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The Journal of Immunology, 2001, 167: 5610-5619.
Copyright © 2001 by The American Association of Immunologists

CD4 Ligation Promotes the IL-4-Independent Development of IL-4-Producing Clones from Naive CD4+ T Cells1

Scott B. Campbell*,{dagger}, Tadashi Komata2,* and Anne Kelso3,*,{dagger},{ddagger}

* Queensland Institute of Medical Research, {dagger} Joint Transplantation Biology Program, University of Queensland, and {ddagger} Cooperative Research Centre for Vaccine Technology, Brisbane, Queensland, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The signals that trigger IL-4-independent IL-4 synthesis by conventional CD4+ T cells are not yet defined. In this study, we show that coactivation with anti-CD4 mAb can stimulate single naive CD4+ T cells to form IL-4-producing clones in the absence of APC and exogenous IL-4, independently of effects on proliferation. When single CD4+ lymph node cells from C57BL/6 mice were cultured with immobilized anti-CD3{epsilon} mAb and IL-2, 65–85% formed clones over 12–14 days. Coimmobilization of mAb to CD4, CD11a, and/or CD28 increased the size of these clones but each exerted different effects on their cytokine profiles. Most clones produced IFN-{gamma} and/or IL-3 regardless of the coactivating mAb. However, whereas 0–6% of clones obtained with mAb to CD11a or CD28 produced IL-4, 10–40% of those coactivated with anti-CD4 mAb were IL-4 producers. A similar response was observed among CD4+ cells from BALB/c mice. Most IL-4-producing clones were derived from CD4+ cells of naive (CD44low or CD62Lhigh) phenotype and the great majority coproduced IFN-{gamma} and IL-3. The effect of anti-CD4 mAb on IL-4 synthesis could be dissociated from effects on clone size since anti-CD4 and anti-CD11a mAb stimulated formation of clones of similar size which differed markedly in IL-4 production. Engagement of CD3 and CD4 in the presence of IL-2 is therefore sufficient to induce a substantial proportion of naive CD4+ T cells to form IL-4-producing clones in the absence of other exogenous signals, including IL-4 itself.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Activated T cells can be polarized toward preferential synthesis of type 1 or type 2 cytokines while expressing diverse combinations of cytokine genes at the single-cell level (1, 2, 3, 4). It is now established that individual CD4+ and CD8+ T cells acquire the ability to synthesize a particular cytokine combination by a process of differentiation that occurs during and after primary activation and is driven by cytokines and other stimuli (5, 6, 7, 8).

IL-4 itself is the best characterized signal promoting the development of IL-4-producing T cells in vitro and in vivo (7, 9, 10). The cellular origin of the IL-4 which initiates this process has been the subject of much discussion and it now seems unlikely there is a single universal source. Mast cells, basophils, eosinophils, {gamma}{delta} T cells, and CD4+NK1.1+ T cells can all produce IL-4 and may play a significant role in certain circumstances (2, 11, 12, 13, 14, 15, 16). There is also strong support for the idea that conventional CD4+ T cells can, under some conditions, produce enough IL-4 to prime the positive feedback loop that leads to expansion of the IL-4-producing T cell pool (17, 18). Previously primed CD4+ T cells can promote IL-4 production by newly activated naive CD4+ T cells (19) and, importantly, repeated stimulation of phenotypically naive CD4+ T cells in accessory cell-dependent systems can also lead to IL-4 synthesis (20, 21, 22). Recent experiments with cells from mice deficient in IL-4R{alpha} or STAT6 have confirmed that naive CD4+ T cells can be activated to synthesize IL-4 in the absence of IL-4R signaling (23, 24).

Several signals have been described that might trigger IL-4-independent IL-4 synthesis by conventional T cells, including the CD28 ligand CD86 (B7-2) (25, 26), agents that cross-link CD40 ligand (27), and IL-6 (28). As yet undefined signals delivered by certain dendritic cell subpopulations also favor the development of IL-4-producing T cells in the apparent absence of IL-4 itself (29, 30). A role for CD4 ligation has been suggested by the enhancing effects of nondepleting anti-CD4 Abs on development of IL-4-producing cells in various rodent models in vivo (31, 32, 33, 34) and in MLRs in vitro (35), and by the effects of CD4 deficiency or mutation on IL-4 responses in several systems (36, 37, 38). In addition, we recently reported that the combination of anti-CD3 and anti-CD4 mAb stimulation induced IL-4 synthesis in bulk cultures of CD4+ T cells (39). The interpretation of some of these studies remains controversial, however, because effects of coactivating signals on cytokine profile development are difficult to dissociate from their effects on the strength of stimulation, cell division number, and the frequency of responding cells, and because dependence on costimulation varies with the T cell activation state (1, 40). For example, in our own studies (39), it remained possible that the IL-4-producing cells were derived from a minor atypical subset of CD4+ T cells that were predisposed to production of this cytokine.

