Induction of type 2 activity in adult human CD8+ T cells by repeated stimulation and IL-4

Luminita A. Stanciu, Kevan Roberts1,, Laurie C. K. Lau1,, Anthony J. Coyle2, and Sebastian L. Johnston

National Heart and Lung Institute, Imperial College School of Medicine, Norfolk Place, London W2 1PG, UK
1 University Medicine, Southampton General Hospital, Southampton SO16 6YD, UK
2 Millennium Pharmaceuticals, Boston, MA 02139, USA

Correspondence to: S. L. Johnston


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
Repeated administration or chronic presence of antigen during CD4+ T cell activation and a cytokine milieu enriched in IL-4 favour the generation and maintenance of a Th2 population. However, there is little data on how these factors affect adult human CD8+ T cell functions. We established in vitro conditions to culture purified human CD8+ T cells, and investigated how repeated stimulation and exogenous IL-4 modulated their functions. Repeated TCR–CD3 stimulation of CD8+ T cells increased the number of CD25-, CD30- and CD40 ligand-expressing cells, and their capacity to secrete IL-4 and IL-5. In addition, repeatedly stimulated CD8+ T cells had cytotoxic activity and provided help to resting B cells for IgE synthesis. The presence of exogenous IL-4 during repeated stimulation further increased the number of CD25+ and CD30+ CD8+ T cells, up-regulated the number of IL-5+ cells, and increased IL-5 levels released. These observations demonstrate that repeated TCR–CD3 stimulation of normal human CD8+ T cells favoured the growth of cells with a type 2 phenotype and that this was further amplified by the presence of IL-4. These mechanisms may be important in virus-induced lung eosinophilic inflammation in healthy subjects and virus-induced exacerbations of asthma.

Keywords: cellular differentiation, cytokines, cytotoxic T lymphocyte


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
Effector CD4+ and CD8+ T cells can be categorized into subsets based on the pattern of cytokines they produce: type 1 producing IL-2 and IFN-{gamma}, type 2 producing IL-4 and IL-5, and type 0 secreting both type 1 and type 2 cytokines (15). Factors that modulate differentiation toward a type 1 or type 2 phenotype include the local cytokine milieu, the nature, mode of administration and dose of antigen, and the type of antigen-presenting cells (APC) and of co-stimulatory molecules (6). Among the relevant cytokines, the presence of IL-4 during TCR–CD3 activation was found to be essential for differentiation of mouse naive CD4+ and CD8+ T cells and type 0 CD4+ T cells into cells with a type 2 phenotype (3,4,711). In mice it has been shown that if type 2 cytokines are present in the lung environment, CD8+ T cell function is skewed toward a type 2 phenotype (12). Ex vivo peripheral blood CD8+ T cells from atopic asthmatic subjects contain higher levels of IL-4 compared with CD8+ T cells from normal subjects (13) and bronchoalveolar lavage CD8+ T cell lines from atopic asthmatic subjects secrete higher levels of IL-5 relative to those from control subjects (14). The increased capacity of CD8+ T cells from atopic asthmatic subjects to produce type 2 cytokines compared with normal subjects may be due to the fact that their stimulation by antigens takes place in an environment enriched in IL-4 (15,16).

Previous reports of IL-4 modulation of human CD8+ T cell function are limited to studies on T cell clones (TCC) (2) and on neonatal T cells (17). However, it is known that neonatal T cells have a type 2 phenotype that is deviated toward type 1 function by environmental stimulation during the first years of life (18). One therefore cannot assume that T cells from adult subjects will behave the same way as those from neonates.

CD8+ T cells are the major defence against viral pathogens and viruses are the major cause of asthma exacerbations (19). Asthma is a disease associated with excess production of IL-4. We therefore hypothesized that adult human CD8+ T cell functions could be deviated towards type 2 function by the presence of repeated stimulation (as occurs in viral infections) and by the presence of excess IL-4 (as occurs in asthma).

