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. 1998 Nov;114(2):284–292. doi: 10.1046/j.1365-2249.1998.00709.x

In vitro differentiation of peripheral blood T cells towards a type 2 phenotype is impaired in rheumatoid arthritis (RA)

S Asselin *, H Conjeaud *, D Fradelizi *, M Breban *
PMCID: PMC1905095  PMID: 9822289

Abstract

We have examined the capacity of peripheral blood T cells from RA patients to be polarized in vitro towards a type 1 (T1) or a type 2 (T2) phenotype. Peripheral blood T cells from RA patients and from healthy donors were primed by 1 week of culture with soluble OKT3 in the presence of polarizing cytokines. The recovered T cells were restimulated and their cytokine secretion profile determined. Priming of T cells from RA patients in the presence of recombinant (r)IL-2 plus rIL-12 induced a shift towards a T1 pattern, characterized by increased production of interferon-gamma, that was more pronounced than in the case of healthy donors. Conversely, priming of T cells from RA patients in the presence of IL-4 failed to induce a shift towards a T2 profile after 1 week, whereas it induced T cells from healthy donors to acquire such a profile characterized by heightened production of IL-4, IL-5 and IL-13. However, a T2 polarization profile emerged in T cells from RA patients that were primed in the presence of rIL-4 and subsequently maintained in culture in rIL-2 alone for 1 or 2 additional weeks. We conclude that in vitro differentiation of peripheral T cells towards a type 2 phenotype is impaired in RA. Nevertheless, conditions required to drive peripheral T cells towards a type 2 phenotype were established. Administration of autologous polyclonal T cells expressing a type 2 cytokine secretion profile is proposed as a therapeutic strategy in RA.

Keywords: rheumatoid arthritis, cytokines, T cell, passive immunotherapy

INTRODUCTION

RA is a chronic inflammatory disease of unknown origin affecting primarily the synovial tissue. Macrophage-derived cytokines are the most abundant in rheumatoid synovitis [1]. However, T cells are thought to play a critical role in the pathogenesis of RA [2]. Mature T helper (Th) cells can be divided into distinct functional subsets according to the array of cytokines they produce: Th1 cells, which produce cytokines such as IL-2, interferon-gamma (IFN-γ) and tumour necrosis factor-beta (TNF-β), favour the development of a DTH reaction, whereas Th2 cells provide help for B cells via the production of IL-4, IL-5 and IL-6 [3]. The Th2 response also involves IL-10 and IL-13 in mice, whereas secretion of these cytokines by human T cell clones is not strictly restricted to a distinct phenotype [3]. A similar dichotomy was also described for CD8+ T cells leading to a broader concept of type 1 (T1) and type 2 (T2) cytokine secretion pattern [3,4].

It has been proposed that an imbalance between mutually antagonistic T1 and T2 polarized T lymphocytes could play a role in the pathogenesis of autoimmune diseases [4]. T1 cells appear to play a central role in the pathogenesis of several animal models of organ-specific autoimmune diseases, whereas T2 cells seem rather to be associated with protection or remission [5–7]. Concerning RA, evidence has accumulated suggesting that T1 cells predominate in RA synovitis [8–12], and increased detection of T1 cytokines has been described in the peripheral blood of RA patients [13]. Taken together, these findings raise the possibility that a persistent state of T1 activation could maintain chronic inflammation in RA, and conversely that T2 cells could be protective in this disease. Indeed, T cell-derived cytokines have been shown to influence synovial inflammation. More specifically, IFN-γ induces the in vitro production of proinflammatory cytokines by RA monocytes and synovial macrophages [14], whereas T2 cytokines such as IL-4 and IL-13 have an opposite effect [15–17]. Moreover, IL-4 was shown to inhibit several features of the joint inflammatory process that develops in RA, such as the production of proinflammatory cytokines by synovial cells and tissue [18–21], the synoviocyte proliferation [22] and the bone resorption [23].

