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The Journal of Immunology, 1999, 162: 209-214.
Copyright © 1999 by The American Association of Immunologists

Presence or Absence of TGF-ß Determines IL-4-Induced Generation of Type 1 or Type 2 CD8 T Cell Subsets1

Francois Erard2,*, Jose A. Garcia-Sanz{dagger}, Richard Moriggl{ddagger} and Marie-Therese Wild§

* Laboratoire d’Immunologie, Institut de Recherches Cliniques de Montréal, Montréal, Canada; {dagger} Department of Immunology and Oncology Centro Nacional de Biotecnologia-CSIC Universidad Autonoma Campus de Cantoblanco, Madrid Spain; {ddagger} Department of Biochemistry, St. Jude Children’s Research Hospital, Memphis, TN 38101; and § Department of Research, Novartis Pharma, Basel, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD8+ T cells often differentiate into highly cytotoxic cells, secreting a Th1-like or type 1 cytokine pattern characterized by the production of IFN-{gamma}. However, cytotoxic, and in some reports, noncytotoxic, type 2 cells that secrete IL-4, IL-5, or IL-10 instead of IFN-{gamma}, can be generated when CD8+ T cells are primed in the presence of IL-4. Here, we show that IL-4 can also generate typical CD8 type 1 responses. Indeed, while presence of TGF-ß biases the development of CD8 T cells that, then, produce little cytolytic activity and IFN-{gamma}, addition of IL-4 results in the recovery of cytotoxicity and IFN-{gamma} production. The cooperative effects of TGF-ß and IL-4 imply dual functions, not only for IL-4, but also for TGF-ß. Indeed, depending on the presence or absence of IL-4, TGF-ß either inhibits or induces the generation of type 1 CD8+ T cells. Physiologically, the ratio of local IL-4/TGF-ß concentration may therefore be a critical element in determining the outcome of T cell responses to pathogen and autoantigens. It allows CD8 T cells to switch from an immunotolerant state in the presence of only TGF-ß or IL-4, to an immunocompetent proinflammatory type 1 state in the absence or presence of both cytokines.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD4+ T cells of the Th phenotype have long been regarded as the major immune regulatory cells. On the other hand, CD8+ T cells were viewed as CD4-dependent, IFN-{gamma}-secreting cytotoxic T cells, whose main function was the MHC class I-restricted lysis of infected or altered host cells (1). Thus, most studies on cytokine production have focused on the activity of CD4+ T cells. These cells were usually classified in two subsets, Th type 1 and type 2, each associated with the production of characteristic patterns of cytokines contributing to the regulation of different types of immunity. While Th type 1 cells produce mainly IL-2, IFN-{gamma}, and TNF, and direct cell-mediated immune responses, Th type 2 cells produce IL-4, IL-5, and IL-10, and provide help for some B cell responses (2, 3, 4). Abnormalities in the balance of these cytokines, as well as deregulated secretion, have been associated with many immunological disorders including allergy and autoimmune diseases (5). Recently, numerous studies have shown that the effector functions of CD8 T cells overlap those of CD4 T cells much more than previously anticipated. Indeed, it has been reported that CD8+ T cells can also develop in culture as polarized type 2 cells (also called Tc2). These cells can be cytotoxic or, in some circumstances, noncytotoxic, CD8- and provide B cell helper activity (6, 7, 8, 9, 10, 11, 12, 13). Such type 2 cells have been isolated from the blood of patients suffering from lepromatous leprosy (14), as well as from HIV patients (15). They have also been recovered from the bronchial biopsies of asthmatics (16).