One reason for the paucity of information regarding adult human CD8+ T cell functions is that conditions to permit survival and expansion of CD8+ T cells of human origin have been difficult to establish, especially in the absence of APC. To test our hypothesis, it was desirable to establish in vitro conditions permissive to the culture of purified adult human CD8+ T cells in an APC-free system. Having done this, we then examined the effects of repeated TCR–CD3 stimulation on human CD8+ T cell surface phenotype and cytokine production. We further investigate how exogenous IL-4 present during repeated stimulation modulated CD8+ T cell surface antigens, cytokine production, cytotoxic activity and the ability to provide help for IgE production by B cells. We found that resting human CD8+ T cells, in an APC-free system, are driven toward a type 2 phenotype by repeated TCR–CD3 stimulation. The presence of exogenous IL-4 during stimulation of CD8+ T cells amplified the type 2-inducing effect of repeated stimulation, increasing the number of CD30+ and IL-5+ cells and IL-5 production.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
mAb, isolation kits and recombinant cytokines
mAb were: anti-CD3 (OKT3; ATCC, Rockville, MD), anti-CD4, anti-CD16, anti-CD19, anti-CD14 and anti-CD11b (Serotec, Oxford, UK), rabbit anti-mouse (Dako, High Wycombe, UK), peridinin chlorophyll protein-conjugated anti-CD3, FITC-conjugated anti-CD4, FITC- and -phycoerythrin (PE)-conjugated anti-CD8, and FITC- and -PE-conjugated anti-CD25 (Becton Dickinson, Mountain View, CA), FITC-conjugated anti-CD30 and PE-conjugated anti-CD154 [CD40 ligand (CD40L)] (PharMingen, San Diego, CA), PE-conjugated anti-IL-4, -IL-5 and -IFN-{gamma} (PharMingen), and FITC-conjugated anti-perforin (Hölzel Diagnostika, Köln, Germany). The `B cell isolation kit' was from Miltenyi (Miltenyi Biotec, Bergisch Gladbach, Germany). Cytokines were rIL-2 and rIL-4 (Genzyme, West Malling, UK).

CD8+ T cell preparation and culture
Peripheral blood mononuclear cells from nine healthy non-atopic donors (mean age 39, seven female and two male) were separated from heparinized peripheral blood by density-gradient centrifugation on Lymphoprep and depleted of adherent monocytes by 37°C adherence to plastic plates. CD8+ T cells were enriched by negative selection using mAb reactive to CD4, CD11b, CD14, CD16 and CD19, and removal of positive cells by panning to rabbit anti-mouse Ig-coated plates. The resulting populations were >97% CD3+ and >95% CD8+ as shown by flow cytometry, and contained no detectable CD4+, CD16+, CD19+ and CD14+ cells. An APC-free system was used for culture in order to avoid (i) the alteration of CD8+ T cell functions by cell–cell surface interactions with APC and by soluble factors released by APC, and (ii) the consumption, catabolism or antagonism of cytokines used in culture (IL-2 and IL-4) or released by CD8+ T cells. A model used to culture neonatal human CD8+ T cells (17) was adapted, by replacing the stimulus (anti-CD3 mAb immobilized on mouse L fibroblasts transfected with both CD32 and human B7 antigens) with plastic-bound anti-CD3 mAb.

In order to avoid stimulating only the CD28+ subset of CD8+ T cells, we elected not to use anti-CD28 antibody as a co-stimulus but supplemented the culture with IL-2 to prevent possible anergy. CD8+ T cells (0.5x106/ml) were stimulated with anti-CD3 mAb (10 µg/ml)-coated plastic 96-well round-bottom microtitre plates in culture medium (RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate and penicillin, streptomycin and amphotericin B) and exogenous IL-2 (50 U/ml) in the absence or presence of exogenous IL-4 (10 ng/ml). After 3 days stimulation, the cells were harvested, washed, counted and adjusted to 0.2x106/ml, and cultured for 4 days in the same culture medium containing 50 U/ml IL-2 (expansion). After the first cycle of stimulation and expansion the cells were harvested, washed, counted and adjusted to 1x106/ml, and tested for cytokine production, or were subjected to another cycle of stimulation and expansion.