These results suggest that modulating the T1/T2 balance of lymphokine secretion by T cells in favour of T2 cytokine production could be beneficial in RA [24]. Indeed, immune-mediated disorders were improved in several animal studies, by shifting in vivo the immune response from a T1 to a T2 profile. In those experiments, immune deviation was achieved in several ways, i.e. by administration of polarizing cytokines at the time of immunization [25]; by administration of T cells primed in vitro in the presence of polarizing cytokines [26]; by reactivation of primed bystander Th2 cells at the time of immunization [27]; or by passively tranferring antigen non-specific Th2-like T cells [28]. Since RA is an autoimmune disease with no established target antigen, we choose to generate in vitro polyclonal T2 cells, that could be passively re-administered to the patient to modify the T1/T2 balance and hopefully ameliorate disease.

Bearing this purpose in mind, we have previously established in vitro conditions that drive bulk peripheral blood T cells from normal healthy donors towards a predominant T2 lymphokine secretion profile [29]. In the present study we applied these conditions to peripheral blood mononuclear cells (PBMC) from RA patients and examined if a T2 phenotype could similarly be induced.

PATIENTS AND METHODS

Patients

PBMC were obtained from seven patients with active RA (Table 1) or from normal donors by density gradient centrifugation and stored in liquid nitrogen until use. All RA patients met the criteria of the American College of Rheumatology [30]. None had received any disease-modifying anti-rheumatic drug for at least 1 month. This study was approved by the institutional Ethics Committee. Written informed consent was obtained from RA patients.

Table 1.

Characteristics of the RA patients

graphic file with name cei0114-0284-t1.jpg

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Human recombinant cytokines, and murine MoAbs

The following cytokines were used in culture at the indicated concentrations: rIL-2 10 ng/ml, kindly provided by Sanofi Recherches (Labège, France); rIL-4 10 ng/ml and rIL-12 2 ng/ml purchased from R&D Systems Europe Ltd. (Abingdon, UK). Murine MoAbs used were: anti-CD3 (OKT3, IgG2a; Cilag, Boulogne, France); neutralizing anti-IL-4R (23463.11, IgG2a; R&D Systems); anti-CD4 (BL4, IgG2a; Immunotech, Marseille, France); anti-CD8 (UCHT4, IgG2a; Sigma Immunochemicals, St-Quentin Fallavier, France); anti-CD14 (B-A8; Innotest, Besançon, France); anti-CD19 (B-C3; Innotest); anti-CD56 (ERIC 1; Bioatlantic, Nantes, France); anti-CD11a (B.B15; Innotest); anti-CD54 (B-H17; Innotest); anti-CD45RA (B-C15; Innotest); anti-CD45RO (B-P2; Innotest); anti-CD49d (HP2.1; Immunotech); anti-HLA-DR (D1.12, a kind gift of Dr D. Charron, Paris, France); anti-CD25 (B-F2; Innotest); anti-CD30 (HRS-4, IgG1; Immunotech); and anti-lymphocyte activation gene 3 (LAG-3) (17B4, IgG1; a kind gift from Dr M. Dreano, Ares Serono, Geneva, Switzerland).

Cell preparations and cultures

Cell cultures were performed in RPMI 1640 with l-glutamine, 5% heat-inactivated normal AB+ serum, penicillin G 100 U/ml, streptomycin 100 μg/ml, and sodium pyruvate 1 mm. PBMC (5 × 105/ml) were primed with soluble OKT3 (sOKT3; 10 ng/ml) in the presence of exogenously added cytokines for 3 days. Cells were washed and maintained with fresh cytokines for another 4 days. At the end of the first week, cells were washed, resuspended at 106 cells/ml and restimulated with phytohaemagglutinin (PHA; 1 μg/ml), or with antigen-presenting cell-independent stimuli, i.e. PHA (1 μg/ml) + phorbol 12-myristate acetate (PMA; 50 ng/ml). In some experiments, cells were maintained with rIL-2 for 1 or 2 additional weeks before being restimulated with PHA or with PHA + PMA.

Quantification of cytokines in T cell culture supernatant by sandwich ELISA

Quantitative determinations of IFN-γ and IL-4 were performed using the anti-IFN-γ MoAbs 350B10G6 and 67F12A8 (Medgenix Diagnostics, Rungis, France) and the anti-IL-4 MoAbs 860A4B3 and 860F10H12 (Medgenix), respectively, following the manufacturer's recommendations.

