the development of this resistant phenotype is an important factor in enriching for committed Th2 effector cells in a mixed population. In this model, cells that are more committed to the Th2 developmental pathway will be correspondingly more resistant to AICD and will have a survival advantage over less committed Th cells. In Th precursor cells that do not signal efficiently through CD4, there is an inappropriate resistance to AICD removing the survival advantage of the more committed Th2 cells. This creates a more mixed population of effector cells after several days of cell expansion (and cell death), with a relative decrease in the proportion of high-rate IL-4 producing Th2 effector cells.

This model is supported by another report describing the paradoxical effect of IFN-{gamma} in promoting Th2 development (19). Bocek et al. found that cells in an IFN-{gamma}-deficient environment were less efficient in developing Th2 effector cells. Similar to our model, the authors proposed that IFN-{gamma} promotes susceptibility to AICD in uncommitted Th precursors, which allows more committed Th2 cells that are relatively resistant to AICD to have a selective advantage. The regulatory role of Fas and AICD in selecting for Th effector cell subsets is also consistent with data in young MRL/lpr (Fas deficient) mice which exhibit a propensity toward Th1 development and IFN-{gamma} production compared with MRL control mice (20).

The defect in AICD seen in the absence of CD4 signaling is similar to that observed in some previous reports; however, the molecular basis for this defect has not been clear (21, 22). In contrast to the results of Hamad et al. (22), we do not find any evidence of a decrease in FasL expression in the CD4 mutant T cells. Instead, our data suggests that CD4 signals are important to render Fas competent to initiate death receptor signaling and initiate caspase activation. It has been known for some time that expression of Fas and FasL is not sufficient for the induction of cell death in T lymphocytes, but that additional signals from the CD3/TCR complex provide a critical competency signal (23, 24). The precise nature of this signal is not yet known; however, it closely correlates with the oligomerization of Fas molecules and Fas residence in lipid raft membrane microdomains (25). Our current data suggest that CD4 signaling provides a necessary part of this competency signal.

Although CD4 signaling may affect other components of death receptor signaling, our hypothesis that CD4 promotes oligomerization of the Fas signaling macromolecular complex, is consistent with our previous data indicating that CD4 signaling is required for the association of the TCR complex with lipid rafts after T cell activation (26). CD4 may play a similar role in promoting Fas association with lipid rafts and/or in promoting Fas oligomerization. The mechanism(s) by which CD4 (and presumably CD4-associated lck) may promote oligomerization and effective Fas signaling are also not yet clear. One possibility is that CD4 signaling provides a positive signal required for DISC oligomerization. For example in the absence of CD4 there may be a defect in protein kinase C activation, which has been suggested to be an important signaling event in developing competence for Fas-mediated cell death following CD4 and/or CD3/TCR cross-linking (24, 27). Alternatively, CD4 signaling may be required to inhibit signaling pathways that constitutively down-regulate Fas signaling pathways. One possibility in this scenario would be inappropriate activation of the ras/raf/ERK pathway due to a requirement of CD4-associated lck for the activation of dok family members and ras-GAP activation, which normally would inhibit ERK activation. Inappropriate activation of ERK has been associated with resistance to Fas-mediated cell death (28); thus CD4-dependent signaling may be normally required to down-regulate ERK activation, which would in turn enhance sensitivity to Fas-mediated cell death.

Regardless of the mechanism by which CD4 promotes AICD, our data suggest that this is a critical step in the development and selection of Th effector populations. These data may have important implications for controlling Th development in vivo. Following initial T cell priming, developing T cells will typically be in a complex microenvironment with a variety of competing signals and cytokines capable of influencing Th1 and Th2 effector cell development. Our finding that dysregulation of AICD in CD4 mutant T cells affects Th2 development, suggests that the differential development of susceptibility and resistance to AICD seen in Th1 and Th2 cells can be a factor in selecting for biased Th1 and Th2 effector populations. Therapeutic strategies that alter susceptibility to AICD during the Th development may be an effective means of biasing outcomes of T cell activation.


