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Medline Plus Health Information
*Joint Disorders
*Rheumatoid Arthritis
The Journal of Immunology, 2004, 173: 2825-2833.
Copyright © 2004 by The American Association of Immunologists

Ex Vivo Homeostatic Proliferation of CD4+ T Cells in Rheumatoid Arthritis Is Dysregulated and Driven by Membrane-Anchored TNF{alpha}1

Ulf Wagner2, Matthias Pierer, Matthias Wahle, Falk Moritz, Sylke Kaltenhäuser and Holm Häntzschel

Department of Medicine IV, University of Leipzig, Leipzig, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The systemic CD4+ T cell compartment in patients with rheumatoid arthritis (RA) is characterized by TCR repertoire contraction, shortened telomere lengths, and decreased numbers of recent thymic emigrants, suggesting a disturbed CD4+ T cell homeostasis. In mice, homeostatic proliferation of peripheral CD4+ T cells is regulated by TCR interaction with self peptide-MHC complexes (pMHC) and can be reproduced in vitro. We have established an ex vivo model of homeostatic proliferation, in which self-replication of human CD4+ T cells is induced by cell-cell contact with autologous monocytes. In healthy individuals, blockade of TCR-pMHC class II contact resulted in decreased CD4+ T cell division. In contrast, homeostatic proliferation in RA patients was not inhibited by pMHC blockade, but increased during the initial culture period. The anti-TNF-{alpha} Ab cA2 inhibited homeostasis-driven ex vivo proliferation in healthy controls and in RA patients. In addition, treatment of RA patients with infliximab decreased the ex vivo rate of homeostatic proliferation of CD4+ T cells. Our results suggest a disturbed regulation of CD4+ T cell homeostasis leading to the repertoire aberrations reported in RA. Membrane-anchored TNF-{alpha} appears to be a cell-cell contact-dependent stimulus of homeostatic proliferation of CD4+ T cells, possibly favoring self-replication of autoreactive CD4+ T cells in patients with RA.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Rheumatoid arthritis (RA)3 is a chronic and disabling autoimmune disease that is characterized by profound impairment of the function of diseased joints, eventually leading to total destruction of articular structures. However, an extra-articular systemic dimension of the ongoing abnormal immune response becomes evident in a subgroup of patients who suffer from disease processes involving connective, glandular, and other tissues and, in the most severe cases, major organ systems (1). Fever, generalized lymphadenopathy, and weight loss are systemic symptoms that can be present in disease courses that are otherwise confined to the involved joints, and which are often already present at the onset of disease.

CD4+ T lymphocytes have been implied in the pathogenesis of the disease due to the profound impact of the presence of distinct MHC class II alleles on disease susceptibility and clinical course of RA (1, 2, 3). In addition, CD4+ T cells are a major cell population in the inflammatory infiltrate in synovitic joints of RA patients, and their topographic distribution in some of the histologically most typical cases is consistent with an involvement in a germinal center reaction comparable to the Ag-specific immune responses in secondary lymphoid organs (4, 5).

The clinical perspective of RA as a systemic disease that is not limited to the joint but involves a generalized abnormal response of the cognitive immune system is supported by a number of pathologic findings in the systemic CD4+ lymphocyte pool of patients with this disease. The peripheral CD4 compartment of RA patients is known to frequently contain expanded clonotypes of CD4+ T cells of considerable clonal size (6, 7), some of which cannot be detected in the synovial membrane of affected joints (8). Peripheral CD4+ T cell clones in RA are autoreactive, have lost the expression of the costimulatory molecule CD28 while gaining MHC class I-recognizing NK cell receptors (9, 10), and frequently occur in the naive CD4+ T cell compartment. In addition, RA patients have less naive CD4+ T cells compared with healthy controls, while their peripheral T cell pool contains atypical hyperresponsive progeny (11). On a functional level, abnormalities of the peripheral CD4+ T cell pool of RA patients became apparent in studies using the MLR (AMLR) as a model system for self-recognitive mechanisms. The proliferative response of T cells toward irradiated autologous APCs in the AMLR is dependent on the expression of MHC class II and costimulatory molecules (12), requires irradiation of APCs for the induction of T cell proliferation (13, 14), and is thought to represent autoreactivity to autoantigens. However, despite the presumably autoreactive T cell repertoire in RA, RA patients have been found to show a profoundly impaired proliferative response as well as impaired generation of high-affinity IL-2Rs in the AMLR (15, 16, 17, 18).

In addition to a limited number of clonal expansions, the majority of T cell specificities randomly selected from the systemic circulation of RA patients also occurs at 10- to 20-fold higher frequencies compared with healthy controls (19), which indicates a considerably contracted TCR repertoire in those patients due to increased self-replication with subsequent loss of diversity in a large fraction of the total repertoire. In accordance with this, telomere lengths of naive and memory CD4+ T cells are prematurely eroded in comparison to age-matched controls indicative of their increased proliferative history (19, 20). When the frequency of recent thymic emigrants as a quantitative measure of thymic output was analyzed in RA patients by determining levels of TCR rearrangement excision circles, a substantial reduction compared with age-matched controls was found even in young patients that was independent of the preceding disease duration (11, 20). Collectively, those results suggest an abnormal peripheral homeostasis and differentiation of CD4+ T cells, possibly due to insufficient thymic output, as an underlying mechanism of autoimmunity in this disease (21).