These confounding factors can be avoided by using simple, single-cell activation systems in which the frequency and magnitude of T cell proliferative and cytokine responses to defined stimuli can be monitored. Using such a system, we show here that anti-CD4 mAb can act directly on a high proportion of single CD4+ T cells of naive phenotype to promote the development of IL-4-synthesizing clones in the absence of any exogenous source of IL-4 and independently of effects on their proliferative response. The relative efficacy of anti-CD4 mAb compared with mAb to CD11a or CD28 in this system provides direct evidence for a role for CD4 signaling in promoting IL-4-independent IL-4 synthesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
mAbs and cytokines

The hamster anti-mouse CD3{epsilon} mAb 145.2C11 (41) and the rat anti-mouse mAb to CD4 (GK1.5 and H129.19) (42, 43), CD11a (I21/7.7) (44), CD28 (37.51.9) (45), CD8 (53-6.7) (46), and CD44 (IM7.8.1) (47) were purified from hybridoma supernatants by their affinity for protein A or G. The rat anti-mouse mAb to IL-4 (11B11) (48) was used as an ammonium sulfate precipitate. The rat anti-mouse mAb to B220 (RA3-6B2) (49), I-Ab,d,q, and I-Ed,k (M1/5114) (50), CD62L (51), and IL-6 (6B4) (52) were used as hybridoma supernatants. Purified human rIL-2 prepared in Escherichia coli was provided by Cetus (Emeryville, CA) (53); titers are expressed in World Health Organization international units. Murine rIL-4 was the supernatant of Sf9 cells infected with an IL-4-expressing recombinant baculovirus (54); titers are expressed in units defined as the concentration-stimulating half-maximal proliferation of the IL-4-responsive cell line CT.4S (54).

CD4+ T lymphocyte preparation

Specific pathogen-free female C57BL/6 mice and BALB/c mice were purchased from the Animal Resources Center (Murdoch, Western Australia) and used at 6–12 wk of age. Pooled axillary, inguinal, iliac, and mesenteric lymph nodes were disaggregated by passage through stainless steel mesh. Viable lymphocytes were isolated by centrifugation over Ficoll-Paque (Pharmacia, Uppsala, Sweden) and incubated on ice, first with anti-CD8, anti-B220, and anti-class II MHC mAb and then, after washing, with FITC-conjugated goat anti-rat Ig (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Cells were enriched for viable CD4+ cells of small lymphocyte size based on forward and 90o scatter properties, exclusion of propidium iodide, and absence of FITC binding using a FACSVantage (BD Biosciences, Sunnyvale, CA). Reanalysis of the purified cells after staining with PE-conjugated GK1.5 mAb (BD Biosciences) revealed them to be 95–97% CD4+ cells. In some experiments, this FITC-CD4+ cell-enriched population was further incubated either with biotinylated anti-CD44 mAb followed by streptavidin-PE (Caltag Laboratories, Burlingame, CA) or with anti-CD62L mAb followed by goat anti-rat Ig-FITC. Cells were then passed through the sorter irrespective of staining or separated into CD44low (lowest 15–25%) and CD44high (highest 4–8%) or CD62Llow (6–10%) and CD62Lhigh (90–94%) populations. Single cells of the desired phenotype were deposited directly into culture wells using an automated cell deposition unit attached to the FACSVantage. Approximately 20% of all wells were checked microscopically and none was found to contain more than one cell.

T lymphocyte culture

Round bottom 96-well microtiter plates (Corning Glass, Corning, NY) were incubated with combinations of the following anti-receptor mAb in PBS, at concentrations previously found to achieve maximal cloning frequency: 145-2C11 (10 µg/ml), GK1.5, H129.19, I21/7.7, and/or 37.51.9 (each at 15 µg/ml) (55, 56). Plates were incubated overnight at 37°C and washed three times with PBS. All cultures were performed in DMEM (Life Technologies, Gaithersburg, MD) modified as described elsewhere (54) and supplemented with L-glutamine (216 mg/L), 2-ME (5 x 10-5 M), 10% heat-inactivated FCS (CSL, Parkville, Victoria, Australia), and 600 IU/ml rIL-2. In some experiments, cultures also received rIL-4. Cultures were incubated at 37°C in a humidified atmosphere of 5% CO2 in air. After 12- 14 days, wells were checked microscopically for the presence of clones and their size was estimated using the following scale: 0, no visible clone; 1, <100 cells; 2, 100-1000 cells; 3, 1000–5000 cells; and 4, >5000 cells. In some experiments, clones were then washed three times in situ and transferred to new microtiter wells coated with 10 µg/ml anti-CD3 mAb for another 36 h.

Cytokine assays

IL-3 was measured using the IL-3-dependent cell line 32D clone 3 in a [3H]TdR uptake assay (57). The response of these cells to activated T cell supernatants was blocked at least 10-fold by neutralizing anti-IL-3 mAb (55, 58). IFN-{gamma} was assayed using the IFN-{gamma}-sensitive cell line WEHI-279 in a colorimetric assay (57). IL-4 was measured by ELISA as described previously (59) using mAb BVD4-1D11 at 5 µg/ml to coat plates and biotinylated BVD6-24G2 at 0.3 µg/ml for detection with streptavidin-HRP (60). Clonal culture supernatants were generally assayed for cytokines at a single concentration (50%) and scored as positive if activity exceeded by >3 SD the mean activity of control wells in which no clone had developed. In some experiments, clonal supernatants were titrated over 6 half-log10 dilutions in the WEHI-279 assay for IFN-{gamma} (in the presence and absence of neutralizing anti-IFN-{gamma} mAb R4-6A2 (ATCC HB170)) and the IL-4 ELISA, and their activities were determined by reference to standard recombinant IFN-{gamma} and IL-4 preparations as described elsewhere (54, 57, 59); IFN-{gamma} titers of all positive supernatants were reduced at least 10-fold by anti-IFN-{gamma} mAb.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Monoclonal anti-CD4 Abs promote the generation of IL-4-producing clones from CD4+ T cells