Cytokine-specific quantitative reverse transcription-polymerase chain reaction

Cellular (cel)-RNA was isolated with the TRI Reagent (Molecular Research Centre, Cincinnati, OH) and reverse transcribed into cDNA using oligo (dT)15 primers (Genosys, Cambridge, UK) and AMV reverse transcriptase (Promega, Madison, WI). cDNA reverse transcribed from 50 ng of cel-RNA was co-amplified by polymerase chain reaction (PCR) in the presence of 100 fg of standard (std)-DNA, consisting of linearized synthetic plasmid containing priming site sequences for IL-4, IL-5, IFN-γ (pQA1), or β-actin and IL-13 (pQB2) (provided by D. Shire, Sanofi Recherches) [29], 100 ng of each primer and 1 U Taq polymerase (A.T.G.C., Noisy Le Grand, France). Primer sequences have been previously described [29]. Samples were overlaid with mineral oil and amplified using a DNA thermal cycler (Perkin Elmer Cetus Instruments, St-Quentin-en-Yvelines, France). The conditions consisted of 5 min at 92°C followed by 35 sequential cycles of denaturation at 92°C for 1 min, annealing at 57°C for 1 min and extension at 72°C for 1 min. PCR was terminated by elongation at 72°C for 10 min. PCR products were electrophoresed on a 3% Nusieve agarose gel in the presence of Sybr Green I. For each gene product amplified, cel-DNA and std-DNA migrated as two bands that were quantified on polaroid negative pictures of the gel with a personal densitometer and the corresponding Image Quant 3.3 software (Molecular Dynamics, Sunnyvale, CA). Results were expressed as ratios of cel-amplicon/std-amplicon. Evaluation of β-actin in parallel with specific cytokine mRNA was used to normalize samples for their cel-mRNA content. Final results were expressed as a copy ratio (CR) of specific cytokine/β-actin transcripts contained in a given amount of cel-RNA.

Flow cytometric detection of cell surface antigen

Briefly, 1–5 × 105 cells were incubated with saturating concentrations of one (for single-colour immunofluorescence) or two (IgG1 and IgG2a, for two-colour staining) primary MoAb(s), then incubated with either monoclonal goat anti-mouse (GAM) IgG-FITC (Eurobio, Les Ulis, France), or with two secondary MoAbs (GAM IgG1-FITC and GAM IgG2a-PE; Southern Biotechnology, Birmingham, AL), respectively. After washing, the cells were fixed in 1% paraformaldehyde and analysed using a FACScan flow cytometer and Lysis software.

RESULTS

Proliferation of T cells in the presence of cytokines is reduced in RA

Peripheral lymphocytes from five normal donors and five RA patients were primed in vitro with sOKT3, in the presence of IL-2, or IL-4, or a combination of IL-2 + IL-12. Analysed by flow cytometry, the proportion of CD3+ T cells at the end of the 1-week priming period was comparable in all conditions of culture, > 90% with negligible (< 4%) natural killer (NK) cells (CD56+), monocytes (CD14+), and B cells (CD19+) (not shown). The proportion of CD4+ and CD8+ T cells was roughly comparable in all conditions, each subset representing close to 50% of total T cells (not shown).

At the end of the first week of culture, the number of live cells recovered from RA patient cultures was consistently less than from normal donors, whatever the cytokines added (Fig. 1a). However, cells stimulated in the presence of IL-2 or IL-2 + IL-12 proliferated more and exhibited less pronounced decreased proliferation in RA patients than those stimulated in the presence of IL-4 (proliferation index of RA patients relative to normal donors: −34% in IL-2; −21% in IL-2 + IL-12, and −59% in IL-4).

Fig. 1.