Peripheral self-replication of T lymphocytes for population maintenance is a physiologic mechanism supplementing the decreasing thymic de novo generation of naive CD4+ and CD8+ T cells during adult life and aging (22, 23, 24). This cell division of T lymphocytes in the absence of exogenous or specific antigenic stimuli has been termed homeostatic proliferation and has been examined in several recently published studies in mouse models (25, 26, 27, 28, 29, 30, 31, 32, 33). This proliferative activity is induced by lymphopenia (27, 28), is dependent on TCR interaction with self peptide-MHC complexes (pMHC) of the appropriate class of MHC molecules (27, 29, 30, 31), and possibly requires coreceptor-MHC interaction as well (32). During the underlying "self-recognition", TCRs of naive T cells are maintained in a state of partial phosphorylation owing to recognition of self-peptide MHC molecule ligands, although the resulting phosphorylation patterns induced by this continuous subthreshold signaling resemble those induced by inhibitory antagonists rather than agonists of the TCR (34). The peptides required for homeostasis-stimulating TCR-pMHC interaction are likely to be self peptides also present in the thymus (29, 32), although some controversy remains (30, 33). In addition, cytokines like IL-7, IL-12, and IL-15 have been found to be involved in the regulation of T cell homeostasis (22, 35, 36), although their role for human T cell maintenance proliferation seems diminished later in adult life (37, 38, 39).

Homeostatic proliferation of naive T cells was reproduced in vitro, when murine TCR-transgenic CD4+ T cells were cultured with syngeneic dendritic cells in the absence of exogenous Ag (40). This in vitro response closely resembles lymphopenia-induced proliferation in mice. In this study, a similar system was used to analyze homeostasis-driven proliferation ex vivo in RA patients and healthy controls. The results show that homeostasis-driven proliferation in human CD4+ T cells from healthy individuals is also regulated by contact with self-peptide MHC II complexes. However, this regulatory mechanism is lost in patients with RA. Although homeostatic proliferation is still dependent on cell-cell contact in RA, membrane-bound TNF-{alpha}, rather than pMHC class II complexes, appear to be the main stimulating molecules.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Study population

Blood samples were obtained from 46 patients with RA who had unequivocal radiological evidence of destructive RA. All patients fulfilled the 1987 American College of Rheumatology criteria for the diagnosis of RA (41), and 40 patients were seropositive for rheumatoid factor (RF) (Table I). Clinical data documented for all patients included duration of morning stiffness, swollen and tender joint count, presence of extra-articular manifestations of the disease, and past and present medications received. As one control group, 25 healthy volunteers were recruited (median age, 47.4 years; range, 26–82 years). A second control group was comprised of 10 patients with psoriatic arthritis and intermediate disease activity (median age, 55.3 years; range, 27–67 years). The study protocol was approved by the University of Leipzig Institutional Review Board, and informed consent was obtained from all patients.


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  		Table I. Characteristics of the patient population

 
From 10 RA patients, blood samples were obtained before and during treatment with infliximab (3 mg/kg body weight i.v.). Pretreatment blood samples were drawn 2 wk before the initiation of treatment, and follow-up samples were obtained after 3 mo of therapy in a 2-wk interval before the following infusion.

Cell separation

PBMC were isolated by Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) density gradient centrifugation. CD4+ T cells were negatively isolated using the MACS CD4+ T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Untouched CD14+ monocytes were isolated with the monocyte isolation kit (Miltenyi Biotec). For both negative selections, MACS columns type LS+ were used in a MidiMACS magnetic cell separator (Miltenyi Biotec). Purity of the separated cell population was consistently above 95%.

Tissue culture

For the coculture experiments, 1.5 x 106 CD4+ T cells were incubated either alone or with 5 x 105 autologous monocytes in 24-well plates in serum-free RPMI 1640 containing 2% TCH (Defined Serum Replacement; MP Biomedicals, Aurora, OH), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Serum supplementation with FCS induced increased proliferative activity compared with serum-free medium, and therefore, was not used in the experiments described. The dosage of IL-2 required in the cocultures was titrated in a series of experiments with addition of different concentration of the cytokine at varying time points (data not shown). Supplementation of the cultures with 25 U/ml IL-2 at the beginning of incubation was sufficient to prevent excessive apoptotic cell death in the cultures (<10% of CD4+ T cells after 7 days of culture) and was found not to stimulate proliferation in CD4+ T cells cultured alone (Fig. 1A). Repeated addition of IL-2 resulted in stimulation of proliferation in the separated CD4+ T cells and was abandoned.