Three-colour flow cytometric analysis of cell-surface antigens
Following the second cycle of stimulation/expansion, the cells were harvested and washed. Cells (0.1x106 cells/tube) were incubated with mAb for 30 min at 4°C, washed, resuspended in 500 µl PBS, and analysed using a FACScan flow cytometer (Becton Dickinson) and Lysys II software. Live lymphocytes were gated by the forward scatter and side scatter pattern, and the percentage and the mean fluorescence intensity (MFI) for different surface antigens were measured.

Evaluation of cytokines in CD8+ T cell culture supernatants by ELISA
At baseline and at the end of the first and second cycle of stimulation/expansion, cells were washed, resuspended at 1x106 CD8+ T cells/ml and stimulated with 20 ng/ml phorbol myristate acetate (PMA), 2 µg/ml ionomycin and 200 U/ml IL-2 or with immobilized anti-CD3 (10 µg/ml) for 24 h. IL-4, IL-5 and IFN-{gamma} levels were evaluated in the culture supernatants using Biosource ELISA detection systems (Biosource Europe, Fleurus, Belgium) following the manufacturer's instructions.

Flow cytometric analysis of intracellular cytokine synthesis
Intracellular cytokine staining was used to determine the frequency of cytokine-producing CD8+ T cells at the single-cell level. After cycle 2, the cells (1x106 cells/ml) were re-stimulated for 4 h with 25 ng/ml PMA, 1 µg/ml ionomycin and 10 µg/ml Brefeldin A. Cells were harvested and stained for surface expression of CD3 and CD8. After washing with staining buffer (1% FBS and 0.1% sodium azide), the cells were fixed and permeabilized using Cytofix/Cytoperm solution (PharMingen). PE-conjugated anti-cytokine or isotype-matched controls mAb were added and allowed to bind for 30 min at 4°C. To demonstrate antibody specificity, incubation with excess unlabelled anti-cytokine antibodies was performed 1 h before the addition of labelled anti-cytokine antibodies. This procedure resulted in >95% inhibition of the cytokine detection. The flow cytometric analysis was performed as above.

Cytotoxic activity
An anti-CD3 mAb-redirected cytotoxicity system was used to measure non-specific cytotoxic activity of CD8+ T cells exposed to two cycles of stimulation/expansion (20). Exponentially growing target cells (FcR-bearing murine mastocytoma cell line P815; ATCC) were labelled with [methyl-3H]thymidine (Amersham, Little Chalfont, UK) 5 µCi/ml for 4 h at 37°C, washed and resuspended at 1x105/ml in RPMI/5% FBS in the presence of mouse anti-human anti-CD3 mAb (OKT3, 1 µg/ml). Then 100 µl aliquots of CD8+ T cells (2. 5x106/ml) were plated in round-bottomed 96-well plates and 100 µl [3H]thymidine-labelled target cells added, and the plates were incubated for 4 h at 37°C. All samples were plated in triplicate. The cells were harvested and a ß plate scintillation counter was used to measure [3H]thymidine incorporation. The percent killing was calculated by subtracting experimentally retained DNA in the presence of effector cells (in c.p.m.) from retained DNA in the absence of effector cells (S = spontaneous), divided by S and multiplied by 100.

The frequency and MFI of perforin+ CD8+ T cells were determined by performing the same protocol as for intracellular cytokine staining, using FITC-conjugated anti-human perforin and matched control mAb.