The minimal cytokine requirement for the generation of CD8 subsets is similar to that required by CD4 T cells for their differentiation (17). Mitogen and Ag-stimulated naive CD8 cells exposed to IL-2 alone or in combination with IL-12 develop a classical type 1 phenotype, whereas the same cells exposed to IL-4 develop a type 2 phenotype (8, 10, 12). Circumstantial evidence indicates that IL-4 may also be necessary to enable the generation of type 1 cells. Indeed, in studies performed with parasite-infected IL-4-deficient mice, IL-4 is needed to mount protective Th type 1 responses (18, 19). Moreover, increased IFN-{gamma} levels are observed in IL-4-transgenic mice (20). IL-4 is capable of both suppressing (9) or enhancing (21) CD8 T cell cytotoxicity in different circumstances. These data imply that other factors may modulate the differentiating effects of IL-4. TGF-ß is another cytokine associated with similar complex regulation of T cell effector functions. TGF-ß, which is released by a wide variety of cell types including APC’s such as macrophages and dendritic cells, and also by some T cells named Th3 (22, 23), has either growth-inhibitory effects (24, 25) or long-term growth-promoting activity when added to resting CD4 and CD8 T cells (26, 27, 28). Recent studies suggest that TGF-ß may either inhibit the generation of Th type 1 cells, or induce type 1 differentiation when a type 2 response is expected (8, 26, 29, 30). A coregulation of T cell differentiation involving TGF-ß and IL-4 is therefore conceivable and is the focus of this work. Here, we provide evidence that TGF-ß and IL-4 can cooperate to regulate the differentiation of CD8 T cells. Indeed, we find that the generation of cytotoxic type 1 CD8 T cells is suppressed when CD8 precursors are primed in the presence of TGF-ß or IL-4, but induced when both TGF-ß and IL-4 are present.

As STAT6 activation has previously been associated with the IL-4-dependent development of Th type 2 cells (31), we investigated whether this activation was prevented when the role of IL-4 was modified by TGF-ß. Our findings reveal similar levels of activated STAT6 in type 2 and type 1 CD8 T cells primed by IL-4 and IL-4/TGF-ß, respectively.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
CD8+ T cell purification and labeling

Mesenteric lymph nodes from mice were homogenized to yield a single cell suspension. In order to remove CD4+ cells, CD44+ memory T cells (32), B lymphocytes, and FcR+ cells by magnetic separation, the cells were stained with phycoerythrin (PE)-conjugated rat anti-mouse CD4 and CD44 Abs and further incubated with a combination of Dynabeads M-450 (Dynal, Great Neck, NY) coated with sheep anti-mouse IgG and anti-rat IgG. The remaining cells were then stained with FITC-conjugated anti-CD8 (53-6.7, PharMingen, San Diego, CA) and purified as small CD8+ T cells to more than 99% purity by FACS. CD4 expression was measured with phycoerythrin-labeled anti-CD4 (GK1.5, PharMingen).

CD8+ T cell cultures

Microcultures were established as previously described (8) in round-bottom microwells from 96-well plates, with 500 purified resting CD8+ T cells in 200 µl medium containing human rIL-2 (200 U/ml) with or without mouse rIL-4 (500 U/ml) and/or human rTGF-ß2 (10 ng/ml). The amount of TGF-ß used was optimized by titration in order to completely inhibit PMA + IL-2-stimulated CD8 T cell proliferation (data not shown). Three different conditions of activation were used: 1) CD8 T cells from BALB/c mice were stimulated by a mitogen [(PMA, 10 ng/ml) and ionomycin (250 ng/ml)]; 2) CD8 T cells from C57BL/6 mice (H-2b) were cultured in the presence of 4000 irradiated (4000 rad) allogeneic P815 mastocytoma cells (H-2d); 3) CD8 T cells derived from lymphocytic choriomeningitis virus (LCMV)3-specific TCR transgenic C57BL/6 mice were stimulated in the presence of the viral peptide (100 ng/ml) corresponding to the glycoprotein epitope 33-41 of LCMV (33), and 5000 irradiated (5000 rad) EL-4 thymoma cells as syngeneic APC. The cells were harvested from each culture after 6 days (or 12 days for the allogeneic stimulation) and their effector functions were analyzed. The Ag specificity of the response was demonstrated by the fact that no cell would grow in the absence of LCMV peptide. The precursor cells used in all experiments were considered as resting based on the fact that they could not proliferate in IL-2 alone.

Cytolytic activity and cytokine production

Cells harvested on day 6 were either incubated for 3.5 h with 2000 51Cr-labeled P815 target cells in the presence of anti-CD3{epsilon} (25 µg/ml) (8) and the release of 51Cr measured, or recultured (105 cells/200 µl) on flat-bottom plates coated with anti-CD3{epsilon} (25 µg/ml) in the presence of IL-2 (200 U/ml) for 24 h, and the production of cytokines determined by ELISA with Abs and standardized to commercial cytokines (Genzyme, Cambridge, MA).