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  		FIGURE 1. Cell division of CD4+ T cells in cocultures after 7 (upper panels) or 14 (lower panels) days of incubation. A, CFSE-labeled, negatively selected CD4+ T cells were incubated alone under nonstimulating conditions in serum-free medium. The representative histogram depicts CFSE intensity in CD4+ T cells from a healthy donor after electronic gating on viable lymphocytes and on CD4+ cells. B, Coincubation with monocytes at a ratio of 3:1 induces cell division of CD4+ T cells. C, Representative dot plot depicting CFSE intensity in CD4+ T cells incubated in unseparated PBMC from a healthy donor. D, FACS tracing from C gated on R2. Percentages of divided cells in CD45RA positive (upper quadrants) and negative (lower quadrants) CD4+ cells are given.

 
For the analysis of homeostatic CD4+ T cell proliferation in cultures of PBMC, 2 x 106 cells were incubated under the same conditions. A mAb blocking MHC class II molecules (anti-HLA-DR, DP, DQ, clone TÜ39; BD Pharmingen, Heidelberg, Germany), the anti-TNF-{alpha} Ab cA2 (infliximab; Essex Chemicals, Lucerne, Switzerland) or an irrelevant mouse IgG Ab (clone 107.3; BD Pharmingen) were added at a concentration of 10 µg/ml. In addition, soluble TNF-{alpha} (Sigma-Aldrich, St. Louis, MO) at concentrations of 1 and 10 ng/ml was tested for stimulation of CD4+ T cell proliferation.

In the transwell experiments, a tissue culture plate insert with a 0.45-µm pore size microporous membrane (Millicell-HA; Millipore, Bedford, MA) was used to separate two communicating culture chambers in one well. A total of 5 x 105 monocytes were placed in the bottom chamber of the transwell, and 1.5 x 106 CD4+ T cells were added to the top chamber. Experiments were performed in 24-well plates using the culture conditions described above.

Flow cytometry

For the analysis of ex vivo cell division, CD4+ T cells or unseparated PBMC were labeled with CFSE (Molecular Probes, Eugene, OR) as described previously (42). Cells were incubated with CFSE for 10 min at 37°C, washed twice, and then cultured as described above. For immunophenotypic analysis of divided cells, the following mAbs were used: anti-CD4-PC5 (clone 13B8.2; Immunotech, Beckman Coulter, Marseille, France), anti-CD25-PE (clone B-B10; IQ Products, Groningen, The Netherlands) and anti-CD45RA-PE (clone HI100; BD Pharmingen). Cells were harvested from the tissue culture plates after 7 or 14 days of culture, incubated with Abs for 20 min at 4°C in the dark, washed, and analyzed on a FACSCalibur (BD Pharmingen). For quantification of T lymphocyte subpopulation in PBMC, the mAbs anti-CD28-PE (clone L293; BD Pharmingen) and CD45RA-PE (clone UCHL1; BD Pharmingen) were used. Flow cytometric quantification of variable element (BV) usage of CD4+ T cells was performed using the mAbs anti-TCRBV1S1/2 (clone BL37.2), anti-TCRBV2 (clone MPB2D5), anti-TCRBV3 (clone CH92), anti-TCRBV5S2 (clone 36213), anti-TCRBV7 (clone ZOE), anti-TCRBV8S1/2 (clone 56C5.2), anti-TCRBV12S1 (clone VER2.32.1), anti-TCRBV14S1 (clone CAS1.1.3), and anti-TCRBV17S1 (clone E17.5F3.15.13; all Immunotech, Beckman Coulter).

Determination of HLA DRB1 alleles was performed by hybridization of DRB1 PCR products to sequence-specific oligonucleotide probes as described previously (43).

For statistical analysis, the Student t test or paired t test were used if data followed a normal distribution, and the Mann-Whitney rank sum test or Wilcoxon signed rank test if normality test failed. Results are given as mean or median ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Spontaneous homeostatic CD4+ T cell proliferation is inhibited by pMHC class II blockade in healthy controls

When negatively selected CD4+ T cells from healthy donors were cultured alone in serum-free medium, no significant cell division was detected after 7 or 14 days of incubation (Fig. 1A). Addition of negatively selected CD14+ monocytes from the same donor at a 3:1 CD4+ T cell:monocyte ratio induced CD4+ T cell proliferation starting at day 7 (mean 16.3% ± SEM 6.7% vs 0.7 ± 0.2% in separated CD4+ T cells alone; p = 0.05), which increased further by 14 days of culture (20.7 ± 5.7% vs 2.1 ± 0.9%; p = 0.05). Proliferating cells underwent up to six cell divisions during 14 days of incubation (Fig. 1B). The percentages of proliferated CD4+ T cells in cocultures from six healthy individuals are given in Fig. 2A.



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  		FIGURE 2. Percentage of ex vivo cell division in CD4+ T cells from healthy controls (A and B), RA patients (C), and patients with psoriatic arthritis (D). A, Immunomagnetically separated CD4+ T cells from six healthy controls were incubated alone ({blacksquare}) or in the presence of negatively selected CD14+ monocytes () for 7 and 14 days. Cell division of CD4+ T cells in cultures of unseparated PBMC is shown for comparison (). Levels of significance: *, p = 0.05; **, p = 0.008; compared with separated CD4+ T cells alone. B, Percentage of divided CD4+ T cells in cocultures of unseparated PBMC in 25 healthy donors in the presence of a control Ab () or anti-MHC class II Ab (). ***, p < 0.001; *, p < 0.05. C, Percentage of divided CD4+ T cells in cultures of PBMC in 46 RA patients in the presence or absence of anti-MHC class II Ab. *, p < 0.01. D, Percentage of cell division in cultures of PBMC from 10 patients with psoriatic arthritis in the presence or absence of MHC class II Ab. *, p = 0.03.