B cell isolation and B–T cell co-culture for IgE production
Highly purified autologous peripheral blood B cells (>90% CD19+, <0.4% CD4+ T cells) were obtained from the same subject on the last day of the CD8+ T cell culture using the `B cell isolation kit' and MACS system from Miltenyi Biotec. After 14 days of culture, CD8+ T cells were harvested, adjusted to 1x105/ml and co-cultured with B cells at 1x106/ml in anti-CD3 mAb (10 µg/ml) precoated 96-well round-bottom plates in RPMI supplemented with 10% FBS in a final volume of 200 µl/well. The supernatants from the triplicate cultures were collected after 10 days, the triplicates pooled and IgE measured by a sensitive `in-house' ELISA. A purified mouse mAb (HB 121 cell line supernatants; ATCC) was bound to the wells of a microtitre plate at pH 9.5. Then 100 µl of supernatants of CD8+ T cell–B cell co-cultures was added to the plates. After incubation, the plates were washed in PBS/0.1% Tween 20 and bound IgE was detected by horseradish peroxidase-labelled rabbit anti-human IgE polyclonal antibody (Dako, Cambridge, UK) using o-phenylenediamine as substrate. Standard curves were constructed using reference sera which were calibrated against a WHO international standard (NIBSC, Pottersbar, UK).

Statistical analysis
Normally distributed data were expressed as mean ± SEM and paired Student's t-tests were used to compare data between conditions. The levels of IL-4, IL-5 and IFN-{gamma} were expressed as median [range] and the non-parametric Wilcoxon signed-rank test was used to compare between conditions. Values of P < 0.05 were considered as statistically significant.


    Results and discussion
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
Repeated TCR–CD3 stimulation induced CD25 and CD30 expression on CD8+ T cells and exogenous IL-4 further increased the frequency of CD25+ and CD30+ CD8+ T cells
We observed a significant increase in the expression of IL-2R{alpha} (CD25) on repeatedly stimulated CD8+ T cells. At baseline only a small number (3.1 ± 0.6%) of CD8+ T cells were activated and positive for CD25. After two cycles of stimulation/expansion a large number of cells were activated, 67 ± 8% of CD8+ T cells being CD25+ (P = 0.001, Table 1Go). The presence of exogenous IL-4 during stimulation significantly further increased the expression of CD25 on repeatedly stimulated CD8+ T cells (80 ± 5% with IL-4 as compared to 67 ± 8% without IL-4, P = 0.02, Table 1Go). The IL-2R {alpha} chain is rapidly and potently induced in response to mitogenic stimuli and by its own ligand IL-2, and together with the ß and {gamma} chains mediates high-affinity IL-2 binding. CD3 stimulation of resting or activated murine CD8+ T cells in the presence of IL-4 has been shown to induce IL-2 responsiveness (21). In our culture conditions of CD8+ T cells, exogenous IL-4 synergized with IL-2 and amplified CD25 expression.


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Table 1. Surface marker expression of human CD8+ T cells repeatedly stimulated in the absence or in the presence of exogenous IL-4
 
We also examined the expression of CD30 at baseline and after two cycles of stimulation/expansion in CD8+ T cells cultured in the presence or absence of IL-4. CD30 was absent on highly purified human CD8+ T cells at baseline but more than one-third of CD8+ T cells exposed to two rounds of stimulation/expansion expressed CD30 (36 ± 8%, P < 0.001) (Table 1Go and Fig. 1Go). Repeated stimulation of CD8+ T cells in the presence of exogenous IL-4 induced a further increase in the frequency of CD30+ cells (49 ± 8% with IL-4 as compared to 36 ± 8% without IL-4, P < 0.01, Fig. 1Go). CD30 antigen is considered a surface marker for type 0 and type 2 Th cell IL-4 responsiveness, because type 1 cells did not up-regulate CD30 on exposure to IL-4 and did not start to produce IL-4 (22,23). Human CD8+ T cell clones that produce IL-4, IL-5 and IFN-{gamma}, i.e. with the Tc0 phenotype, have also been shown to express CD30 (24). The appearance of a CD30+ population of CD8+ T cells following repeated stimulation in our culture conditions is likely to reflect the endogenous production of IL-4 and the outgrowth of cells with a Tc0/Tc2 phenotype. This hypothesis is supported by our findings of a further increase in CD30 expression if this stimulation occurred in the presence of exogenous IL-4.