Fig. 1

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Expansion of sOKT3-stimulated T cells in the presence of exogenously added cytokines in normal donors and RA patients. Peripheral blood mononuclear cells (PBMC) from normal donors (▪) and RA patients (□) were stimulated with sOKT3 and cultured in the presence of rIL-2, rIL-4 or rIL-2 + rIL-12 for 1 week. At the end of the first week of culture, live cells were numbered, washed and cultured for 1 additional week in the presence of rIL-2 alone. Data are expressed as proliferation index (mean + s.e.m.), i.e. the ratio of live cells (> 90% CD3+ T lymphocytes) at the end of the first (a) or second (b) week of culture to the number of cells seeded at a density of 5 × 105 cells/ml at the beginning of each week.

In some experiments, T cells from RA patients and normal donors that had been primed for 1 week as described above in the presence of IL-2, or IL-4, or IL-2 + IL-12, were subsequently maintained in culture supplemented with exogenous rIL-2. During this additional period, T cells from RA patients expanded several fold, at levels comparable to normal donors and without differences between priming conditions (Fig. 1b).

Lack of induction of a T2 profile after primostimulation of RA T cells in the presence of IL-4

To examine if priming in the presence of exogenous cytokines could drive T cells from RA patients to differentiate towards T1 or T2 phenotype, T cell cytokine production was assayed at the end of the 1-week priming period after restimulation with PHA.

IL-4 and IFN-γ were assayed at the protein level by ELISA in culture supernatants (SN) of primed cells that had been restimulated for 18 h (Fig. 2). In normal donors, the production of IL-4 by T cells was consistently increased when the priming conditions were with rIL-4 (Fig. 2a), whereas production of IFN-γ was enhanced after priming in the presence of rIL-2 + rIL-12 (Fig. 2b), confirming our previous findings [29]. In contrast, T cells from RA patients primed in the presence of rIL-4 produced minute amounts of IL-4, that were not different from those produced under other conditions (Fig. 2a). Experiments in which restimulation was performed in the presence of a neutralizing anti-IL-4R MoAb indicated that this low level of IL-4 was not secondary to cellular consumption of IL-4 (not shown). Conversely, we observed a greater production of IFN-γ by T cells from RA patients than from normal donors in all culture conditions. The greatest production of IFN-γ was reproducibly observed in SN from cells that had been primed in the presence of IL-2 + IL-12 (Fig. 2b).

Fig. 2.

Fig. 2

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Comparison of type 1 and type 2 cytokine protein synthesis by T cells primed in the presence of exogenously added cytokines after 1 week of culture. Peripheral blood mononuclear cells (PBMC) from normal donors (▪) and RA patients (□) were primed with sOKT3 in the presence of rIL-2, rIL-4 or rIL-2 + rIL-12 and secondarily challenged for 18 h with phytohaemagglutinin (PHA). IL-4 (a) and IFN-γ (b) were assayed by ELISA in the culture supernatant. Results are expressed as mean + s.e.m.

Evolution of the cytokine secretion profile of primed T cells subsequently cultured in rIL-2 alone

Our previous studies in normal donors have shown that the polarization towards a T2 profile, induced as early as 1 week after priming T cells in the presence of IL-4, further increased after additional culture in rIL-2 alone [29]. Priming of RA T cells in the presence of IL-4 failed to induce a T2 polarization after 1 week. However, those T cells were subsequently maintained in culture supplemented with rIL-2 for 1 or 2 additional weeks, with the object of achieving their T2 differentiation.

Evolution of the cytokine production profile was assessed by periodic restimulation of the T cell populations with PHA, both at mRNA (Fig. 3) and protein (Fig. 4) levels. Cytokine mRNA synthesis was examined 6 h after restimulation with PHA (Fig. 3). This evaluation confirmed the lack of induction of T2 cytokines (IL-4, IL-5 and IL-13; Fig. 3a–c), 1 week after priming T cells in the presence of IL-4, whereas IFN-γ (Fig. 3d) was increased in T cells primed in the presence of IL-2 + IL-12. Interestingly, after weeks 2 and 3 of culture, a clear polarization towards a T2 phenotype emerged specifically in the RA T cell population originally primed in the presence of IL-4. This evolution was characterized by increased synthesis of IL-4, IL-5 and IL-13 mRNA (Fig. 3a–c) and by increased release of IL-4 in culture SN upon restimulation (Fig. 4a). In parallel we observed a decline of T1 cytokine production by T cells upon restimulation. Indeed, the levels of IFN-γ mRNA synthesis (Fig. 3d) and protein production (Fig. 4b) decreased in all combinations from week 2 after priming. Comparable results were observed with other restimulation protocols, i.e. PMA + PHA (not shown).