RNA preparation and Northern blot analysis

Total RNA was prepared using the guanidinium thiocyanate/acid phenol method as described (34). Aliquots of the RNA samples were separated on 1.2% denaturing formaldehyde-agarose gels, followed by RNA staining and transfer to nylon membranes. Northern blots were hybridized using random-primed 32P-labeled cDNA with probes specific for mouse CD8{alpha}, CD8ß (both obtained from P. Cosson, Basel Institute for Immunology, Basel, Switzerland), perforin (35), Fas-L/CD95 (obtained from P. Krammer, DKFZ, Heidelberg, Germany), and ß-actin (36) as described (37).

STAT6 analysis: preparation of cell extracts, electrophoretic mobility shift assays (EMSA) and supershift analysis

Whole cell extracts were prepared from CD8 type 1 and type 2 cell populations by suspension of cell pellets in a buffer containing 400 mM NaCl, 50 mM KCl, 20 mM HEPES (pH 7.9), 1 mM EDTA, 20% glycerol, 1 mM DTT, and the following protease inhibitors: 0.2 mM PMSF, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 100 µM of sodium orthovanadate, and 20 µM phenyl arsine oxide. After freeze-thawing three times, lysates were centrifuged for 15 min at 4°C and 20,800 x g. The supernatants were recovered for bandshift experiments. The protocol for the bandshift assays has been described elsewhere (38). In the bandshift experiments, the following high affinity STAT6-binding site was used: 5'-{gamma}TCAACTTCCCAAGAACAGAA-3' from the Ig {kappa} heavy chain germ-line {epsilon}-promoter (I{epsilon}) (39, 40). The STAT probe was endlabeled with polynucleotide kinase to a specific activity of 8000 cpm/fmol. Equal amounts of proteins from whole cell extracts were introduced into bandshift assays. Proteins were measured at a wave length of 595 nm with the Bradford Protein assay kit (Bio-Rad, Richmond, CA) according to the manufacturer’s instructions. The bandshift signals were quantitated with a PhosphorImager (BIO-IMAGER BAS 2000; Fuji). Three individual experiments were carried out. For supershift analysis, the following Ab was used: polyclonal chicken antiserum directed against the carboxyl-terminal domain of STAT6 (41). The antiserum used for supershift experiments recognize the STAT6 protein only and is not cross-reactive to any other STAT family member as examined with recombinant STAT expressed in COS-7 cells followed by EMSA and supershift assays. Supershifting of STAT6-containing complexes was achieved by adding 5 to 10 µl of antiserum against STAT6 to the whole cell extracts 10 min prior to the start of the binding reaction.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TGF-ß suppresses the development of type 1 CD8 subset in the absence of IL-4 and induces this subset in the presence of IL-4

To study the effects of TGF-ß and IL-4 on the differentiation of CD8+ T cell precursors, we used a culture system that limits T cell activation resulting from endogenously produced lymphokines and accessory cell contacts. Such a system facilitates the analysis of both the activation requirements of the CD8 T cells and the identification of accessory signals that do not act through the TCR. With this culture system, a very high efficiency of CD8 cell activation is observed as confirmed by limiting dilution analysis (8). FACS-sorted small CD8+ T cells (500 cells/200 µl) were primed with PMA, ionomycin, and IL-2, in the presence or absence of TGF-ß and/or IL-4. On day 6, the cells reached a density of about 5 to 8 x 105 cells/ml (corresponding to an average doubling time of 18 h for the total cell population), regardless of the presence or absence of TGF-ß and IL-4, suggesting that the activated cells did not represent the outgrowth of minor subpopulations. Examples of cell recoveries under these conditions are shown in Table IGo.