 
Coincubation of separated lymphocytes with monocytes was chosen as an experimental setting because it mimics the cell-cell interaction between Th cells and APCs thought to be relevant for CD4+ T cell homeostasis in vivo. CD4+ T cell division was also analyzed in cultures of unseparated PBMC and was found not to be significantly different from the cocultures of separated CD4+ T cells with purified CD14+ monocytes from identical blood samples (Figs. 1C and 2A).

Analysis of CD45 expression of CD4+ T cells in the cocultures revealed that both naive and memory CD4+ T cells were undergoing homeostatic cell division (Fig. 1D). It has been shown in several mouse systems that lymphopenia-induced maintenance proliferation of naive Th cells is dependent on interaction of the TCR with MHC class II molecules presenting autologous peptides. To investigate pMHC contact requirements of human ex vivo homeostatic CD4+ T cell division, mAbs blocking TCR-pMHC class II interaction were added to the cultures. In PBMC from 25 healthy individuals, the mean percentage of divided cells was 9.7 ± 1.0% after 7 days, and 20.9 ± 4.6% after 14 days of incubation (Fig. 2B). pMHC class II blockade significantly decreased this percentage to 5.8 ± 0.5% after 7 days (p < 0.001) and to 13.0 ± 2.2% after 14 days (p < 0.05, Fig. 2B).

To differentiate between soluble and cell-cell contact-dependent factors, purified CD14+ and CD4+ cells from three healthy donors were coincubated in transwell experiments preventing direct cell-cell contact of the two populations (see Materials and Methods). Ex vivo cell division of CD4+ T cells was completely abrogated by separation from the monocytes (Fig. 3, A and B). In those experiments, the inhibitory effect of pMHC class II blockade was also seen in control cultures of CD4+ T cells with CD14+ monocytes without the tissue culture insert (Fig. 3C).



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  		FIGURE 3. Direct cell-cell contact is necessary for the induction of CD4+ T cell proliferation in cocultures with autologous monocytes in healthy controls. A, Negatively selected CD4+ T cells were incubated with autologous monocytes at a ratio of 3:1. B, Coculture in the transwell system with 5 x 105 CD14+ monocytes in the bottom chamber and 1.5 x 106 CD4+ T cells in the top chamber. C, Addition of anti-MHC II Ab to the coculture of CD4+ T cells and monocytes. Shown is one experiment representative for three experiments in healthy controls.

 
Homeostatic CD4+ cell proliferation is independent of pMHC class II contact in RA patients, but not in psoriatic arthritis

Comparable to the healthy controls, CD4+ T cells isolated from the peripheral circulation of RA patients (n = 6) did also not undergo cell division when incubated alone in serum-free medium. Addition of autologous CD14+ monocytes to the cultures induced proliferation of CD4+ T cells that started around day 7 and increased further during the second week of incubation. The percentages of divided cells and the number of cell generations detectable after 7 and 14 days in six patients was not significantly different from that seen in controls (mean ± SEM, 8.0 ± 3.6% at day 7, and 21.9 ± 8.8% at day 14).

Ex vivo homeostatic proliferation of CD4+ T cells in cultures of unseparated PBMC was analyzed in a group of 46 RA patients fulfilling the 1987 American College of Rheumatology criteria (clinical data are given in Table I). The mean percentage of divided CD4+ T cells in PBMC from RA patients was 7.4 ± 0.8% at day 7, and 17.3 ± 2.8% at day 14, and was not significantly different from the healthy controls (Fig. 2C). In contrast to the controls, cell division after 14 days of incubation was not inhibited by addition of MHC class II Abs in RA patients (16.6 vs 17.3%, p = 0.57). Furthermore, the percentage of proliferated cells at day 7 in cultures from RA patients containing anti-MHC class II Ab was even increased compared with cultures containing a control Ab (9.1 ± 1.1% vs 7.4 ± 0.8%; p < 0.01; Fig. 2C). The difference between patients and controls in HLA dependency of the proliferation was also reflected in the higher percentage of proliferated CD4+ T cells at day 7 under blockade of TCR-pMHC interaction in RA patients compared with the control group (9.1 ± 1.1% vs 5.8 ± 0.5%; p < 0.05).

As a control group of patients with a chronic inflammatory condition other than RA, 10 patients with psoriatic arthritis were analyzed for ex vivo homeostatic proliferation. After 7 and 14 days of incubation with autologous APCs, the percentage of divided CD4+ cells from psoriatic arthritis patients was found to be comparable to both the healthy controls and the RA patients (10.6 ± 2.1% after 7 days, and 18.3 ± 3.0% after 14 days). However, in contrast to RA patients and undistinguishable from the healthy controls, ex vivo homeostatic proliferation in psoriatic arthritis patients was also inhibited by TCR-pMHC blockade (7.9 ± 1.8%, p = 0.03 after 7 days; and 14.6 ± 2.6%, p = 0.1 after 14 days; Fig. 2D).