Fig. 3.

Fig. 3

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Evolution of T1 and T2 cytokine mRNA synthesis by T cells from RA patients primed in the presence of exogenous cytokines and maintained in culture. Peripheral blood mononuclear cells (PBMC) from RA patients were primed for 1 week with sOKT3 in the presence of rIL-2, rIL-4 or rIL-2 + rIL-12. One week after priming, T cells were either immediately restimulated with phytohaemagglutinin (PHA), or kept in culture in rIL-2 alone and restimulated with PHA 2 weeks or 3 weeks after priming. Cytokine-specific mRNA levels were assessed by quantitative reverse transcription-polymerase chain reaction (RT-PCR) 6 h after restimulation and expressed as copy ratio (CR) of cytokines to β-actin. Results from three RA patients are shown for cells restimulated 1 or 2 weeks after priming and from two RA patients for cells challenged 3 weeks after priming (mean + s.e.m.).

Fig. 4.

Fig. 4

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Evolution of T1 and T2 cytokine production by T cells from RA patients primed in the presence of exogenous cytokines and maintained in culture. Peripheral blood mononuclear cells (PBMC) from RA patients were primed for 1 week with sOKT3 in the presence of rIL-2, rIL-4, or rIL-2 + rIL-12. One week after priming, T cells were either immediately restimulated with phytohaemagglutinin (PHA), or kept in culture in rIL-2 alone and restimulated with PHA 2 weeks or 3 weeks after priming. IL-4 (a) and IFN-γ (b) were assayed by ELISA in culture supernatant, 18 h after restimulation. Results obtained from four RA patients are shown for cells restimulated 1 or 2 weeks after priming and from three RA patients for cells challenged 3 weeks after priming (mean + s.e.m.).

Expression of LAG-3 and CD30 at the surface of T cells

LAG-3 and CD30 are T cell surface activation markers with selective expression on T1 and T2 cells, respectively [31]. To explore further the polarized phenotype of the cells, we analysed expression of these markers by flow cytometry at the surface of RA and normal donor T cells after priming.

In normal donors, the level of expression of LAG-3 was increased at the surface of both CD4+ (both frequency of LAG-3+ cells and the intensity of labelling were increased on this subset) and CD8+ T cells (only fluorescence intensity level was increased on this subset) 1 week after priming in the presence of IL-2 + IL-12, compared with priming in the presence of IL-2 (Fig. 5). This result is consistent with LAG-3 being a differentiation marker for T1 lymphocytes. In contrast, the level of LAG-3 expression was markedly reduced at the surface of both CD4+ and CD8+ T cells primed in the presence of IL-4, indicating the inhibitory effect of IL-4 upon T1 differentiation (Fig. 5). Similar variations were observed with RA patients, although a trend towards higher expression of LAG-3 after priming with IL-2 + IL-12, and conversely towards lower expression after priming with IL-4, was occasionally observed in RA patient compared with normal donors (Fig. 5).

Fig. 5.

Fig. 5

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T cell surface expression of type 1 differentiation-associated lymphocyte activation gene (LAG)-3 antigen. Peripheral blood mononuclear cells (PBMC) from normal donors and RA patients were primed for 1 week with sOKT3 in the presence of rIL-2, rIL-4, or rIL-2 + rIL-12. One week after priming, expression of LAG-3 was evaluated by two-colour staining and flow cytometry on the surface of CD3+, CD4+ and CD8+ T cells. Shown here are histograms obtained with anti-CD4/anti-LAG-3 staining. This picture was precisely mirrored by anti-CD8/anti-LAG-3 staining. Percentages in quadrants refer to the proportion of positive cells as defined by the binding of irrelevant IgG1 and IgG2a MoAbs, and MFI to the mean fluorescence intensity. Results shown are representative of three separate experiments.