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  		Table I. Effects of TGF-ß on cell proliferationa

 
CD8+ T cells primed in the presence of IL-2 developed a type 1 phenotype, producing IFN-{gamma} but no IL-4, IL-5, and IL-10, upon restimulation with anti-CD3/IL-2 (Fig. 1a). Expression of this phenotype was compromised by the addition of TGF-ß. Indeed, presence of TGF-ß resulted in the development of cells with a suppressed phenotype, producing only small amounts of IFN-{gamma} and no type 2 cytokines (Fig. 1b). On the other hand, resting CD8+ T cells primed in the presence of IL-2 and IL-4 developed a type 2 phenotype, releasing IL-4, IL-5, and IL-10, and only low levels of IFN-{gamma} (Fig. 1c). When TGF-ß was also added during priming, no switch to a type 2 phenotype was observed. Instead, CD8 T cells developed a "classical" type 1 cytokine profile, releasing very low levels of IL-4, IL-5, and IL-10, but substantial amounts of IFN-{gamma} (Fig. 1d). In fact, these cells expressed the same cytokine pattern as cells primed in the absence of IL-4 and TGF-ß (Fig. 1a).

We further investigated whether IL-4 and TGF-ß could exert similar control of phenotypic differentiation on CD8 cells activated through allogeneic stimulation. FACS-sorted CD8+ T cells isolated from C57BL/6 mice were stimulated by allogeneic P815 mastocytoma cells (4000 irradiated cells per 200 µl) and IL-2, in the presence or absence of IL-4 and/or TGF-ß. The cells showed similar behavior in response to TGF-ß/IL-4 as those primed by PMA/ionomycin: while IL-4 in the absence of TGF-ß induced the generation of type 2 CD8 T cells that produced IL-5 and decreased levels of IFN-{gamma}, TGF-ß generated cells producing only small amounts IFN-{gamma} and no IL-5; addition of both TGF-ß and IL-4 induced a strong type 1 response characterized by IFN-{gamma} secretion (Fig. 2a).

Similar results were also obtained with CD8+ T cells from mice expressing a transgenic H-2Db-restricted TCR specific for the glycoprotein epitope 33-41 of LCMV (33), and primed by the viral peptide. In this system, the CD8 T cell populations were rapidly expanding, regardless of the presence or absence of IL-4 or TGF-ß (Table IGo). Cells that were generated in the absence of TGF-ß exhibited a type 1 phenotype whereas those primed in the presence of TGF-ß released reduced amounts of cytokines (Fig. 2b). As expected, cells primed in the presence of IL-4 developed a type 2 phenotype, whereas those primed with both IL-4 and TGF-ß generated type 1 cells (Fig. 2b). Thus, the differentiating effects of IL-4 and TGF-ß appeared to be independent of the mode of stimulation, i.e., mitogenic, allogenic, or antigenic.

The effects hereby described were obtained using TGF-ß2 but a comparable phenomenon was observed using the other members of the TGF-ß family found in mammals, i.e., TGF-ß1 and TGF-ß3 (data not shown).

The cytotoxicity of CD8 T cells can be inhibited by IL-4 or TGF-ß and induced by IL-4 and TGF-ß

The large majority of the described Ag-specific type 2 CD8 T cells were shown to be highly cytotoxic (10, 12, 13). However, we previously observed that type 2 CD8 T cells that were generated in the presence of PMA/ionomycin and IL-4 were noncytolytic (8) and resembled those found in HIV-infected individuals (15). Therefore, we wondered whether their cytotoxicity could be coregulated by TGF-ß and IL-4. Using a quantitative anti-CD3 redirected lysis assay on P815 target cells, we observed that the cytotoxicity that developed in the presence of IL-2 alone (Fig. 3a) was abolished by IL-4 (Fig. 3b) or TGF-ß (Fig. 3c), but strongly enhanced in the presence of both IL-4 and TGF-ß (Fig. 3d). The cytolytic activity reached levels observed with cells stimulated by PMA plus IL-2, in the absence of ionomycin (data not shown). Therefore TGF-ß and IL-4 together were able to overcome not only inhibitory signals of TGF-ß or IL-4, but also the one mediated by ionomycin costimulation (8).

Cytotoxicity of CD8 T cells is known to be mediated by the induction of apoptosis in target cells through the action of perforin and Fas ligand (42). Already aware of the fact that perforin mRNA is lacking in noncytotoxic CD8 type 2 cells (8), we wanted to know whether TGF-ß and IL-4 could control the transcription of genes coding for perforin and Fas-L/CD95. Northern blot analysis showed that this is most likely the case since the change in cytotoxicity mediated by the two cytokines correlated with similar change in the steady state levels of perforin and Fas-ligand mRNA (Fig. 3e).