CD25 expression on CD4+ T cells was determined by flow cytometry after 7 and 14 days of incubation of PBMC in 20 RA patients and in 10 healthy controls. The rate of proliferation was higher in CD4+CD25+ T cells compared with the CD4+CD25– population at both time points analyzed (data not shown). However, proliferative activity was also clearly detectable in CD4+CD25– cells.

In parallel to the analysis of CD25 expression, cell division was analyzed in the naive, CD45RA-positive T cell population. Homeostatic proliferation was detectable in CD45RA+ cells and was not significantly different from that of CD4+ T cells during the first week of incubation (data not shown). However, after 14 days of incubation the percentage of divided naive cells was lower than in total CD4+ T cells both in healthy controls and in the RA patients (p = 0.002 and p = 0.036, respectively).

Ex vivo homeostatic proliferation is polyclonal and does not change the observed frequencies of TCR BV families in PBMC

Homeostatic proliferation, which serves the maintenance of the peripheral T cell pool, by definition has to involve a wide spectrum, if not all, of the TCR specificities. In vitro artifacts of proliferation, on the contrary, are likely to be caused by stimuli driving clonal outgrowth of selected TCR specificities. Possible stimuli include heat shock proteins that have been shown to become up-regulated during prolonged in vitro culture of T lymphocytes, and other cell surface molecules that could be involved in stimulating oligoclonal T cell outgrowth masquerading as homeostatic proliferation. Such oligoclonal outgrowth should be distinguishable from broad maintenance proliferation by the BV usage of the proliferated cells.

To analyze the clonality of in vitro proliferated CD4+ T cells, a panel of mAbs recognizing the BV elements of different TCR BV families was used. TCR BV usage was surprisingly stable and remained essentially unchanged during the incubation period from the distribution found in vivo in corresponding PBMCs (Fig. 4A).



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  		FIGURE 4. BV usage in separated and in in vitro proliferated CD4+ T cells. A, Percentages of CD4+ T cells positive for the BV elements indicated in PBMCs () and in corresponding cultures after 14 days of incubation () are given for 10 healthy controls (left panel) and for 10 RA patients (right panel). No significant changes were observed neither during the incubation period nor between the patient and control group. B, Proliferation in CD4+ T cells using the indicated BV elements in cultures of PBMCs from controls (left panel, n = 10) and RA patients (right panel, n = 10) after 7 days () and 14 days () of incubation. Significant cell division was found in all BV families analyzed indicating a polyclonal proliferation with no discernible differences between patients and controls.

 
The stable distribution of TCR BV elements in the cultures in comparison to BV usage in PBMC from the same patients indicates that no BV families were specifically expanded or contracted during 14 days of incubation. Moreover, analysis of proliferation in the different BV families showed that significant cell division occurred in all 10 BV families analyzed, indicating a truly polyclonal proliferative activity (Fig. 4B). Comparison of proliferation in the different BV families did not reveal differences between RA patients and healthy controls.

TNF-{alpha} blockade inhibits CD4+ cell division in vitro, and treatment with infliximab leads to decreased ex vivo proliferation

Although the rapid response of a subset of RA patients to treatment with TNF-{alpha} inhibitors emphasizes the crucial role of this proinflammatory cytokine in RA, it has also been suggested to exert regulatory effects in autoimmune diseases when present in increased levels over prolonged periods of time (44, 45). Therefore, the ex vivo assay was used to analyze the effect both of TNF-{alpha} inhibition in vitro and of in vivo treatment with infliximab on homeostatic expansion of CD4+ T cells in RA.

When infliximab was added to culture assays from healthy controls, the percentage of divided cells was significantly inhibited both after 7 (mean ± SEM, 3.0 ± 0.4 vs 5.4 ± 0.84%; p = 0.006), and 14 days (mean ± SEM, 11.3 ± 2.5 vs 25.0 ± 7.5%; p = 0.05) of incubation (Fig. 5A). Similarly, in vitro TNF-{alpha} blockade also significantly decreased ex vivo proliferation of CD4+ cells in cultures from RA patients at those time points (4.8 ± 0.7% vs 6.0% ± 0.9%, p = 0.03 at day 7; and median 11.9 ± 1.4% vs 18.6 ± 3.5%, p = 0.002 at day 14; Fig. 5A).



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  		FIGURE 5. Impact of TNF-{alpha} blockade on ex vivo cell division of CD4+ T cells. A, Inhibition of CD4+ T cell proliferation in cultures from healthy controls (n = 25) and RA patients (n = 46) in the presence of the monoclonal anti-TNF-{alpha} Ab cA2 after 14 days of incubation. The proliferation in control cultures with an isotype-matched mouse IgG1 Ab is shown as clear gray bars, and the proliferation in the presence of cA2 is shown as gray hatched bars. The white hatched bars show proliferation in cultures from 10 controls and 10 patients with exogenously added soluble TNF-{alpha} in the indicated concentrations. Levels of significance for comparison of cultures containing anti-TNF-{alpha} Abs to control cultures: *, p < 0.05; **, p = 0.002; comparison of cultures with exogenous soluble TNF-{alpha} to control cultures NS. B, In vitro homeostatic proliferation of CD4+ T cells in 10 patients in whom therapy with infliximab was initiated. The decrease of the percentage of divided CD4+ T cells under therapy was significant both after 7 and 14 days of incubation (levels of significance are given).

