MRL/Mp CD4+,CD25− T cells show reduced sensitivity to suppression by CD4+,CD25+ regulatory T cells in vitro: A novel defect of T cell regulation in systemic lupus erythematosus

Authors


Abstract

Objective

To investigate the hypothesis that loss of suppression mediated by peripheral CD4+,CD25+ regulatory T cells is a hallmark of systemic lupus erythematosus (SLE).

Methods

Mice of the MRL/Mp strain were studied as a polygenic model of SLE. Following immunomagnetic selection, peripheral lymphoid CD25+ and CD25− CD4+ T cells were cultured independently or together in the presence of anti-CD3/CD28 monoclonal antibody–coated beads. Proliferation was assessed by measuring the incorporation of tritiated thymidine.

Results

While MRL/Mp CD4+,CD25+ regulatory T cells showed only subtle abnormalities of regulatory function in vitro, syngeneic CD4+,CD25− T cells showed significantly reduced sensitivity to suppression, as determined by crossover experiments in which MRL/Mp CD4+,CD25− T cells were cultured with H-2–matched CBA/Ca CD4+,CD25+ regulatory T cells in the presence of a polyclonal stimulus.

Conclusion

Our findings highlight a novel defect of peripheral tolerance in SLE. Identification of this defect could open new opportunities for therapeutic intervention.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by intrinsic T and B cell defects (1–3). Recent studies examining the role of CD4+,CD25+ regulatory T cells in the pathogenesis of SLE have revealed a deficiency of regulatory T cell numbers in 2 murine models, (NZB × NZW)F1 (NZB/NZW) and (SN)F1 (4), and in human patients (5, 6). While a numerical deficiency of CD4+,CD25+ regulatory T cells may contribute to the pathogenesis of SLE, functional regulatory T cell abnormalities may also exist. In the present study, we therefore examined the functional properties of CD4+,CD25+ regulatory T cells selected from the peripheral lymphoid organs of MRL/Mp mice, which form the susceptible background strain (MRL/Mp-++) for the lupus-accelerating gene lpr. We found that, while MRL/Mp CD4+,CD25+ regulatory T cells display only subtle abnormalities of function, syngeneic CD4+,CD25− T cells show significantly reduced sensitivity to suppression. This result thus discloses a novel defect of peripheral tolerance in this disease.

MATERIALS AND METHODS

Mice.

Female MRL/Mp (H-2k), NZB/NZW (H-2d/z), and C57BL/6.129 (D1Mit105-223) mice (7) were examined at the age of 6–10 weeks, prior to disease onset. Age- and sex-matched control strains included CBA/Ca (H-2k), BALB/c (H-2d), and C57BL/6 (H-2b). All mice except the BL/6 congenic strain, which was developed in-house, were purchased from Harlan Olac (Bicester, UK) and were maintained under specific pathogen–free conditions.

Selection of CD4+,CD25+ regulatory T cells and coculture with CD4+,CD25− T cells.

All assays were performed with RPMI 1640 (Life Technologies, Paisley, UK) supplemented with 100 units/ml penicillin/streptomycin (Gibco, Paisley, UK), 2 mML-glutamine (Gibco), 10 mM HEPES (Gibco), and 10% (volume/volume) heat-inactivated fetal calf serum (Helena BioSciences, Sunderland, UK). Spleens and selected lymph nodes were macerated through a 70-μm cell strainer and then treated with Red Cell lysis buffer (Sigma-Aldrich, Dorset, UK). Negative magnetic selection of CD4+ T cells was performed with sheep anti-rat DynaBeads (Dynal, Oslo, Norway) to capture cells labeled with anti–class II major histocompatibility complex (M5/114.15.2), anti-CD8 (53.6.72), and anti-CD32 (2.4.G2) monoclonal antibodies (mAb). CD4+,CD25+ T cells were positively selected on MiniMACS columns (Miltenyi, Bergish Gladbach, Germany), using Streptavidin MicroBeads (Miltenyi) to capture CD4+ cells labeled with biotinylated anti-CD25 mAb (clone 7D4; BD Biosciences, Heidelberg, Germany). Purity of both CD25+ and CD25− T cells was >93%.

Purified CD4+,CD25+ T cells (1 × 105/well) were cultured, in 96-well round-bottomed plates, with CD25− T cells in the presence of Epoxy DynaBeads (1 bead/5 cells; Dynal) coated with anti-CD3 and anti-CD28 mAb. After 3 days, the incorporation of tritiated thymidine (3H-TdR; Amersham Biosciences, Roosendaal, The Netherlands) over 16 hours was measured.

Flow cytometry.