   Acknowledgments
 
We thank Drs. Mark Williams, Achsah Keegan, Stan Vukmanovic, and Stephanie Constant for critical review of the manuscript and for helpful discussions.


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


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

1 This work was supported in part by grants from the Arthritis Foundation and the National Institutes of Health (AI42963). Back

2 Address correspondence and reprint requests to Dr. David Leitenberg, Department of Immunology, The George Washington University, 2300 I Street, NW, Washington, DC 20037. E-mail address: dleit{at}gwu.edu Back

3 Abbreviations used in this paper: AICD, activation-induced cell death; FasL, Fas ligand; DISC, death-inducing signaling complex. Back

Received for publication November 22, 2004. Accepted for publication July 27, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Leitenberg, D., K. Bottomly. 1999. Regulation of naive T cell differentiation by varying the potency of TCR signal transduction. Semin. Immunol. 11:283.-292. [Medline]
  2. Murphy, K. M., S. L. Reiner. 2002. The lineage decisions of helper T cells. Nat. Rev. Immunol. 2:933.-944. [Medline]
  3. Ho, I.-C., L. H. Glimcher. 2002. Transcription: tantalizing times for T cells. Cell 109:S109.-S120.
  4. Farber, D., M. Luqman, O. Acuto, K. Bottomly. 1995. Control of memory CD4 T cell activation: MHC class II molecules on antigen presenting cells and CD4 ligation inhibit memory but not naive CD4 T cells. Immunity 2:249.-259. [Medline]
  5. Stumbles, P., D. Mason. 1995. Activation of CD4+ T cells in the presence of a non-depleting monoclonal to CD4 induces a Th2-type response in vitro. J. Exp. Med. 182:5.-13. [Abstract/Free Full Text]
  6. Chirmule, N., A. Avots, S. M. Lakshmitamma, S. Pahwa, E. Serfling. 1999. CD4-mediated signals induce T cell dysfunction in vivo. J. Immunol. 163:644.-649. [Abstract/Free Full Text]
  7. Fowell, D. J., J. Magram, C. W. Turck, N. Killeen, R. M. Locksley. 1997. Impaired Th2 subset development in the absence of CD4. Immunity 6:559.-569. [Medline]
  8. Brown, D. R., N. H. Moskowitz, N. Killeen, S. L. Reiner. 1997. A role for CD4 in peripheral T cell differentiation. J. Exp. Med. 186:101.-107. [Abstract/Free Full Text]
  9. Leitenberg, D., Y. Boutin, S. Constant, K. Bottomly. 1998. CD4 regulation of T cell receptor signaling and T cell differentiaion following stimulation with peptides of different affinites for the T cell receptor. J. Immunol. 161:1194.-1203. [Abstract/Free Full Text]
  10. Kaye, J., M.-L. Hsa, M.-E. Sauron, S. C. Jameson, N. Gasciogne, S. M. Hendrik. 1989. Selective development of CD4+ T cells in transgenic mice express a class II MHC restricted antigen receptor. Nature 341:746.-749. [Medline]
  11. Killeen, N., D. R. Littman. 1993. Helper T-cell development in the absence of CD4–p56lck association. Nature 364:729.-732. [Medline]
  12. Yelon, D., K. L. Schaefer, L. J. Berg. 1999. Alterations in CD4-binding regions of the MHC class II molecule I-Ek do not impede CD4+ T cell development. J. Immunol. 162:1348.-1358. [Abstract/Free Full Text]
  13. Matechak, E. O., N. Killeen, S. M. Hedrick, B. J. Fowlkes. 1996. MHC class II-specific T cells can develop in the CD8 lineage when CD4 is absent. Immunity 4:337.-347. [Medline]
  14. Miller, A. T., H. M. Wilcox, Z. Lai, L. J. Berg. 2004. Signaling through Itk promotes T helper 2 differentiation via negative regulation of T-bet. Immunity 21:67.-80. [Medline]