TGF-ß inhibits IL-4-dependent down-regulation of CD8 expression

We have previously observed that, when CD8 T cell precursors are stimulated in the presence of IL-4 by PMA/ionomycin, they switch to a CD8- phenotype through the loss of surface CD8{alpha}/ß heterodimer (8). We have now determined whether the generation of this physiologically minor subset, detected in HIV-infected individuals in the late stages of AIDS and in hypereosinophilia (15, 43, 44, 45), could be prevented by TGF-ß. We found that TGF-ß not only prevented the IL-4-dependent generation of CD8- subset, but produced cells expressing higher levels of CD8 than cells stimulated in the absence of both TGF-ß and IL-4 (Fig. 4a). Northern blot analysis showed that the loss of cell surface CD8 induced by IL-4 correlated with the disappearance of CD8{alpha} but not CD8ß chain mRNA whereas the enhancing effects of TGF-ß involved increased levels of both CD8{alpha} and CD8ß mRNA (Fig. 4b).

Type 1 CD8 T cells generated in the presence of TGF-ß/IL-4 express high steady state levels of active STAT6

IL-4 signaling through its receptor leads to tyrosine phosphorylation, homodimerization, nuclear translocation of STAT6, and subsequent transcriptional activation or repression of IL-4 responsive gene (46, 47, 48). Experiments on STAT6-deficient mice demonstrated the importance of this transcription factor for the IL-4-dependent development of type 2 cells (31, 49). To examine whether the IL-4-dependent generation of type 1 CD8 subset could be due to a TGF-ß-driven inhibition of STAT6 activation, we performed in vitro DNA-binding assays with a specific binding element from the Ig {kappa} heavy chain germ-line {epsilon}-promoter (I{epsilon}), which does not bind STAT family members other than STAT6. Whole cell extracts were prepared from the different CD8 T cell subsets obtained after 6-day cultures and introduced to EMSA assays.

CD8 T cells stimulated by PMA, ionomycin, and IL-2, and which developed a type 1 phenotype (see Fig. 1a), contained trace amounts of activated STAT6 able to form DNA complexes (Fig. 5Go, lane 1), whereas no complex was observed for cells stimulated in the presence of TGF-ß (Fig. 5Go, lane 3), which produce almost no cytokine (see Fig. 1b). Following IL-4 priming, CD8 T cells that developed a type 2 phenotype (see Fig. 1c) contained much higher amounts of activated STAT6 (Fig. 5Go, lane 5); quantitative analysis by phosphor imaging revealed an approximately 20-fold higher amount of DNA-bound STAT6 compared with the CD8+ Th1 cells (lane 1). Similar amounts of activated STAT6 were found in type 1 CD8 T cells derived from cultures in the presence of IL-4 and TGF-ß (Fig. 5Go, lane 7) and which produce IFN-{gamma} and almost no type 2 cytokines (see Fig. 1d). The composition of the DNA-binding complexes was confirmed by supershift analysis: The anti-STAT6 antiserum induced complete supershift (Fig. 5Go, lanes 2, 6, and 8). These data show that STAT6 activation during T cell priming does not necessarily correlate with type 2 differentiation. We also observed that STAT1, STAT3, STAT5a, and STAT5b, which are known to be induced by IL-2 (48), were activated to similar extents in cells from every culture condition, irrespective of type 1 or type 2 CD8 differentiation (data not shown). Neither activated STAT4, known to be expressed in type 1 cells induced by IL-12 (50), nor activated STAT2 were detected (data not shown).



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  		FIGURE 5. Activated STAT6 is expressed in both CD8 Th1 and Th2 type cells generated from IL-4/TGF-ß and IL-4 cultures, respectively. Whole cell extracts were prepared from the different subpopulations of CD8 T cells obtained from 6-day cultures in conditions described in Fig. 1Go legend. Extracts were incubated with the high affinity STAT6 32P-labeled I{epsilon} probe and analyzed by EMSA (lanes 1, 3, 5, and 7), and, adding STAT6-specific Ab, by supershift analysis (lanes2, 4, 6, and 8).

