Originally published online as doi:10.1189/jlb.0607436 on October 10, 2007

Published online before print October 10, 2007

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(Journal of Leukocyte Biology. 2008;83:112-121.)
© 2008 by Society for Leukocyte Biology

Identification and monitoring of effector and regulatory T cells during experimental arthritis based on differential expression of CD25 and CD134

Esther N. M. Nolte-’t Hoen^*,1, Elmieke P. J. Boot^,,1,2, Josée P. A. Wagenaar-Hilbers, Jolanda H. M. van Bilsen^,3, Ger J. A. Arkesteijn, Gert Storm, Linda A. Everse^,,4, Willem van Eden and Marca H. M. Wauben^*,5

Departments of
^* Biochemistry and Cell Biology and
Infectious Diseases and Immunology, Faculty of Veterinary Medicine, and
Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands

⁵ Correspondence: Department of Biochemistry & Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 2, 3584CM Utrecht, The Netherlands. E-mail: m.h.m.wauben{at}vet.uu.nl

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Major problems in the analysis of CD4⁺ effector cell and regulatory T cell (Treg) populations in an activated immune system are caused by the facts that both cell types can express CD25 and that the discriminatory marker forkhead box p3 can only be analyzed in nonviable (permeabilized) cells. Here, we show that CD134 (OX40) can be used as a discriminatory marker combined with CD25 to isolate and characterize viable CD4⁺ effector cells and Tregs. Before and during adjuvant arthritis in rats, coexpression of CD134 and CD25 identified activated Tregs consistently, as these T cells proliferated poorly to disease-associated antigens and were suppressive in vitro and in vivo. Depending on the time of isolation and location, CD4⁺ T cell populations expressing CD134 or CD25 contained effector/memory T cells. Analysis of the function, phenotype, and amount of the CD4⁺ T cell subsets in different lymph node stations revealed spatiotemporal differences in effector cell and Treg compartments during experimental arthritis.

Key Words: FoxP3 • immune regulation • kinetics • lymph nodes

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The immunological balance between autoaggressive T cells and regulatory T cells (Tregs) is crucial for prevention of autoimmune diseases, such as rheumatoid arthritis (RA) [¹ , ² ]. Regulatory CD4⁺CD25⁺ T cells, which are key players in the control of autoimmunity, are hypoproliferative to TCR stimulation in vitro and effectively suppress the response of other T cells upon activation in vitro as well as in vivo (reviewed in refs. [² , ³ ]). Numerous studies have attempted to characterize Tregs phenotypically. High intracellular expression of the forkhead box p3 transcription factor (FoxP3) [⁴ ] is, as yet, the most specific feature of such CD4⁺CD25⁺ Tregs. However, functional analysis of FoxP3⁺ T cells is not possible, as this marker can only be analyzed in permeabilized (nonviable) cells. The cell surface marker expression on cells within the CD4⁺CD25⁺ T cell population is heterogeneous. Besides reported heterogeneity in expression of, for example, CD103, CD62 ligand (CD62L), and CD134 [^{5 6 7} ], we demonstrated previously that the Treg population can be subdivided into functionally different subsets based on expression of CD134 (OX40) [⁸ ]. We showed in naïve rats that the CD134-expressing subpopulation of CD4⁺CD25⁺ T cells represented in vivo-activated Tregs, which suppressed T cell responses without further stimulation in vitro. CD4⁺CD134^–CD25⁺ T cells, on the contrary, needed to be activated in vitro to become suppressive.

Methods to identify in vivo-activated Tregs would facilitate the monitoring of Treg activation during an autoimmune response. However, the identification of Tregs induced during ongoing immune responses is complicated, as activation markers such as CD25 are also expressed on activated effector T cells [⁷ , ^{9 10 11} ]. Here, we show that analysis of CD25 expression in combination with CD134 expression can be used to identify and discriminate between effector cells and Tregs during experimental arthritis in rats. This allows simultaneous monitoring of effector cell and Treg dynamics during the disease process. We studied these dynamics in the rat adjuvant arthritis (AA) model, in which a monophasic arthritic syndrome is evoked by injection of Mycobacterium tuberculosis (Mt) in adjuvant [¹² ] and in which several studies have indicated the presence of disease-inducing and disease-regulating T cells [^{13 14 15 16} ]. In addition, T cell activation during the initiation phase of AA and during the active arthritis phase can be monitored in two distinct lymph node (LN) stations. In inguinal LN (ILN), which drain the Mt-injection site, Mt-specific T cell responses are generated during the initiation phase of AA. In contrast, popliteal LN (PLN) mainly drain the inflamed arthritic joints, where tissue damage takes place during AA. Using our CD25/CD134 phenotyping method, we show that the numbers of activated Tregs and effector T cells responding to disease-associated antigen change extensively at these sites during AA.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Rats, T cell clone Z1a, and antigens
Male inbred Lewis rats (7–10 weeks old) were obtained from the University of Limburg (Maastricht, The Netherlands) and were housed under conventional conditions. The Utrecht University Animal Ethics Committee (The Netherlands) approved all animal experiments.