Fig. 1Go summarizes results obtained in three experiments in which CD4+ T cells were purified from lymph nodes of normal C57BL/6 mice and cultured as single cells with anti-CD3 mAb and various combinations of mAb to CD4, CD11a, and CD28 in the presence of IL-2. Previous work showed that combinations of these mAbs were the most effective of a number tested for stimulating primary CD4+ T cell clone formation in the absence of accessory cells (55, 56). Negative selection of CD4+ T cells was used throughout the studies reported here to avoid any effects of anti-CD4 mAb binding on subsequent responses. Mean cloning efficiencies assessed at day 12 ranged from 66 to 78% and were not consistently affected by the presence of any coactivating mAb. No clones were obtained when either anti-CD3 mAb or IL-2 was omitted (data not shown).



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  		FIGURE 1. Effect of anti-receptor Abs on the activation of single CD4+ T cells to form clones and synthesize cytokines. Different symbols represent data obtained in three independent experiments in which single negatively selected CD4+ T cells from lymph nodes of C57BL/6 mice were cultured with the indicated immobilized mAb and IL-2 (96 wells per combination in each experiment). After 12 days, cloning efficiencies were assessed microscopically (displayed as the percentage of wells containing viable cells, upper panel) and culture supernatants were assayed for IL-3, IFN-{gamma}, and IL-4 (displayed as the percentage of clones positive for each cytokine, lower panels).

 
By contrast, assays of clonal culture supernatants at day 12 revealed marked differences in the frequencies of clones that secreted the type 1/2-unrestricted cytokine IL-3, the type 1 cytokine IFN-{gamma}, and the type 2 cytokine IL-4 under different coactivating conditions. Frequencies of IL-3 and IFN-{gamma} producers were low among clones obtained with anti-CD3 mAb alone, reflecting their low average clone size (see below), and were enhanced by every combination of coactivating mAb. Anti-CD28 mAb caused the smallest enhancement over anti-CD3 mAb alone. The great majority (92%) of IL-4-producing clones arose in cultures containing the anti-CD4 mAb GK1.5. Similar results were obtained in parallel cultures in the same experiments using a different anti-CD4 mAb, H129.19 (data not shown).

When all experiments performed throughout this study were compared, a clear hierarchy was noted. Development of IL-4-producing clones was promoted most strongly and consistently by anti-CD4 mAb; these frequencies were not increased when higher anti-CD4 mAb-coating concentrations were used. Significant numbers of IL-4-producing clones were obtained in some but not all experiments with anti-CD11a mAb and were modestly elevated when the anti-CD11a mAb-coating concentration was increased from 15 to 30–50 µg/ml. IL-4-producing clones were rarely detected in cultures coactivated with anti-CD28 mAb at any concentration up to 50 µg/ml.

The effect of anti-CD4 Abs on IL-4 production is not due to increased clone size

We have previously observed a positive correlation between detection of cytokine synthesis in primary T cell clones and their size (cell number) (55, 56, 61). Fig. 2Go shows the distributions of cell numbers among the clones described in Fig. 1Go. On average, clones obtained with anti-CD3 mAb with or without anti-CD28 mAb alone were smaller (<4% contained >103 cells) than those obtained with the other coactivating mAb (34% contained >103 cells). Among the latter groups of clones, size distributions were similar whether or not the cultures contained anti-CD4 mAb. Frequencies of IL-3- and IFN-{gamma}-producing clones were closely correlated with clone size, being highest among clones obtained with anti-CD4 and/or anti-CD11a mAb. This was not the case for IL-4-producing clones. Although clone size distributions were similar for all combinations of mAb to CD4 and CD11a, IL-4 producers were found at significantly higher frequency in the presence of anti-CD4 mAb than in its absence. The preferential effect of anti-CD4 mAb on IL-4 production therefore was not simply due to enhancement of clone size.



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  		FIGURE 2. Relationship between cytokine production and clone size among CD4+ T cell clones generated with various anti-receptor mAbs and IL-2. The sizes of the clones shown in Fig. 1Go were assessed microscopically after 12 days of culture using the following scale: 0, no visible clone; 1, <100 cells, 2, 100-1000 cells; 3, 1000–5000 cells; and 4, >5000 cells. Panels in the top row show the distribution of clone sizes obtained with each mAb combination. Lower panels show the percentage of clones of each size that produced IL-3, IFN-{gamma}, or IL-4 as indicated.