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Fig. 1. Frequencies of CD30+ cells following repeated stimulation with and without exogenous IL-4. CD30 expression is induced and is further increased by the presence of exogenous IL-4 during repeated stimulation of human CD8+ T cells. CD8+ T cells were exposed to two cycles of stimulation with or without IL-4. CD8+ T cells were harvested and stained for CD3, CD8 and CD30. Lymphocytes were gated based on their forward scatter/side scatter profile, and gated further based on CD3 and CD8 expression. Percentages of CD30+ CD8+ T cells in eight subjects are shown (bars represent mean + SEM values). Incubation with IL-4 significantly increased surface expression of CD30 compared to 14 day culture without IL-4 (*P < 0.01).

 
Repeated stimulation of human CD8+ T cells increased type 2 cytokine production
To test this hypothesis, at baseline and after the first and second cycles of stimulation/expansion, CD8+ T cells were stimulated for 24 h with PMA, ionomycin and IL-2 or with anti-CD3 mAb, and the levels of IL-4, IL-5 and IFN-{gamma} released into supernatants were determined by ELISA (Fig. 2Go). Because the results obtained with both kinds of stimulation were similar, we will present the results obtained with PMA, ionomycin and IL-2 stimulation. The exposure of CD8+ T cells to two cycles of stimulation/expansion induced higher levels of IL-4 (median [range] 196 [88–547] pg/ml) as compared with both baseline levels (60 [52–92] pg/ml, P = 0.07) and levels after one cycle of stimulation (62 [40–123] pg/ml, P = 0.04, Fig. 2AGo). Similarly, the exposure of CD8+ T cells to two cycles of stimulation/expansion induced much higher levels of IL-5 (537 [125–1851] pg/ml as compared with baseline levels (111 [87–249] pg/ml, P = 0.07) or the levels after one cycle of stimulation (78 [65–142] pg/ml, P = 0.04, Fig. 2BGo). The capacity to release IFN-{gamma} in culture increased slightly but not significantly after one cycle of stimulation/expansion (23.5 [3.3–47.0] ng/ml) as compared with baseline (6.2 [5.9–23.0 ng/ml] ng/ml, P = 1), but remained stable after two cycles (23.0 [6.8–36.0 ng/ml] ng/ml, Fig. 2CGo). Thus the capacity to release IL-4 and IL-5, but not IFN-{gamma}, in culture supernatants was increased by repeated TCR–CD3 stimulation of CD8+ T cells.



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Fig. 2. Levels of IL-4 (A), IL-5 (B) and IFN-{gamma} (C) released following repeated stimulation with and without exogenous IL-4. Repeated TCR–CD3 stimulation of human CD8+ T cells increased their capacity to release IL-4 and IL-5, and the presence of exogenous IL-4 during stimulation further increased IL-5 production. CD8+ T cells were stimulated in the presence or absence of IL-4 for two cycles. At baseline (day 0) and after the first (day 7) and second cycle (day 14) of TCR–CD3 stimulation, 1x106/ml CD8+ T cells were further stimulated with PMA, ionomycin and IL-2 for 24 h. The cytokine levels in culture supernatants were measured by ELISA. The data represent the median and inter-quartile range of five subjects. *P = 0.2 when compared with the levels without exogenous IL-4.