All mAb and their isotype controls were purchased from BD Biosciences, except phycoerythrin (PE)–conjugated glucocorticoid-induced tumor necrosis factor receptor superfamily member 18 (GITR) (108619; Caltag, Burlingame, CA). Cells were stained with PE-conjugated cholera toxin B (Molecular Probes, Eugene, Oregon) in phosphate buffered saline (PBS) containing 0.5% (weight/volume) piscine gelatin (Sigma-Aldrich), following fixation in 0.75% (v/v) paraformaldehyde and permeabilization with PBS–0.05% (w/v) saponin.

Electron microscopy.

CD4+,CD25+ and CD25− T cells (1:1 ratio) were cultured for 16 hours in complete medium (0.75 × 106/ml) containing anti-CD3/CD28 Dynabeads in 24-well plates (Techno Plastic Products, Trasadingen, Switzerland), before routine fixation for electron microscopy.

Statistical analysis.

Data were analyzed using 2-tailed, unpaired t-tests (2 columns) or one-way analysis of variance with Bonferroni adjustment for multiple comparisons (≥3 columns). P values less than 0.05 were considered significant.

RESULTS

Normal numbers of peripheral lymphoid CD4+,CD25+ regulatory T cells in MRL/Mp mice.

The mean ± SEM proportion of CD25+,CD4+ T cells in MRL/Mp mice was 6.2 ± 0.7% (n = 8), which was not significantly different from the proportions in CBA/Ca mice (4.9 ± 0.4%; n = 8), BALB/c mice (6.2 ± 0.6%; n = 4), C57BL/6 mice (7.4 ± 0.7%; n = 4), or congenic C57BL/6 mice (5.3 ± 0.7%; n = 3). NZB/NZW mice had a lower proportion of peripheral CD4+,CD25+ T cells (mean 2.9%; n = 2). Thus, a numerical deficiency of regulatory T cells may play a role in the pathogenesis of lupus in some murine strains (4), but not in others.

Similar phenotype of CD4+,CD25+ regulatory T cells in MRL/Mp mice and in CBA/Ca mice.

Expression of CD25, CD26, CD69, CD44, CD45RB, CD152 (CTLA-4), CD62 ligand (CD62L), and GITR by CD4+,CD25+ regulatory T cells was similar in MRL/Mp and CBA/Ca mice. Expression of these markers by CD4+,CD25− T cells was also similar among the strains. Recent reports have described increased expression of CD40L (CD154) (8), lymphocyte function–associated antigen 1 (9), and glycosphingolipid 1 (10, 11) by lupus T cells. However, expression of these markers by MRL/Mp and CBA/Ca CD4+,CD25+ regulatory T cells was similar, as was their expression by CD4+,CD25− T cells (data not shown).

Reduced sensitivity to suppression in MRL/Mp CD4+,CD25− T cells in vitro despite intimate cellular associations with CD4+,CD25+ regulatory T cells.

MRL/Mp CD4+,CD25+ T cells exhibited regulatory function in cocultures with syngeneic CD4+,CD25− T cells: the mean proportion of CD4+,CD25+ regulatory T cells required to effect 50% suppression of 3H-TdR incorporation (IC50) was 11.4% (n = 4), with a mean maximal suppression (Smax) of 66.2% (Figure 1B). However, similar syngeneic cocultures of CD25+ and CD25− CD4+ T cells selected from control strains (CBA/Ca, BALB/c, C57BL6) all showed ∼10-fold lower mean IC50 values and ∼15–30% higher mean Smax values (Figure 1B), suggesting significantly more potent suppression. In common with the findings in MRL/Mp cocultures, NZB/NZW cocultures demonstrated high IC50 (mean ± SEM 17.5% ± 1.5%) and low Smax values (59.0 ± 1.5%) (Figure 1B). Interestingly, a C57BL/6 congenic line carrying the telomeric region of chromosome 1 from the 129 strain also showed high IC50 (14.9 ± 0.9%) (Figure 1B), suggesting that genetic defects facilitating the loss of peripheral tolerance reside within this chromosomal region (12, 13).

Figure 1.