  15. Cannons, J. L., L. J. Yu, B. Hill, L. A. Mijares, D. Dombroski, K. E. Nichols, A. Antonellis, G. A. Koretzky, K. Gardner, P. L. Schwartzberg. 2004. SAP regulates T(H)2 differentiation and PKC-{theta} mediated activation of NF-{kappa}B1. Immunity 21:693.-706. [Medline]
  16. Davidson, D., X. Shi, S. Zhang, H. Wang, M. Nemer, N. Ono, S. Ohno, Y. Yanagi, A. Veillette. 2004. Genetic evidence linking SAP, the X-linked lymphoproliferative gene product to src-related kinase FynT in T(H)2 cytokine regulation. Immunity 21:707.-717. [Medline]
  17. Zhang, X., T. Brunner, L. Carter, R. W. Dutton, P. Rogers, L. Bradley, T. Sato, J. C. Reed, D. Green, S. L. Swain. 1997. Unequal death in T helper cell (Th) 1 and Th2 effectors: Th1, but not Th2, effectors undergo rapid Fas/FasL-mediated apoptosis. J. Exp. Med. 185:1837.-1849. [Abstract/Free Full Text]
  18. Varadhachary, A. S., S. N. Perdow, C. Hu, M. Ramanarayanan, P. Salgame. 1997. Differential ability of T cell subsets to undergo activation-induced cell death. Proc. Natl. Acad. Sci. USA 94:5778.-5783. [Abstract/Free Full Text]
  19. Bocek, P., G. Foucras, W. E. Paul. 2004. Interferon {gamma} enhances both in vitro and in vivo priming of CD4+ T cells for IL-4 production. J. Exp. Med. 199:1619.-1630. [Abstract/Free Full Text]
  20. Zhang, X. R., L. Y. Zhang, S. Devadas, L. Li, A. D. Keegan, Y. F. Shi. 2003. Reciprocal expression of TRAIL and CD95L in Th1 and Th2 cells: role of apoptosis in T helper subset differentiation. Cell Death Differ. 10:203.-210. [Medline]
  21. Maroto, R., X. Shen, R. Konig. 1999. Requirement for efficient interactions between CD4 and MHC class II molecules for survival of resting CD4+ T lymphocytes in vivo and for activation-induced cell death. J. Immunol. 162:5973.-5980. [Abstract/Free Full Text]
  22. Hamad, A. R., A. Srikishnan, P. Mirmonsef, C. P. Broeren, C. H. June, D. Pardoll, J. P. Schneck. 2001. Lack of coreceptor allows survival of chronically stimulated double-negative {alpha}/{beta} T cells: implications for autoimmunity. J. Exp. Med. 193:1113.-1121. [Abstract/Free Full Text]
  23. Hornung, F., L. Zheng, M. J. Lenardo. 1997. Maintenance of clonotype specificity in CD95/Apo-1/Fas-mediated apoptosis of mature T lymphocytes. J. Immunol. 159:3816.-3822. [Abstract]
  24. Wong, B., J. Arron, Y. Choi. 1997. T cell receptor signals enhance susceptibility to Fas-mediated apoptosis. J. Exp. Med. 186:1939.-1944. [Abstract/Free Full Text]
  25. Muppidi, J. R., R. M. Siegel. 2004. Ligand-independent redistribution of Fas (CD95) into lipid rafts mediates clonotypic T cell death. Nat. Immunol. 5:182.-189. [Medline]
  26. Balamuth, F., D. Leitenberg, J. Unternaehrer, I. Mellman, K. Bottomly. 2001. Distinct patterns of membrane microdomain partitioning in Th1 and Th2 cells. Immunity 15:729.-738. [Medline]
  27. Algeciras, A., D. H. Dockrell, D. H. Lynch, C. V. Paya. 1998. CD4 regulates susceptibility to Fas ligand- and tumor necrosis factor-mediated apoptosis. J. Exp. Med. 187:711.-720. [Abstract/Free Full Text]
  28. Holmstrom, T. H., I. Schmitz, T. S. Soderstrom, M. Poukkula, V. L. Johnson, S. C. Chow, P. H. Krammer, J. E. Eriksson. 2000. MAPK/ERK signaling in activatied T cells inhibits CD95/Fas-mediated apoptosis downstream of DISC assembly. EMBO J. 19:5418.-5428. [Medline]




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