 
In 10 of the patients analyzed in the initial experiments, treatment with infliximab was started subsequently due to the clinical situation of previous disease-modifying antirheumatic drug failure and persistent high disease activity. Blood samples were again obtained from those patients, and ex vivo cell division was determined after 3 mo of therapy and compared with the pretreatment values. In all but one patient, the percentage of proliferated CD4+ T cells decreased under the influence of therapy both after 7 and 14 days of incubation in the ex vivo assay (p < 0.05 and p = 0.02, respectively; Fig. 5B).

To differentiate between the effects of soluble and membrane-anchored TNF-{alpha}, purified CD14+ and CD4+ cells from RA patients were coincubated in transwell experiments as described above. As in the healthy controls, ex vivo cell division of CD4+ T cells was also completely abrogated by separation from the monocytes. The results obtained in experiments with CD4+ T cells and monocytes from six RA patients were identical with those shown in Fig. 3 for the healthy controls.

To further distinguish between the effects of soluble and membrane-anchored TNF-{alpha}, the cytokine was added to cocultures containing PBMC from 10 RA patients and 10 controls in two different concentrations. Although cell division was somewhat increased in the wells containing the higher concentration of soluble TNF-{alpha}, no significant rise in the rate of proliferation could be detected, compared with the control culture, without addition of external TNF-{alpha} (Fig. 5A).

Relation to disease activity, HLA-DRB1 genotyping, and circulating lymphocyte subpopulations

Laboratory results and flow cytometric data were available from all patients in the study and analyzed for associations with the ex vivo homeostatic proliferative activity. No correlation between parameters of acute phase response (C-reactive protein and erythrocyte sedimentation rate) was detected. The decrease of ex vivo cell division of CD4+ T cells in patients treated with infliximab was also not related to the clinical response of the patients. In addition, levels of autoantibody production (RF IgM, RF IgA, antinuclear factors), circulating immune complexes, and serum Ig concentrations were analyzed without discernible correlation with ex vivo cell division. The disease duration and the history of previous disease-modifying antirheumatic drug therapy were also not related to homeostatic CD4+ T cell proliferation. No influence of the presence or absence of RA-associated DRB1 alleles or of the shared epitope sequence on a DRB1*04 allele was detected. The influence of age on ex vivo homeostatic proliferation was analyzed by comparing healthy individuals above and below the control group’s median age of 47.4 years (n = 12 and n = 13, respectively). The results given in Fig. 6 show that immunosenescence in the elderly is accompanied by a trend toward an increased rate of homeostatic proliferation (11.5 vs 8.7% after 7 days, and 18.0 vs 9.0% at day 14), although this did not reach statistical significance (p = 0.171 and p = 0.174, respectively). However, in contrast to the RA patients, homeostatic proliferation was inhibited both in younger and older individuals by addition of Abs blocking the TCR-pMHC class II interaction (Fig. 6).



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  		FIGURE 6. The influence of age on ex vivo homeostatic CD4+ T cell proliferation. Percentage of in vitro divided cells in healthy individuals above and below the median age of all controls analyzed (47 years) after 7 and 14 days of incubation (p = NS between age groups). Spontaneous in vitro proliferation is inhibited in both groups by addition of MHC class II-blocking Ab. Level of significance: *, p < 0.05; ***, p < 0.005.