 
Human peripheral blood CD8+ T cells are a mixture of naive and memory cells, and of type 0, 1 and 2 cells. The requirement of IL-4 for inducing naive uncommitted T cells to differentiate into cells with a type 2 phenotype is a well-established concept (8,25). It is now accepted that naive human and mouse CD4+ and CD8+ T cells are capable of producing low levels of IL-4 (and other type 2 cytokines), either spontaneously or induced by an IL-4-independent pathway (2630), and CD4+ T cells have been shown to acquire a type 2 phenotype upon repeated stimulation (6,26,31). In vitro prolonged TCR–CD3 stimulation of naive and memory murine CD4+ T cells resulted in a Th0-type phenotype (IFN-{gamma}, IL-4 and IL-5 production) (32). In a mixed population of Th1 and Th2 cells, prolonged TCR–CD3 ligation leads to Th2 predominance (33). We have demonstrated that in our culture conditions, repeated stimulation of CD8+ T cells induced autocrine production of IL-4 during the second cycle of stimulation and expansion (Fig. 2AGo). We believe that this production of IL-4 selectively promoted the expansion of Tc0 and Tc2 cells, the only phenotypes that respond to IL-4 (22,23), and altered the balance between type 1 and type 2 cytokine in the favour of type 2 cytokine production. Our findings of increased production of IL-5 by CD8+ T cells following the second cycle of stimulation and expansion supports this view, as does the induction of expression of CD30. In contrast, the levels of IFN-{gamma} released after one cycle of stimulation (when autocrine IL-4 production had not occurred) were not further increased by the second cycle of stimulation, indicating that IFN-{gamma} production is not increased by repeated stimulation, and that the further increase that would have been expected following the second cycle of stimulation and expansion may have been actively suppresses by the autocrine IL-4 production.

Exogenous IL-4 increased type 2 cytokine production induced by repeated stimulation of human CD8+ T cells, the frequency of IL-5-expressing cells and the intracellular levels of IL-5
Having observed that induction of autocrine production of IL-4 was associated with induction of type 2 activity in human CD8+ T cells, we then wished to determine whether the addition of exogenous IL-4 during stimulation would further augment this induction. The capacity of exogenous IL-4 to influence the cytokine production of CD8+ T cells was investigated by comparing the levels of cytokine released by CD8+ T cells exposed to TCR–CD3 stimulation in the presence and absence of exogenous IL-4. IL-4 and IFN-{gamma} levels released in culture by repeatedly stimulated CD8+ T cells after one or two cycles of stimulation/expansion in the absence of IL-4 were similar with the levels released by the CD8+ T cells stimulated in the presence of exogenous IL-4 (Fig. 2A and CGo). The production of IL-5 by CD8+ T cells after the second cycle of stimulation in the presence of IL-4 (2034 [322–2768 pg/ml] pg/ml) was 4-fold higher compared with the production of CD8+ T cells stimulated in the absence of IL-4 (537 [125–1851 pg/ml] pg/ml, P = 0.2, Fig. 2BGo).

Having observed that the presence of exogenous IL-4 during repeated TCR–CD3 stimulation of CD8+ T cells further increased the levels of IL-5 released in culture supernatants, we then investigated if the presence of IL-4 during repeated stimulation modulated cytokine production at the single-cell level. The presence of IL-4 had no significant effect on either the frequency or MFI of IL-4-staining cells (data not shown). The frequency of IL-5+ CD8+ T cells in repeatedly stimulated CD8+ T cells in the presence of exogenous IL-4 was significantly increased compared with the frequency in repeatedly stimulated CD8+ T cells in the absence of exogenous IL-4 (3.7 ± 0.5% with IL-4 as compared with 2 ± 0.3% without IL-4, P = 0.002, Fig. 3AGo). In addition, MFI of intracellular IL-5 levels was significantly increased when exogenous IL-4 was present during stimulation (35 ± 6 versus 28 ± 5.7, P = 0.05, Fig. 3CGo). There was a trend toward a reduction in MFI of IFN-{gamma}+ CD8+ T cells when exogenous IL-4 was present (430 ± 144 with IL-4 as compared with 702 ± 209 without IL-4, P = 0.06, Fig. 3DGo) as well as a trend towards reduced frequency of IFN-{gamma}+ cells (35 ± 8% with IL-4 versus 41 ± 9% without IL-4, P = 0.2, Fig. 3BGo). These results confirmed at the single-cell level that the presence of exogenous IL-4 during TCR–CD3 stimulation of human CD8+ T cells selectively augments the number of IL-5-staining cells and decreases IFN-{gamma} production.