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Lymph node CD8 T cells activated in the presence of IL-4 can develop into the cytotoxic type 2 CD8+ T cells that have been described in numerous studies (8, 10, 12, 13) and into the less common noncytotoxic type 2 CD8-CD4- T cells (8, 15). Using a culture system designed to ensure a high efficiency of CD8 T cell activation, we hereby show that TGF-ß and IL-4 can coregulate CD8 T cell polarization. While type 1 priming (without IL-4) in the presence of TGF-ß leads to a suppressed CD8 T cell phenotype, i.e., cells secreting neither type 1 nor type 2 cytokines, priming in the presence of IL-4 leads to the two type 2 phenotypes described above. Moreover, the presence of both TGF-ß and IL-4 directs the cells toward a cytotoxic type 1 phenotype. In this latter case, TGF-ß seems to deliver a critical signal that alters the normal effect of IL-4, leading to the generation of type 1 rather than type 2 CD8 cells.

We still do not know how the distinct signal-transducing pathways induced by TGF-ß and IL-4 converge or interact to modify the effects of each cytokine taken individually. We only know that STAT6, which is an essential transducing signal involved in the IL-4-dependent development of type 2 cells (31, 47, 49), is still activated during the type 1 differentiation induced by IL-4/TGF-ß. This indicates, in particular, that STAT6, although necessary, is not sufficient to induce a type 2 phenotype. STAT6 might, in fact, also mediate the IL-4-dependent generation of IFN-{gamma}, since a STAT6-binding site is localized in transcriptional regulatory elements within the IFN-{gamma} locus (51). The conversion of IL-4 and TGF-ß activities, when both cytokines cooperate, might be the result of a mutual inhibition of specific genes under the control of each individual cytokine. This would explain why similar phenotypes could be generated in the presence or absence of both cytokines. Alternatively, IL-4/TGF-ß might be inhibiting costimulatory signals required for the generation of type 2 subsets. This assumption is supported by the fact that 1) the inhibitory effect of ionomycin on the cytotoxicity of PMA-derived CD8 T cells is abolished by TGF-ß/IL-4, and that 2) ionomycin is a cosignal required for the shift of CD8 T cells triggered by PMA, IL-2, and IL-4 toward Th2 (8).

Cytokines are known to display synergistic, additive, and antagonistic biological activities. Here, the combination of TGF-ß and IL-4 results in activities that differ from those displayed by each cytokine alone. This may represent a sensitive mechanism enabling the deviation of the immune responses. Indeed, secretion of TGF-ß by APCs and some regulatory T cells (22) in the microenvironment of CD8 cells could possibly redirect them from IL-4-dependent type 2 phenotype differentiation and expansion. Conversely, a competent type 1 response could also be reactivated by IL-4 under conditions in which CD8 cells are suppressed by TGF-ß. Speculation of such an escape mechanism involving also the control of immune reaction by CD4 cells can be made, supported by evidence demonstrating that TGF-ß can either inhibit or induce Th type-1 generation (26, 29, 52).

While IL-12 has often been described as the Th2 to Th1 switch factor (53), its activity differs from that of TGF-ß. Whereas TGF-ß prevents the IL-4-dependent development of CD4 and CD8 type 2 cells, IL-12 does not (54, 55) (F. Erard and M. T. Wild, unpublished observation). IL-12 appears more as a dominant factor directing the development of T cells that produce elevated IFN-{gamma} levels (56), whereas TGF-ß only inhibits the IL-4-mediated drop in IFN-{gamma}.

Circumstantial evidence suggests that coregulation of T cell differentiation by TGF-ß/IL-4 is physiologically relevant. In several infectious disease models involving CD8 T cells, IFN-{gamma}-dominated responses have been shown to be protective while IL-4- or TGF-ß-dominated responses lead to enhanced disease state (57, 58). However, the critical role of IL-4 in disease susceptibility is challenged in models in which direct IL-4 administration reduced parasitemia and rendered animals resistant to reinfection. IL-4 prevented mortality during acute Toxoplasma infection, by enhancing IFN-{gamma} production and thus parasite killing (18). IL-4 induced also protective type 1 responses in IL-4-deficient mice infected by Candida albicans (19). Furthermore, IL-4 induced tumor rejection in syngeneic hosts, partially mediated through IFN-{gamma} induction (59). Concerning TGF-ß, its most potent activity on lymphocytes is immunosuppression (24, 25), and TGF-ß1-deficient animals tend to develop inflammatory disease (60). Nevertheless, enhanced CD8 T cell responses to infectious agents and inflammation induced by TGF-ß was observed (61). Therefore, the phenomenon described here reveals one fundamental mechanism by which cytokines can delicately counterbalance protective and damaging functions. We wonder whether such dual cytokine activities are an exceptional hallmark of TGF-ß and IL-4, or might be a more general feature of cytokine function.