 
In 20 patients analyzed for ex vivo proliferation of CD4+ T cells, the distribution of activated, naive, and memory cells, and of CD4+CD28-negative T cell populations was determined. No correlation of the percentage of CD4+CD25+ T cells in the peripheral circulation with the ex vivo proliferation of CD4+ cells at day 7 or 14 was discerned (R = –0.134, p = 0.62; and R = 0.0604, p = 0.835). However, the size of the CD4+CD28-negative T cell compartment was significantly related in the Spearmen rank order correlation to the percentage of divided CD4+ cells after 14 days of incubation (R = 0.515, p = 0.01). In addition, we found a significant correlation between the percentage of CD4+CD45RO+ memory T cells and the rate of ex vivo cell division both at day 7 and 14 (R = 0.671, p = 0.02; and R = 0.644, p = 0.04, respectively). The percentage of CD4+ T cells from all CD3+ lymphocytes, which can serve as a surrogate marker for the frequency of CD4+ T cells in the peripheral circulation (46), showed a significant negative correlation with the rate of ex vivo proliferation after 7 and 14 days (R = –0.39, p = 0.05; and R = –0.468, p = 0.03, respectively). This was reconcilable with the absolute lymphocyte counts obtained from the differential blood counts. The group of patients with peripheral lymphocytopenia (n = 10, 33.3%) according to standard reference values (<1.0 x 109 lymphocytes per liter) was found to show significantly higher rates of proliferation after 7 and 14 days of incubation compared with patients with normal lymphocyte numbers (8.7 ± 2.4% vs 4.0 ± 0.9%, p = 0.04; and 26.3 ± 7.0% vs 9.8 ± 2.1%, p = 0.03; respectively).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Homeostasis-driven expansion of peripheral T lymphocytes is a physiologic mechanism for replenishment of T cells in case of declining thymic output or under conditions of lymphopenia. It has been shown to be reproducible in vitro in a mouse system by coculture of T cells with syngeneic dendritic cells (40). We have established a serum-free culture system for human T cells in which CD4+ T cells are induced to undergo homeostatic cell division by cell-cell contact with cocultured CD14+ autologous monocytes. The percentage of divided cells after 14 days of coculture was comparable to the percentages reported for the murine coculture experiments with dendritic cells (40). It has been reported, in contrast to our findings, that autologous monocytes are not able to stimulate significant proliferation of T lymphocytes in the AMLR (12, 17) or in the in vitro model of mouse homeostatic proliferation (40). A drawback of the [methyl-3H]TdR incorporation assay used in previous studies is that only cells proliferating during the final 12 h can incorporate the radioactive marker. In contrast, the decrease in fluorescence intensity of CFSE-labeled cells accumulates over the whole incubation period. In addition, AMLR studies differ most profoundly from the system described in this study because they require irradiated, apoptotic APCs for induction of the T cell proliferation, which is therefore qualitatively and quantitatively different from in vitro homeostatic proliferation. Finally, mixed lymphocyte reactions were usually incubated for 7 days only (12, 17, 47), and the homeostatic in vitro proliferation reported to be induced by dendritic cells but not by monocytes in the mouse system was determined after 5 days of incubation (40). We saw the majority of cell divisions to take place beyond day 7 during the second week of culture. Taken together, the increased sensitivity of the proliferation assay and prolonged determination of cell division over 14 days are contributing to the previously undetected monocyte-induced CD4+ T cell division reported in this study.

In agreement with the results from murine studies, we found ex vivo homeostatic proliferation in human CD4+ T cells from healthy donors also to be dependent on TCR-pMHC contact, although inhibition of this contact only partially inhibited CD4+ T cell division. In patients with rheumatoid arthritis, the regulation of homeostatic proliferation was found profoundly altered. In contrast to healthy controls, MHC class II blockade did not lead to a significant reduction of homeostatic proliferation, but even enhanced the CD4+ T cell division during the first week of incubation significantly.

Numerous studies of TCR {beta}-chain usage and TCR sequence analysis have provided evidence for the relevance of cognate TCR engagement for T cell autoreactivity in RA. Our finding of MHC class II independence of homeostatic CD4+ T cell proliferation in RA patients suggests that, in contrast to healthy controls, TCR engagement has no influence on peripheral T cell expansion in RA. However, interpretation of this phenomenon has to take into account that the resulting proliferative activity is likely to be the net result of the requirements of homeostasis-induced cell division and the counterbalancing effects exerted by regulatory lymphocytes (48, 49). In this scenario, cognate TCR engagement might still influence both of these processes, while the net effect of MHC class II blockade of TCR engagement is uninhibited homeostatic cell division in RA. In fact, the significantly increased proliferative activity in RA patients induced by MHC class II blockade after 7 days might be an indication for simultaneous stimulatory and inhibitory effects of this interaction.

In the murine system, cytokines signaling through the common {gamma}-chain play an important role in the regulation of T cell survival and maintenance proliferation. However, the role of IL-2, IL-4, IL-7, and IL-15 in human T cell homeostasis is most pronounced in umbilical cord and neonatal T lymphocytes (37, 39). IL-7 has also been found to be an in vitro survival factor for human adult peripheral CD4+ T cells, but is not able to induce proliferation, either alone (38) or in combination with IL-2 (37). The complete abrogation of homeostatic proliferation by physical separation of CD4+ T cells and monocytes in the transwell system excludes stimulation of T cell division by soluble factors produced in the cocultures in our system, and the exogenous IL-2 required to prevent excessive apoptosis was also not found to induce proliferation in separated CD4+ cells. In contrast, in vitro analysis of homeostatic T cell proliferation in the murine T cell-dendritic cell coculture system has shown a requirement for soluble factors such as IL-15 for this proliferation (40). However, the IL-15 was produced by the dendritic cells used in this system and might reflect a unique capability of those cells in stimulating homeostasis-driven T cell division.

The pivotal role of the cytokine TNF-{alpha} in rheumatoid arthritis has been highlighted by the clinical success of TNF-{alpha} inhibiting therapies in clinical practice (50). Recently TNF-{alpha} has also been shown to exert stimulatory effects when present as a membrane-anchored molecule. Cell-cell contact-mediated monocyte activation with prefixed stimulated T cells was shown to be mediated in part by membrane-bound TNF-{alpha} molecules (51). Transgenic mice that overexpress a mutant transmembrane form of the murine TNF-{alpha} protein, which remains membrane-bound, have been found to develop a chronic inflammatory arthritis similar to the one seen in other TNF-{alpha} transgenics (52).