Expression of CD30 at the surface of T cells was only detected at low levels, 1 and 2 weeks after priming, without any detectable influence of priming conditions (not shown).

Expression of T cell adhesion molecules and activation markers

Expression of several markers at the surface of T cells was studied by flow cytometry, both in RA patients and normal donor, 1 and 2 weeks after priming. Markers such as LFA1 (CD11a), intercellular adhesion molecule-1 (ICAM-1; CD54) and CD45RO were expressed on most stimulated T cells in all conditions of priming as early as 1 week after priming (not shown). There was no consistent difference in the level of expression between priming conditions.

Although CD49d (VLA-4-specific α4-integrin) was also expressed on the majority of T cells 1 week after priming, its expression level was duller on T cells primed with IL-4 after 1 week, but reached similar intensity levels after 2 weeks in all conditions (Fig. 6). HLA-DR expression also increased between 1 and 2 weeks after priming, but remained lower on T cells primed in IL-4 than in other conditions (Fig. 6). Finally, the frequency of T cells expressing CD25 (IL-2Rα chain) was markedly lower 1 week after priming in the presence of IL-4 than after priming in the two other conditions (Fig. 6). However, the frequency of T cells expressing this marker was stable after 2 weeks in those T cells initially primed with IL-4, whereas it decreased in the two other conditions. There was no remarkable difference in the surface expression of any of those markers tested between normal donors and RA patients (not shown).

Fig. 6.

Fig. 6

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(See next page.) T cell surface expression of activation markers CD49d (VLA-4), HLA-DR and CD25. Peripheral blood mononuclear cells (PBMC) from RA patients were primed for 1 week with sOKT3 in the presence of rIL-2, rIL-4, or rIL-2 + rIL-12. One week after priming, T cells were either immediately stained or kept in culture in rIL-2 alone before being analysed for activation marker expression by flow cytometry, 2 weeks after priming. Percentages refer to the proportion of positive cells as defined by the binding of the control MoAb. Results are representative of two separate experiments.

DISCUSSION

We have previously established culture conditions that drive PBMC from normal donors towards a T2 profile after one round of stimulation with sOKT3 in the presence of exogenously added cytokines [29]. Addition of rIL-4 alone was shown to be sufficient to induce this T2 profile, characterized by heightened production of IL-4, IL-5 and IL-13 upon restimulation. Combining exogenous rIL-2 with rIL-4 partially opposed T2 differentiation. Conversely, addition of rIL-2 + rIL-12 induced a predominant T1 profile characterized by high levels of IFN-γ production. These studies were conducted with cell therapy in autoimmune diseases in mind, based on a strategy of autologous T cell administration to shift in vivo the T1/T2 balance towards a T2 profile. To achieve this goal, several parameters still needed to be set. Indeed, in the present study we examined whether resting peripheral T cells from RA patients could be also shifted towards a T2 polarization profile.

Reproducible in vitro conditions required to induce a T2 cytokine profile in T cells from RA patients were established in the present study. This result was documented both at cytokine protein and mRNA levels. Interestingly, after 1 week of priming with sOKT3 in the presence of rIL-4, differentiation towards a T2 profile was not yet detectable in RA T cells, although it was readily detected in T cells from normal donors. However, when T cells from RA patients where maintained in rIL-2 for 1 or 2 additional weeks, conversion towards a T2 profile finally appeared only in those T cells that had been primed in rIL-4.

Both naive and memory T cells were present in the T cell populations used in this study. Although both subsets of T cells can be polarized in vitro towards a T2 phenotype [32], they may exhibit distinct differentiation capacities. Therefore, variations in the respective proportions of those T cell subsets between normal donors and RA patients could potentially have influenced our results. However, such a hypothesis is unlikely to hold true, since the proportions of peripheral blood CD45RA+ CD45RO− naive T cells and CD45RO+ memory T cells were found similar in RA patients and normal donors ([33–36] and our own unpublished data).