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Fig. 3. Frequency of IL-5+ (A) and IFN-{gamma}+ (B) cells and MFI of IL-5 (C) and IFN-{gamma} (D) intracellular levels following repeated stimulation with and without exogenous IL-4. The frequency and MFI of IL-5+ CD8+ T cells are increased by the presence of exogenous IL-4 during repeated TCR–CD3 stimulation. Cells were washed after the second cycle of stimulation and re-stimulated for 4 h with PMA and ionomycin in the presence of Brefeldin A. After surface staining for CD3 and CD8, the cells were permeabilized and stained with mAb against IL-4, IL-5 and IFN-{gamma}. Lymphocytes were gated based on their forward scatter/side scatter profile, and gated further based on CD3 and CD8 expression. Percentages of CD8+ T cells positive for each cytokine and MFI were determined. The bars represent the mean and SEM values (n = 9). *P = 0.05, **P = 0.002 when compared with the levels without exogenous IL-4.

 
The capacity of IL-4 to induce and amplify type 2 cytokine IL-5 production in CD8+ T cells has been reported in studies of murine T cells, human neonatal T cells and TCC (24,8,17,25,34). However, no previous studies have studied the effect of autocrine or exogenous IL-4 on human adult CD8+ T cells in an APC-free system. Human CD8+ TCC generated in vitro in the presence of IL-4 displayed a Tc0/Tc2 phenotype (35) and a proportion of CD8+ TCC supported IgE synthesis in B cells in the presence of exogenous IL-4 (36). In our culture conditions the presence of IL-4 during TCR–CD3 stimulation induced a bias of CD8+ T cells toward a Tc0/Tc2 phenotype with a higher frequency of CD30+ cells and IL-5+ cells, and higher levels of IL-5 at the single-cell level and in culture supernatants. It is possible that exogenous IL-4, a growth factor for type 0 and 2 cells, but not for type 1, helped the preferential outgrowth of pre-existing Tc0/Tc2 cells and/or induced differentiation of naive, undifferentiated Tc precursors (5,33). This is the first demonstration that repeated stimulation of purified CD8+ T cells from healthy non-atopic adult subjects in a milieu enriched in IL-4 favours the Tc0/Tc2 phenotype.

Cytotoxic capacity of repeatedly stimulated human CD8+ T cells is not altered by the presence of exogenous IL-4 during TCR–CD3 stimulation
Having demonstrated that the presence of exogenous IL-4 during two cycles of stimulation/expansion increased the frequency of CD30+ and IL-5+ CD8+ T cells, we wished to determine whether exogenous IL-4 modulated the cytotoxic capacity of repeatedly stimulated human CD8+ T cells by investigating levels of perforin by intracellular staining and by measuring the cytotoxic activity using an anti-CD3-redirected cytotoxic assay.

The frequency and MFI of perforin+ CD8+ T cells measured by intracellular staining and flow cytometry were not significantly altered by the presence of IL-4 during repeated TCR–CD3 stimulation; 97.5 ± 2% of IL-4-untreated and 95.5 ± 2% of IL-4-treated CD8+ T cells were perforin+, and the MFI of perforin was 76 ± 3 in IL-4-untreated cells and 72 ± 7 in IL-4-treated cells (data not shown). Anti-CD3-redirected cytotoxic activity of CD8+ T cells exposed to repeated stimulation was not significantly altered by the presence of exogenous IL-4 (percent specific killing at an E:T cell ratio of 25:1 was 17 ± 17% in IL-4-untreated and 29 ± 14% in IL-4-treated cells) at this or any other E:T cell ratio (data not shown).

In murine studies, both Tc1 and Tc2 were found to be highly cytolytic (3,4,37), mainly through perforin exocytosis (38), and in vivo both subtypes induced similar inflammatory reactions (39). In one study murine CD8+ T cells stimulated in vitro in the presence of IL-4 have been shown to down-regulate cytotoxic function (34), but in other studies IL-4 did not reduce and even enhanced cytotoxic activity of murine CD8+ T cells (4,40). Both Tc1 and Tc2 allospecific human CD8+ T cells were capable of cytotoxicity when stimulated through the TCR (41). In our culture conditions, repeatedly TCR-stimulated CD8+ T cells were perforin+ and exhibited a degree of anti-CD3-mediated cytotoxicity. Exogenous IL-4 present during TCR stimulation did not significantly alter perforin expression or cytolytic capacity of repeatedly stimulated CD8+ T cells. These data suggest that the presence of IL-4 does not profoundly influence cytolytic activity in adult human CD8+ T cells in the conditions studied.