Reduced sensitivity of MRL/Mp CD4+,CD25− T cells to suppression by CD4+,CD25+ regulatory T cells, a defect of the nonregulatory population. A, Syngeneic coculturing of MRL/Mp CD25+ and CD25− CD4+ T cells revealed titratable, but less robust, suppression than was observed in syngeneic cocultures of cells from CBA/Ca mice. In both cases, no suppression was observed in control wells containing irradiated (Irr.) CD4+,CD25− T cells. Values are the mean and SEM. B, Suppression expressed in relation to the ratio of CD4+,CD25+ regulatory T cells:total CD4+ T cells (top), highlighting the more potent suppression in CBA/Ca cocultures, also measured by the proportion of CD4+,CD25+ regulatory T cells required to effect 50% suppression (IC50) (middle) and the maximal suppression observed (Smax). Data summarizing the IC50 and Smax values for all strains suggest that regulatory T cells in lupus-prone strains in general manifest significantly less potent suppression than those of control strains (P < 0.001, IC50 in MRL/Mp, NZB/NZW, and C57BL/6 congenic mice versus controls; P < 0.01, Smax in MRL/Mp mice versus controls; P < 0.001, Smax in NZB/NZW mice versus controls; P < 0.05, Smax in C57BL/6 or C57BL/6 congenic mice versus CBA/Ca and BALB/c mice). Bars show the means. C, CBA/Ca CD4+,CD25+ regulatory T cells (C+) showed poor suppression of MRL/Mp CD4+,CD25− T cells (M−), yielding a high IC50 value (P = 0.01) and a low Smax value, (P = 0.01), but MRL/Mp regulatory T cells (M+) were able to suppress CBA/Ca CD4+,CD25− T cells (C−) normally. Anti-CD3/CD28 beads were used as stimulus in all cultures. Bars show the means.

Crossover experiments revealed that the MRL/Mp CD4+,CD25+ T cells exhibited normal regulatory activity when cultured with CBA/Ca cells, but that MRL/Mp CD4+,CD25− T cells showed reduced sensitivity to suppression mediated by the CBA/Ca CD4+,CD25+ regulatory T cells (Figure 1C). Interestingly, the IC50 values were intermediate between those in syngeneic CBA/Ca and MRL/Mp cultures, suggesting that when challenged with “poorly suppressible” CD4+,CD25− T cells, MRL/Mp CD4+,CD25+ regulatory T cells show complementary regulatory defects. Crossover cocultures could have resulted in multiple minor histocompatibility responses, but these were likely to be “dwarfed” by the polyclonal stimulus and were thus ignored.

We hypothesized that reduced sensitivity of the MRL/Mp CD4+,CD25− T cells to regulatory T cell suppression could be referable to robust proliferation. However, careful titration of beads revealed that MRL/Mp CD4+,CD25− T cells failed to proliferate more than control cells over a range of stimulation intensities. Rather, there was a trend toward marginally less proliferation in the MRL/Mp cultures, which would have rendered them intrinsically more susceptible to suppression, if other factors were equal (Figure 2). Transmission electron microscopy revealed intimate plasma membrane associations between MRL/Mp CD25+ and CD25− CD4+ T cells (Figure 3). However, despite the demonstration of intact physical interactions between these cells, their functional interactions in vitro were clearly abnormal.

Figure 2.

MRL/Mp CD4+,CD25− T cells are not hyperproliferative in vitro. A, CD4+,CD25− T cells from MRL/Mp mice (light gray bars) proliferated marginally less than those from CBA/Ca mice (dark gray bars) when exposed to a diminishing intensity of anti-CD3/CD28 bead stimulation, as measured by 3H-thymidine incorporation at 24, 48, and 72 hours. Values are the mean and SEM. B, Overall proliferation calculated from the area under the curve (AUC) of proliferation against bead ratio, with ratios of cells:beads ranging from 1:1 (50%) to 20:1 (4.8%).

Figure 3.

Transmission electron microscopy demonstrating the presence of intimate cellular associations between MRL/Mp CD25+ and CD25− CD4+ T cells. This result suggests that the reduced sensitivity of the CD25− T cells to suppression was not due to a physical inability of the cells to interact with regulatory T cells. CD4+,CD25+ regulatory T cells were identified by the presence of Microbeads (Miltenyi, Bergish Gladbach, Germany) on their surface; in this case, the cell on the right is the regulatory T cell. Bar = 1 μm.

DISCUSSION

MRL/Mp CD4+ T cells are hyperresponsive to T cell receptor engagement with low-affinity peptide antigens (14) and resist the induction of anergy in vivo (15). Furthermore, human lupus T cells also show resistance to anergy, associated with severely impaired phosphorylation of Cbl-b (8). A third mechanism can now be added to the increasing repertoire of known abnormalities of peripheral tolerance in this disease—namely, reduced sensitivity of CD4+,CD25− T cells to suppression, reminiscent of the behavior of effector T cells in both NOD mice (16) and Cbl-b−/− mice (17). We suggest that peripheral tolerance fails at various points in individuals who are genetically predisposed to lupus, with resultant activation and unrestrained expansion of autoreactive T cells. Future treatment modalities should aim to redress this balance, perhaps by the adoptive transfer of expanded or activated regulatory T cells to overcome the reduced sensitivity of the CD4+,CD25− T cells characterizing this disease.

Acknowledgements

The authors would like to thank Dr. Jill Moss and Mr. Ian Shore (Department of Histopathology, Imperial College London, Charing Cross campus) for expert assistance with electron microscopy.

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