 
To investigate the relationship between cytokine detection and clone size more directly, clones were generated using the protocol described above, scored for size, then washed and restimulated for 36 h. Supernatants from these secondary cultures were titrated over several dilutions in IFN-{gamma} and IL-4 assays to obtain individual titers for each clone. Data are shown in Fig. 3GoA, segregated according to anti-CD4 mAb stimulation. As in previous assays of primary culture supernatants, frequencies of cytokine-positive clones rose with clone size. However, individual titers were highly variable and the mean titers of positive supernatants were not significantly different between clones of different sizes. The main effect of clone size was therefore to increase the probability that a clone secreted detectable levels of IFN-{gamma} and/or IL-4.



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  		FIGURE 3. Quantitative relationship between clone size and cytokine production in secondary culture of CD4+ T cell clones. Single negatively selected CD4+ T cells from lymph nodes of C57BL/6 mice were cultured with IL-2 and immobilized anti-CD3 mAb and all combinations of anti-CD4, anti-CD11a, and anti-CD28 mAb as shown in Fig. 1Go (48 wells per combination). After 12 days of culture, clone sizes were assessed microscopically and clones were washed and recultured with immobilized anti-CD3 mAb without cytokines. Secondary culture supernatants were collected after 36 h and assayed by titration for IFN-{gamma} and IL-4. Data are displayed for all mAb combinations either lacking anti-CD4 mAb (left panels) or including anti-CD4 mAb (right panels). Lines within the panels represent thresholds of detection in each assay. Numbers within the panels, percentage of clones positive for cytokine; horizontal bars, geometric mean titer of positive clones.

 
Production of IL-3, IFN-{gamma}, and IL-4 is correlated among anti-CD4 mAb-stimulated T cell clones

The relationships between IL-4 and IFN-{gamma} production levels among clones from one of the three experiments described in Figs. 1Go and 2Go are shown in Fig. 4Go, segregated according to anti-CD4 mAb stimulation. In all three experiments, most IL-4-producing clones coproduced IFN-{gamma}; in fact, IL-4 secretion was generally associated with the highest IFN-{gamma} secretion levels (and largest clone size). Apart from triple-negative clones (25%, the great majority of which contained fewer than 103 cells), the most common phenotypes found in anti-CD4 mAb-stimulated cultures in the three experiments were: IFN-{gamma}+IL-3+IL-4- (39%), IFN-{gamma}-IL-3+IL-4- (18%), and IFN-{gamma}+IL-3+IL-4+ (13%). Analysis of the data by {chi}2 contingency table testing showed that, in almost all instances, each possible pair of these cytokines was coexpressed more frequently than expected by chance (Table IGo), suggesting that their production was coregulated.



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  		FIGURE 4. Cytokine profiles of CD4+ T cell clones generated with and without anti-CD4 mAb. IFN-{gamma} and IL-4 coexpression patterns are shown for clones generated in one of the experiments described in Figs. 1Go and 2Go ({circ} in Fig. 1Go), with all mAb combinations either lacking anti-CD4 mAb (left panels) or including anti-CD4 mAb (right panels). Each circle represents a single clone. The axes show the activity of each clonal supernatant in the IFN-{gamma} and IL-4 assays; the scale for the IFN-{gamma} assay is inverted since this is an inhibition assay in which low OD reflects high activity. Lines within the panels represent thresholds of detection in each assay.

 

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  		Table I. Cytokine coexpression patterns among CD4+ T cell clones generated in the presence and absence of anti-CD4 mAb1

 
Analysis of the relationship between IFN-{gamma} and IL-4 titers in secondary culture supernatants (Fig. 3GoB) also showed that most IL-4-producing clones coproduced IFN-{gamma} at a higher frequency than expected by chance (probability of independence <0.001). Although there was a broad association between high titers of the two cytokines, no quantitative relationship was detected by linear regression (r2 = 0.14). Since both cytokines were detected in secondary culture supernatants, it is likely that they were synthesized simultaneously rather than sequentially by most clones.

IL-4 exerts a qualitatively similar effect to anti-CD4 mAb and contributes to the anti-CD4 mAb-mediated generation of IL-4-producing clones

IL-4 is the best known inducer of IL-4 synthesis in newly activated CD4+ T cells. To compare the efficacy of IL-4 and coactivating mAb in inducing IL-4 synthesis, single CD4+ T cells were cultured with anti-CD3 mAb and various combinations of coactivating mAb in the presence and absence of IL-4 at 20 U/ml, a concentration previously found to support maximal stimulation of IL-4 production in bulk cultures of CD4+ T cells with these mAbs. Since IL-4 addition precluded measurement of secreted IL-4 in these primary cultures, clones were washed and recultured without IL-4 for 36 h before supernatant harvest. For reasons that are not yet understood, exogenous IL-4 reduced cloning efficiencies and average clone size in most mAb combinations (Fig. 5Go). Inclusion of IL-4 in primary cultures nevertheless stimulated formation of IL-4-producing clones in the presence of all tested mAb combinations at frequencies up to 46% (up to 62% in another similar experiment). All IL-4-producing clones obtained by anti-CD4 mAb or by IL-4 stimulation coproduced IFN-{gamma} (data not shown). In each case, the frequency of IL-4/IFN-{gamma} double producers was significantly higher than predicted for random association of independent variables (anti-CD4 mAb: 10.4% observed cf 6.9% expected, p < 0.001; IL-4: 29.1% observed cf 21.8% expected, p < 0.001). Therefore, although IL-4 was a more potent inducer of IL-4 synthesis than anti-CD4 mAb, the responses were qualitatively similar in promoting coproduction of IL-4 with IFN-{gamma}.