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  		FIGURE 1. TGF-ß and IL-4 coregulate the development of polarized CD8 T cell subsets triggered by mitogens. FACS-sorted small CD8+ T cells (500 cells with >99% purity/200 µl) from lymph nodes of BALB/c mice were primed with PMA, ionomycin, and IL-2, either in the absence of IL-4 and TGF-ß (a), in the presence of TGF-ß (b), in the presence of IL-4 (c), or in the presence of IL-4 and TGF-ß (d). On day 6, the cells were recultured (105 cells/200 µl) for 24 h, in anti-CD3{epsilon}-coated wells in the presence of IL-2 and the cytokines released in the supernatant quantified by ELISA. Results shown are the mean of two independent cultures ± SE.

 


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  		FIGURE 2. TGF-ß and IL-4 coregulate also the differentiation of alloantigen and Ag-specific CD8 T cells. Microcultures were established in the presence or absence of IL-4 and TGF-ß2 with 500 FACS-sorted CD8+ T cells from lymph nodes of C57BL/6 mice (H-2b) primed for 12 days by 4000 allogeneic P815 mastocytoma cells (H-2d) plus IL-2 (a), or 500 FACS-sorted CD8 T cells derived from lymph nodes of LCMV-specific TCR transgenic C57BL/6 mice primed for 6 days by the LCMV peptide and IL-2 (b). The cells were then recultured for 24 h in anti-CD3{epsilon}-coated wells in the presence of IL-2 and the supernatants analyzed by ELISA. Results shown are the mean of two independent cultures ± SE.

 


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  		FIGURE 3. TGF-ß and IL-4 can affect CD8 T cell cytotoxicity. CD8 T cells derived from 6-day cultures of precursors primed by PMA, ionomycin, and IL-2 in the presence or absence of IL-4 or TGF-ß were incubated for 3.5 h with 2000 51Cr-labeled P815 cells in the presence of 25 µg/ml anti-CD3{epsilon} Ab and the specific 51Cr released was measured. (a—d) RNAs were prepared from cells cultured for 6 days and subjected to Northern blot analysis by hybridization with 32P-labeled cDNA probes specific for perforin, FasL/CD95, and ß-actin (e).

 


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  		FIGURE 4. IL-4 and TGF-ß affect the phenotype of mitogen-stimulated CD8 T cells. FACS-sorted small CD8+ T cells (500 cells with >99% purity/200 µl) were primed with PMA/ionomycin and IL-2, in the presence or absence of IL-4 and TGF-ß. CD8 and CD4 expression was determined on day 6, by staining with FITC-conjugated anti-CD8 and PEconjugated (a) anti-CD4 and subjected to flow cytometric analysis. (a). RNAs from 6-day cultures were prepared as described in Materials and Methods and were analyzed by Northern blot using 32P-labeled cDNA probes specific for CD8{alpha}, CD8ß, and ß-actin (b).

 

   Acknowledgments
 
We thank Hanspeter Pircher, Hans Hengartner, and Rolf Zinkernagel for generously providing the TCR transgenic mice; Pierre Cosson and Peter Kramer for providing cDNA probes; Andre Kiefer for flow cytometry; and Theophil Staehelin, Rafick P. Sekaly, Mohammed Amine Abina, and Patrick Champagne for discussions and critical review of the manuscript.


   Footnotes
 
1 The Department of Immunology and Oncology was founded and is supported by the CSIC and Pharmacia and Upjohn. Back

2 Address correspondence and reprint requests to F. Erard, Laboratoire d’Immunologie, Institut de Recherches Cliniques de Montréal, 110 avenue des Pins Ouest, Montréal H2W 1R7, Canada. E-mail address: Back

3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; EMSA, electrophoretic mobility shift assays; PE, phycoerythrin. Back

Received for publication June 12, 1998. Accepted for publication September 18, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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