Several components of the RA inflammatory state could interfere with T2 differentiation in our system and explain the delay in the establishment of a T2 profile compared with normal donors. Increased detection of T1 cytokines has been described in the peripheral blood of RA patients [13], and we observed that the production of IFN-γ upon restimulation of RA T cells after 1 week was increased in all conditions of differentiation compared with normal donors. This increased production of IFN-γ could indicate either that RA peripheral T cells are biased towards an excess of T1 differentiation, or could hamper T2 differentiation [37]. However, these hypotheses are not supported by the LAG-3 expression study. This molecule is induced on antigen-stimulated T cells differentiating towards a T1 phenotype and in T1 clones upon activation, but not in T2 clones [38]. Its expression is also induced by exposure to IFN-γ and reciprocally inhibited by IL-4 [31]. We observed high expression levels of LAG-3 in both CD4+ and CD8+ T cells primed in the presence of IL-2 + IL-12. This expression level was lower in IL-2-stimulated cells and further reduced in T cells primed with IL-4. However, there was no remarkable difference between normal donors and RA patients. These results suggest that neither the production of IFN-γ nor the degree of T1 polarization are strikingly modified during in vitro differentiation of RA T cells, in our culture conditions. Furthermore, the absence of T2 cytokine production by RA T cells primed with IL-4 is unlikely to be explained by a decreased effect of IL-4 on T cells.

As previously described, we found a decreased proliferative capacity of RA T cells upon priming that has been attributed to a deficiency in the production of IL-2 and a depressed expression of IL-2R early after activation [39,40]. Altered T lymphocyte calcium signalling has been implicated in this defective proliferative response in RA [35], the origin of which remains unclear but could involve chronic systemic production of TNF-α [41] or of IL-10 [42,43]. Interestingly, in our experiments this phenomenon was more pronounced in T cells primed with IL-4 alone than in those primed with IL-2 or IL-2 + IL-12. This observation is consistent with the capacity of exogenous IL-2 to partially correct the defective proliferative response of RA T cells [44], whereas IL-4 is likely to exacerbate it [45,46]. Indeed IL-2R expression level was comparable in T cells stimulated with IL-2 or IL-2 + IL-12 at the end of the first week of culture, but was lower in T cells stimulated with IL-4 alone. The defective proliferative response was completely overcome when the cells were subsequently maintained in culture, confirming the reversibility of this phenomenon [40]. Commitment of T cells towards a T2 phenotype appears to be a very early event after priming in the presence of IL-4 [47]. However, altered T cell signalling could be involved in the delayed progression of RA T cells towards a fully differentiated T2 phenotype for several reasons: (i) a lack of endogenously produced IL-2 could impair T2 differentiation [48]; (ii) even in the presence of appropriate differentiating signals, progression towards a T2 phenotype may implicate several rounds of division that were slowed down in RA; and (iii) transcription of both IL-2 and IL-4 gene is dependent on calcium signalling through nuclear factor of activated T cells [49].

Expression of activation markers CD25, VLA-4 and HLA-DR was delayed in T cells primed in the presence of IL-4, both in normal controls and RA T cells. Interestingly however, fully differentiated T2 T cells obtained after 2 weeks of culture expressed high levels of adhesion molecules such as LFA-1, ICAM-1 and VLA-4, which are thought to be important for T cell migration towards synovial inflammatory sites [50–52]. This point is critical in view of the prospect of cell-mediated immune therapy.

In this study, conditions required to generate fully differentiated polyclonal T2 T cells from peripheral T cells were set in RA patients. Notably, differentiation towards a T2 phenotype was hampered. This finding may be relevant to the pathogenesis of RA and support the concept of impaired T1/T2 balance in this disease. Hence, it can be speculated that recirculating T cells forced to differentiate in vitro into T2 effector cells will gain the capacity to down-regulate the uncontrolled immune response, once re-administered to the patient.

Acknowledgments

This work was supported by a DRC grant. S.A. was supported in part by a grant from ARP10/96. We thank Dr C. Fournier for critical review of the manuscript, and Dr M. Dreano (Ares Serono, Geneva, Switzerland) for kindly providing us with the anti-LAG-3 MoAb, 17B4.

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