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  		FIGURE 5. Comparison of coactivating mAb and IL-4 as inducers of IL-4-producing clones. Single CD4+ T cells from C57BL/6 mice were cultured in microtiter wells with the indicated immobilized mAb in the presence of IL-2 with or without 20 U/ml IL-4 (48 wells per combination). After 14 days, cultures were scored microscopically to determine cloning efficiencies. Clones were washed and recultured with immobilized anti-CD3 mAb without cytokines. Supernatants were collected after 36 h and assayed for IFN-{gamma} and IL-4. Upper panel, Percentages of clones containing >103 cells ({blacksquare}) are overlaid on total cloning efficiencies ().

 
To investigate whether endogenous IL-4 contributed to the development of IL-4-producing clones in response to anti-CD4 mAb coactivation, a series of experiments was performed in which single CD4+ T cells were cultured with various combinations of anti-CD3, anti-CD4, and anti-CD11a mAb and IL-2, with or without 10 µg/ml neutralizing anti-IL-4 mAb (11B11) in soluble form. After 10 days, cultures were scored and washed to remove the soluble mAb, then restimulated with anti-CD3 mAb and IL-2 for another 2 days. Assays of secondary culture supernatants showed that anti-IL-4 mAb markedly inhibited the development of IL-4-producing clones (data not shown). For example, in one experiment, frequencies of IL-4+ clones were reduced from 12 to 0% (p < 0.01) among clones generated with anti-CD3 and anti-CD4 mAb and from 23 to 2% (p < 0.001) among clones generated with anti-CD3, anti-CD4, and anti-CD11a mAb, whereas cloning efficiency was not significantly affected. The data indicate that the IL-4 response to anti-CD4 mAb coactivation is significantly amplified by IL-4 produced by the clone itself. In contrast, other experiments showed that soluble neutralizing anti-IL-6 mAb had no discernible effect on the frequency of IL-4-producing clones that developed in response to anti-CD3 and anti-CD4 mAb (data not shown).

CD4+ T cells from C57BL/6 and BALB/c mice respond similarly to anti-CD4 mAb coactivation

C57BL/6 and BALB/c mice differ in the IL-4/IFN-{gamma} bias of their CD4+ T cell responses to some immunogens (62, 63, 64, 65). We therefore compared the ability of purified CD4+ T cells from normal mice of these two strains to form IL-4- and IFN-{gamma}-producing clones in response to coactivation with mAb to CD4 or CD11a (Fig. 6Go). Whereas both mAb increased the average size of clones, anti-CD4 mAb preferentially enhanced the development of IL-4-producing clones from both mouse strains. Under these culture conditions, BALB/c CD4+ cells did not show any predisposition to IL-4 production compared with C57BL/6 CD4+ cells, in either the presence or absence of anti-CD4 mAb.



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  		FIGURE 6. Comparison of the responses of CD4+ T cells from C57BL/6 and BALB/c mice. CD4+ T cells from the two mouse strains were cultured with the indicated immobilized mAb and IL-2 (192 wells per combination). After 12 days, cultures were scored microscopically to determine cloning efficiencies and supernatants were collected for the indicated cytokine assays. The total percentage of wells containing viable cells and percentage of clones positive for each cytokine are recorded within the panels. Similar results were obtained in a second experiment.

 
IL-4-producing clones can develop from CD4+ T lymphocytes of naive phenotype

It was possible that the IL-4-producing clones obtained in cultures stimulated with anti-CD4 mAb were derived from T cells that had been primed to synthesize IL-4 in vivo and preferentially formed clones under these culture conditions. To test this possibility, CD4+ T cells were purified from normal C57BL/6 lymph nodes then separated further on the basis of high or low expression of CD44 or CD62L to yield cells of naive (CD44low or CD62Lhigh) or activated (CD44high or CD62Llow) phenotype. The distribution of CD44 expression was trimodal, with 15–25% of CD4+ cells in the CD44low fraction and 4–8% in the CD44high fraction; cells with intermediate expression were excluded. The CD62L distribution was bimodal with 90–94% in the positive fraction and the remainder in the negative fraction; no cells were excluded. Double staining of CD4+ cells for the two markers showed that activated cells defined by low expression of CD62L were divided about equally between the CD44high and CD44int populations; 94% of CD44low cells were CD62Lhigh and 76% of CD44high cells were CD62Llow (data not shown).

Unfractionated populations and cells of naive phenotype defined by either marker formed clones with similar efficiency (Fig. 7Go). Cells of activated phenotype formed clones at significantly lower frequency; this was not due to inhibition by bound mAb since it was observed whether the activated cells were selected by high anti-CD44 mAb binding or absence of anti-CD62L mAb binding. IL-4-producing clones arose at similar frequencies in all populations irrespective of activation status. No IL-4-producing clones were detected in parallel cultures in which each of these populations was cultured with anti-CD3 mAb and IL-2 alone (n = 2617; data not shown). We conclude that naive CD4+ T cells can give rise to IL-4-producing clones in anti-CD4 mAb-stimulated cultures and, because they clone at higher frequency than activated cells, are the origin of most of the IL-4-producing clones detected in cultures of unfractionated CD4+ cells.