Repeatedly TCR–CD3-stimulated human CD8+ T cells provide help to B cells for IgE
Following the observation that repeatedly stimulated CD8+ T cells express increased levels of CD40L [12 ± 3% of CD8+ T cells exposed to repeated stimulation without exogenous IL-4 and 9 ± 3% of CD8+ cells exposed to repeated stimulation in the presence of exogenous IL-4 (Table 1Go), as compared with only 2.8 ± 1.3% of CD8+ T cells at baseline, P < 0.01] and produced IL-4, we investigated whether repeatedly stimulated CD8+ T cells were able to induce B cell activation, Ig switching and IgE production. Repeatedly stimulated human CD8+ T cells co-cultured with resting autologous B cells in precoated anti-CD3 plates provided help to B cells for IgE synthesis. There were no significant differences in the levels of IgE secreted by B cells in co-culture with IL-4-untreated CD8+ T cells as compared with co-culture with IL-4-treated CD8+ T cells (2.8 ± 1.1 respectively 2 ± 0.7 versus 0.092 ± 0.07 ng/ml in the B cell cultured alone) (data not shown). This was not surprising because we found that the presence of exogenous IL-4 during TCR–CD3 stimulation did not significantly amplify either CD40L expression or the levels of IL-4 released in culture of repeatedly stimulated human CD8+ T cells.

IL-4 released by T cells together with the signals induced by the interactions between CD40L on activated T cells and CD40 on B cells may induce B cell activation and Ig class switching (42,43). It has previously been reported that stimulation of murine CD8+ T cells with anti-CD3 mAb combined with IL-4 induced CD8+ T cells that produced IL-4 and helped B cells to secrete IgG1 in the presence of exogenous IL-4 (34). Also, murine CD8+ TCC producing IL-4 and expressing CD40L induced small resting B cells to secrete IgM and IgG1 (44). Murine cytolytic Tc2 cells have recently been reported to provide substantial B cell help for Ig production; the killing of B cells by CD8+ T cells in this system was strongly inhibited in the presence of plate-bound anti-CD3 antibodies, a fact which the authors suggest resulted from perforin-mediated cytotoxic activity being focused away from the B cells (45). By using a similar system (with coated anti-CD3) and an adequate ratio between T cells and B cells we have shown that adult human CD8+ T cells can also provide help for IgE synthesis. This is the first demonstration that repeatedly stimulated CD8+ T cells from healthy adult subjects express CD40L, produce IL-4 and provide help to B cells for IgE production.

In summary, this work demonstrates that repeated TCR–CD3 stimulation of normal human CD8+ T cells in an APC-free system induces the growth of human CD8+ T cells with a Tc0/Tc2 phenotype in terms of surface protein expression, cytokine production and helper activity. The presence of exogenous IL-4 during stimulation further increases the frequency of Tc2 (CD30+ and IL-5+) cells and IL-5 production, but does not alter the cytotoxic or helper capacities of repeatedly stimulated CD8+ T cells. The demonstration of these properties implicates CD8+ T cells in diseases associated with excess type 2 activity such as allergy and asthma, and suggests that the development of type 2 function by CD8+ T cells may play an important role in virus-induced exacerbations of asthma.


    Acknowledgments
 
This work was supported by National Asthma Campaign, UK (grant no. 331).


    Abbreviations
 
APC antigen-presenting cell
CD40L CD40 ligand
MFI mean fluorescence intensity
PE phycoerythrin
PMA phorbol myristate acetate
TCC T cell clones

    Notes
 
Transmitting editor: R. L. Coffman

Received 1 September 2000, accepted 4 December 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 

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