In addition, however, TNF-{alpha} has also been shown to possess regulatory effects on T cells besides its proinflammatory action. In TCR-transgenic mice, repeated injections of exogenous TNF-{alpha} over prolonged periods of time were found to attenuate TCR signaling upon cognate interaction with the Ag-MHC complex (44). The underlying mechanism has been shown to be a disruption of proximal TCR signaling leading to hyporesponsiveness of CD4+ T cells (45). In this system, an increase of proliferative activity after addition of anti-TNF-{alpha} Abs was found, which at first glance contradicts the results presented in the current study. This inhibition was interpreted by the authors as a reversion of the down-regulation of TCR signaling through TNF-{alpha} blockade, which then led to an increase of Ag-specific proliferation of transgenic cells in vitro upon rechallenge with their cognate Ag. Apparently, enough endogenous TNF-{alpha} had been produced in those cultures to inhibit TCR signaling of the transgenic cells, leading to the observed effect of TNF-{alpha} blockade.

Two findings presented in Cope et al. (44, 45) indicate that a different regulatory mechanism is in effect in the culture assays used in the current study, however. Soluble TNF-{alpha} did not have a significant effect on homeostatic proliferation in this assay, and the diminished effect of MHC class II blockade in RA patients shows that the regulatory mechanisms are only partially, if at all, dependent on TCR signaling. Therefore, the results reported by Cope et al. (44, 45) are not contradictory to the antiproliferative effect of anti-TNF-{alpha} Abs reported in this study.

The results presented in this study show an important role for TNF-{alpha} in the regulation of homeostasis-driven CD4+ T cell proliferation, because blockade with the mAb cA2 was persistently able to significantly inhibit this proliferation ex vivo both in healthy controls and in RA patients. This was confirmed by the unequivocal decrease of ex vivo proliferation induced by TNF-{alpha}-blocking therapy in 9 of 10 patients. No influence of secreted TNF-{alpha} was discernible from the transwell experiments, and addition of exogenous soluble TNF-{alpha} did not significantly increase homeostatic proliferation. Therefore, we conclude that membrane-anchored TNF-{alpha} plays a crucial role in the regulation of homeostasis-driven ex vivo CD4+ T cell proliferation.

We found several indirect lines of evidence that relative CD4 lymphopenia in RA patients is one of the stimuli required for homeostatic ex vivo CD4+ T cell proliferation. The rate of ex vivo CD4+ T cell division in RA patients correlated inversely with the percentage of CD4+ cells from total peripheral lymphocytes. In addition, a correlation with the size of the patient’s memory CD4+ T cell compartment in vivo was found. The size of the memory CD4+ T cell pool can be expected to increase as a compensatory mechanism in case of insufficient thymic output or disease-related loss of naive cells. Finally, patients with lymphopenia according to standard laboratory results (<1.0 x 109 lymphocytes per liter) were found to have the highest rate of ex vivo homeostatic proliferation. This indicates that the homeostatic signals received by CD4+ T cells in vivo are present, at least in part, in the coculture system with autologous monocytes. Taken together, our results indicate that the expression of membrane-anchored TNF-{alpha} on monocytes might be one of the signals regulating lymphopenia-induced homeostatic proliferation of CD4+ lymphocytes, and that competition for membrane-bound TNF-{alpha} might be a regulatory factor of homeostatic expansion in vivo, too.

The TCR repertoire aberrations in CD4+ T cells in RA, the emergence of CD4+CD28– T cell clones, and the decreased levels of TCR-rearrangement-excision-circles-positive, recent, thymic emigrants in RA have all been attributed to premature immunosenescence in those patients and are phenomena also associated with aging in healthy individuals (21). Our finding of a close correlation between the percentage of CD4+CD28– T cells in the peripheral circulation of RA patients with the extent of homeostasis-driven ex vivo proliferation provides a possible link between the immunosenescences ascribed to those patients and their rate of homeostatic proliferation required for CD4+ T cell maintenance. It is not clear yet whether the abnormal regulation of CD4+ T cell homeostasis described herein is a consequence of the long-standing chronic autoimmune process in these patients or a primary defect contributing to the pathogenesis of the disease. Accordingly, it remains to be determined whether the described effect of TNF-{alpha}-inhibiting agents to reduce homeostatic proliferation is related to the beneficial effects observed under such therapy.


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

1 This work was supported by grants from the German Ministry for Education and Science (Interdisziplinäres Zentrum für Klinische Forschung Leipzig, Teilprojekt A 15 and Kompetenznetzwerk Rheuma, Entzündlich-rheumatische Systemerkrankungen, Teilprojekt C2.7). Back

2 Address correspondence and reprint requests to Dr. Ulf Wagner, Department of Medicine IV, University of Leipzig, Liebigstrasse 22, 04103 Leipzig, Germany. E-mail address: wagu{at}medizin.uni-leipzig.de Back

3 Abbreviations used in this paper: RA, rheumatoid arthritis; AMLR, autologous MLR; pMHC, self peptide-MHC complexes; RF, rheumatoid factor; BV, variable element. Back

Received for publication September 11, 2003. Accepted for publication May 25, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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