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  		FIGURE 7. Comparison of the responses of CD4+ T cells of naive and activated/memory phenotype. Negatively selected CD4+ T cells from C57BL/6 mice were used without further staining (bar 1) or stained with mAb against CD44 or CD62L and used without further separation (bars 2 and 5) or after separation on the basis of low or high mAb binding as indicated. Single cells were cultured with immobilized anti-CD3 and anti-CD4 mAb and IL-2 (192 wells per group). After 12 days, cultures were scored microscopically to determine cloning efficiencies and supernatants were collected for the indicated cytokine assays. Upper panel, Percentages of clones containing >103 cells ({blacksquare}) are overlaid on total cloning efficiencies (). Similar results were obtained in one other experiment with CD62L and four other experiments with CD44.

 
Frequencies of IL-4-producing clones increase with prolonged culture

In the above experiments, cytokines were usually measured at day 12 when clone sizes were estimated to be maximal. To investigate whether this was also the time of peak cytokine accumulation, primary cultures were assessed microscopically and their supernatants were sampled repeatedly for IL-4 assay at intervals up to 26 days after single-cell deposition. In the experiment shown in Fig. 8Go, cloning frequencies were maximal by day 10 and remained high until death commenced at ~3 wk. Mean clone size peaked at about day 13. By contrast, both the frequency of IL-4-producing clones and the mean IL-4 titer of the positive clones (as assessed by OD in an ELISA) continued to rise over most of the 3 wk of clone survival. At all times throughout this period, both frequency and OD were positively correlated with clone size. With prolonged culture, as many as 40% of clones produced detectable IL-4. Comparable results were obtained in a second experiment in which the frequency of IL-4-producing clones rose from 10% at day 14 to 35% at day 26 in cultures stimulated with anti-CD3 and anti-CD4 mAb. This experiment further showed that anti-CD3 mAb alone did not support any increase in the frequency of IL-4-producing clones over 26 days (maximum frequency, 1.5%). The sustained accumulation of IL-4-producing clones therefore depended on coactivation.



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  		FIGURE 8. The effect of prolonged culture on the development of IL-4-producing clones. Negatively selected CD4+ T cells from C57BL/6 mice were stained with anti-CD44 mAb. Single cells from the lowest 25% of the CD44 distribution were cultured with the indicated immobilized mAb and IL-2. At the indicated times, cultures were scored microscopically to determine total cloning efficiencies (•) and the percentage of clones containing >103 cells ({circ}) (upper panels). Twenty microliters of supernatant was collected for IL-4 assay (lower panels), shown as the percent IL-4-positive clones (•) and the mean OD of all positive cultures ({triangleup}).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we show that CD4 coreceptor ligation can promote the development of IL-4-producing clones from single CD4+ T cells activated with anti-CD3 mAb in the absence of exogenous IL-4 or any other cell type. There are two notable implications of this finding. First, since as many as 40% of normal lymph node CD4+ T cells of naive phenotype could generate IL-4-producing clones under these conditions, the response cannot be attributed to an unusual T cell subset. Second, since CD11a (LFA-1{alpha}) engagement exerted a similar potentiating effect on clone growth but not on IL-4 production, the data suggest a specific role for CD4 in promoting IL-4-independent IL-4 synthesis.

We used an activation system in which individual CD4+ T cells were cultured with immobilized Abs to CD3 and various coactivating receptors in the presence of IL-2. This system has several features that distinguish it from bulk culture and in vivo systems used in earlier studies of anti-CD4 and other Ab effects on IL-4 production. First, the absence of accessory cells ensured that the cultured T cell and its progeny were the direct targets of each stimulus and the only possible source of measured cytokines. Second, the use of a single-cell culture system enabled the frequency of responsive precursors to be measured directly. Finally, the ability to monitor clone size over time allowed effects on cytokine synthesis to be dissociated from effects on proliferation and total cell number.

Each of the three groups of coactivating Abs examined exerted distinct effects on clonal expansion and cytokine synthesis in this system. The anti-CD4 mAb GK1.5 and H129.19 both enhanced clonal expansion over that seen with anti-CD3 mAb and IL-2 alone. This is consistent with many earlier studies demonstrating that CD4 can serve a coreceptor function in TCR-dependent activation by forming part of the TCR complex and augmenting signal transduction (66, 67). The greatest enhancement of clonal expansion was usually observed with the anti-CD11a mAb I21/7.7, which we previously found to be the most potent of the coactivating Abs assayed for activation and expansion of both CD4+ and CD8+ T cells in clonal and bulk cultures (39, 55, 56). Others have also reported significant costimulatory effects of Abs and natural ligands of LFA-1 in other T cell activation systems (68, 69, 70). The widely used costimulatory anti-CD28 mAb 37.51 (45) achieved the least enhancement of clonal expansion in our hands, probably because effects of CD28 stimulation on responses to low-affinity ligands and on IL-2 synthesis (70, 71) were bypassed by anti-CD3 mAb and exogenous IL-2 in these experiments. A more pronounced effect on proliferation compared with the other mAb was observed when anti-CD28 was coimmobilized with anti-CD3 mAb in bulk cultures without added IL-2 (S. B. Campbell and A. Kelso, unpublished observations).

Abs to all three coactivating receptors supported the expansion of clones that produced IL-3 and/or IFN-{gamma}. As in our previous studies of primary T cell clones (55, 56, 61), there was a positive association between detection of cytokines and clone size over the 2-wk expansion period and differences in the frequencies of IL-3 and IFN-{gamma} producers between clones stimulated by Abs to CD4, CD11a, and CD28 could not be dissociated from differences in their average clone size. This association probably reflects two factors. One is the increasing probability as clones expand and differentiate that some cells within the clone have been activated to secrete cytokines (61, 72, 73). The other is that many cells may need to be activated for cytokine titers to reach the detection thresholds of the conventional cytokine assays used here. The latter is suggested by our ability to detect cytokine transcripts by PCR in developing clones well before the corresponding proteins are measurable in the supernatant (P. Groves and A. Kelso, unpublished observations; Ref. 61). In the present study, the titers of IFN-{gamma} and IL-4 produced by individual clones were highly heterogeneous, even among clones of similar size. Since some size 1 clones (<100 cells) were strongly positive and many larger clones produced enough cytokine to be detectable after 10- to 100-fold dilution, assay sensitivity was not the only factor underlying the relationship with clone size.

The striking and distinctive effect of CD4 ligation was the stimulation of IL-4 production. This was not a rare event. About 10–40% (mean, 27%; n = 1675 clones) of normal CD4+ T cells gave rise to IL-4-producing clones over 12–14 days in the presence of anti-CD4 mAb. Moreover, kinetic studies showed that the frequency and average production levels of IL-4-producing clones continued to rise for a week or more after clones ceased to expand. It seems likely that this rise was due both to accumulation of IL-4 produced by each IL-4-switched cell and to spread of this phenotype throughout cells of the clone as a result of continued stimulation and exposure to IL-4 secreted by neighboring cells. The development of IL-4-producing clones in response to anti-CD4 mAb also did not appear to be due to selective expansion of in vivo-primed T cells: cells with an activated phenotype (high expression of the stable activation marker CD44 or absence of CD62L) (74) were infrequent in the starting CD4+ lymph node population (<10%) and cloned relatively poorly, whereas cells of naive phenotype yielded IL-4-producing clones at similar frequencies to the starting population.

Most of the experiments reported here were performed with T cells from C57BL/6 mice, a strain which raises an IFN-{gamma}-polarized response to Leishmania major infection (62, 63). CD4+ T cells from the "Th2 strain," BALB/c, responded similarly to those from C57BL/6 mice both in the frequency of IL-4-producing clones generated and their preferential induction by anti-CD4 mAb. Thus, no intrinsic predisposition of BALB/c CD4+ cells to synthesize IL-4 was revealed under these activation conditions, in contrast to the finding of Bix et al. (64) in another system. The frequency at which IL-4-producing clones were obtained from BALB/c cells in the present study also argued against a significant contribution of the minor population of LACK-reactive IL-4-producing V{beta}4+V{alpha}8+CD4+ T cells reported to prime type 2 polarization in L. major-infected BALB/c mice (75).

Previous work in a number of systems has shown effects of anti-CD4 Abs on IL-4 synthesis. For example, prolongation of allograft survival in animals administered nondepleting anti-CD4 mAb has been associated with lowered IFN-{gamma} synthesis and unchanged or elevated IL-4 synthesis in vivo (31, 32), and addition of soluble anti-CD4 mAb to primary mixed lymphocyte culture enhanced IL-4 production upon restimulation (35). In vivo and in vitro, however, these effects have not been dissociated from the inhibitory effects of the Abs on CD4+ T cell numbers and activation which alter the composition of the responding population as well as the strength of signal delivery to T cells. It therefore remains unresolved whether their enhanced IL-4 responses were due to increased involvement of IL-4-producing bystander cells, selective survival of previously activated IL-4-producing T cells, preferential activation of T cells to synthesize IL-4 under suboptimal stimulation conditions, or delivery of an IL-4-promoting signal to T cells via CD4.

By contrast, the present study provides direct evidence for the last of these pathways. Any role for bystander cells was eliminated in this single-cell activation system and, as discussed above, preferential activation of a minor previously primed IL-4-producing subset was unlikely on the grounds of frequency. Most significantly, the approach used here also allowed the IL-4-promoting effects of anti-CD4 and the other strongly coactivating mAb, to CD11a, to be distinguished from effects on clone size. Anti-CD28 mAb supported formation of very few large clones and frequencies of IL-4 producers were no higher in the presence of this mAb than observed with anti-CD3 mAb alone. Anti-CD4 and anti-CD11a mAb, on the other hand, supported development of clones of comparable size yet anti-CD4 mAbs were consistently more effective at promoting development of IL-4 producers. The hierarchy of IL-4 induction by these Abs (anti-CD4 > anti-CD11a > anti-CD28) was observed with two different anti-CD4 mAbs and was not altered by elevating mAb dose. Assuming net clone size reflects cell division number and provides a functional measure of total signal strength received by the T cell under the defined conditions used here, the preferential effect of CD4 engagement on IL-4 synthesis can be dissociated from both of these parameters.

The mechanisms by which the anti-CD4 mAb promote this differentiative pathway in CD4+ T cells are not known. CD4 is believed to strengthen the signal delivered by the TCR both by increasing the affinity of interaction of the TCR complex with class II MHC and by forming an integral part of the TCR recognition and signaling complex (76). In the absence of class II MHC as in the present system, anti-CD4 mAb might enhance TCR-dependent signaling in two ways: at the cell surface by increasing TCR cross-linking and intracellularly by activation of the protein tyrosine kinase p56lck and the adaptor protein LAT, both of which are associated with CD4 and contribute to proximal and downstream events in the TCR signaling cascade, respectively (67, 76, 77). The importance of the extracellular functions of CD4 has been indicated by the observation that CD4-deficient mice generated a diminished IL-4 response to Nippostrongylus brasiliensis infection compared with wild-type littermates and that this response could be restored by reconstituting the mice with T cells expressing CD4 molecules that lacked a cytoplasmic tail (36). Whether this mutant CD4 could also contribute to TCR activation and IL-4 synthesis in the absence of class II MHC engagement remains to be established. Other studies suggest a link between CD4 signal transduction and IL-4 synthesis. In particular, T cells expressing CD4 molecules without a cytoplasmic tail failed to synthesize IL-4 in response to various stimuli that induced this cytokine in wild-type CD4+ cells in vitro or in vivo (37, 38) and overexpression of catalytically inactive Lck inhibited development of type 2 but not type 1 cytokine-producing CD4+ T cells (78).

The experiments reported here add to the recent evidence that normal CD4+ T cells can be activated to synthesize IL-4 in the absence of any exogenous source of this regulator (23, 24). Contrary to some but not other studies (28, 79), IL-6 does not appear to play a role in stimulating this response since the development of IL-4-producing clones was not inhibited by anti-IL-6 mAb. On the other hand, blocking by anti-IL-4 mAb showed that IL-4 produced by the clones themselves promoted further production by a positive autocrine loop. We did not see any evidence for negative regulation of IFN-{gamma} production in IL-4-producing clones but instead observed a highly significant positive association between synthesis of these two cytokines. This was true whether IL-4-producing clones were obtained by culture with anti-CD4 mAb or with exogenous IL-4, and whether cytokine assays were performed on cumulative 12-day primary supernatants or on 36-h secondary supernatants. It therefore seems likely that the two cytokines were synthesized simultaneously rather than sequentially by most clones. Although polarization to an IL-4+IFN-{gamma}- Th2 phenotype may possibly have occurred with more prolonged culture or in the presence of APC, it is notable that IFN-{gamma} synthesis was induced and maintained over 2 wk in these clones despite the absence of any source of IL-12. Factors which might have favored IFN-{gamma} synthesis in this system include the IL-2 added to support high-efficiency cloning, persistent high-avidity TCR ligation, and endogenous IFN-{gamma} itself (80, 81, 82). Anti-IFN-{gamma} mAb does not alter the development of IL-4- and/or IFN-{gamma}-producing CD8+ T cell clones in a similar Ab-driven APC-free system (K. Buttigieg and A. Kelso, unpublished observations) but the effect of IFN-{gamma} neutralization on the CD4+ T cell response studied here has not yet been tested.

The stimulation system used here has revealed the potential of normal CD4+ T cells to synthesize IL-4 in response to CD4 ligation. It is not known whether a similar phenomenon occurs when CD4 molecules interact with class II MHC on APC. The design of experiments to test this possibility, for example by blocking CD4-class II interaction (35), is confounded by the extracellular and intracellular effects of CD4 on the strength of the peptide/MHC-dependent signal delivered through the TCR which may in turn alter Th differentiation. If CD4 engagement also promotes IL-4 synthesis under physiological conditions, however, this pathway is one likely mechanism linking the strength of T cell stimulation with the differentiation of IL-4-producing cells.


   Acknowledgments
 
We thank Penny Groves and Kathy Buttigieg for provision of Abs and other technical support, Grace Chojnowski and Paula Hall for assistance with flow cytometry, and Dr. David Fitzpatrick and Dr. Norbert Kienzle for valuable comments.


   Footnotes
 
1 This work was supported by grants from the National Health and Medical Research Council of Australia and the Queensland Liver Transplant Trust Funds and by a National Health and Medical Research Council Postgraduate Research Scholarship to S.B.C. Back

2 Current address: Department of Neurosurgery, Brain Research Institute, Niigata University, 1-757 Asahimachi, Niigata 951-8585, Japan. Back

3 Address correspondence and reprint requests to Dr. Anne Kelso, Cooperative Research Center for Vaccine Technology, Queensland Institute of Medical Research, Post Office Royal Brisbane Hospital, Queensland 4029, Australia. E-mail address: anneK{at}qimr.edu.au Back

Received for publication May 10, 2001. Accepted for publication September 11, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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