The isolation, maintenance, and properties of the rat CD4⁺ T cell clone Z1a have been described previously [¹⁷ ]. This T cell clone recognizes amino acid sequence 72–85 of guinea pig myelin basic protein (MBP) [¹⁸ ].

Heat-killed Mt, strain H37RA, was obtained from Difco Laboratories (Detroit, MI, USA). For immunization, Mt was suspended in IFA (Difco Laboratories). Peptides Mt heat-shock protein (HSP)60_176–190 (EESNTFGLQLELTEG) and acetylated MBP_72–85 (Ac-MBP_72–85; Ac-QKSQRSQDENPV) were obtained from Isogen Bioscience (Maarssen, The Netherlands).

mAb and second-step reagents
The anti-CD134 (OX40) hybridoma was obtained from the European Collection of Cell Cultures (Salisbury, UK) [⁹ ]. The 12CA5 hybridoma-producing IgG2b isotype control was kindly provided by Dr. Ger. J. Strous (Department of Cell Biology and Institute of Biomembranes, University Medical Center, Utrecht University). The mAb were isolated from hybridoma supernatant by affinity chromatography using GammaBind Plus Sepharose (Roche Pharmacia, Uppsala, Sweden) and biotinylated using D-biotinoyl--aminocaproic acid-N-hydroxy-succinimide ester (Roche Molecular Biochemicals, Basel, Switzerland). Anti-CD4 (OX35), anti-CD25 (OX39), anti-MHC-II RT1-B^L (OX6), and IgG1 isotype control (A112-2/MOPC-31C) and streptavidine were purchased from BD PharMingen (San Diego, CA, USA). FITC-conjugated anti-FoxP3 mAb (FJK-16 s) was obtained from eBiosciences (San Diego, CA, USA). The cross-reactivity of this -mouse mAb with rat FoxP3 has been demonstrated recently [¹⁹ ].

Induction of AA and adoptive transfer of CD4⁺ T cell subsets
Rats were injected intradermally with Mt in 100 µl IFA. For in vitro assays using sorted CD4⁺ T cell subsets, rats were immunized with 10 mg/ml Mt. For adoptive T cell transfer, CD4⁺ T cell subsets were isolated from donor rats on Day 10 or Day 35 after Mt immunization and purified as described hereafter. Recipient rats were immunized with 5 mg/ml Mt (also yielding 100% disease incidence but lower maximum disease scores as compared with 10 mg/ml Mt) and were injected i.v. with 2 x 10⁵ sorted cells in 100 µl PBS immediately thereafter. Rats were weighed and examined for clinical signs of arthritis. The severity of arthritis was scored by grading each paw from 0 to 4, based on erythema, swelling, and immobility of the joints, resulting in a maximum score of 16 per animal [¹² ].

Flow cytometric analysis and cell sorting
On Day 0, 10, or 35 after Mt immunization, rats were killed, and PLN, ILN, submandibular LN (SLN), mesenteric LN (MLN), spleen, and blood were isolated and pooled per organ type. On Day 0, PLN and ILN were pooled, and brachial LN were included to obtain sufficient cell numbers. Single cell suspensions were prepared by mechanically forcing the organs through a 70-µm mesh. Erythrocytes were removed from spleen cell suspensions and blood by Ficoll-Isopaque gradient centrifugation. Cells were washed and incubated in PBS (Cambrex Bio Science, Verviers, Belgium) containing 4% heat-inactivated, naive rat serum, 1% fraction V BSA (Sigma-Aldrich Chemie, Zwijndrecht, The Netherlands), and 0.1% NaN₃. Cells (7x10⁵ per sample) were stained for 30 min on ice for CD4, CD134, and CD25. Where indicated, cells were additionally stained for MHC class II or were subsequently stained for intracellular FoxP3 expression. Briefly, after cell surface staining, cells were incubated in Fixation/Perm solution for 2 h at 4°C, washed with permeabilization buffer, and incubated with anti-CD32 (BD PharMingen) to block FcRs. Anti-FoxP3-FITC was added, and the cells were incubated a further 30 min at 4°C. After washing, cells were analyzed on a FACSCalibur^TM using CellQuest software (Becton Dickinson, Brussels, Belgium).

For cell sorting, PLN and ILN of 15–30 rats were isolated on Day 10 or Day 35 after Mt immunization and pooled per organ type, and single cell suspensions were prepared as described above. Cells were stained for CD4, CD25, and CD134 and sorted into CD25^–CD134^–, CD25⁺CD134⁺, CD25⁺CD134^–, and CD25^–CD134⁺ fractions using a FACSVantage^TM and CellQuest software.

Ex vivo proliferation of and suppression by sorted CD4⁺ T cell subsets
Cells (2.5x10⁴ per well) were washed and incubated for 72 h with antigen (1 µg/ml Mt HSP60_176–190 or Ac-MBP_72–85) and APC (30-Gy-irradiated naïve thymocytes, 5x10⁵ per well) in 200 µl medium in round-bottom, 96-well plates (Corning Costar, Schiphol Rijk, The Netherlands). Finally, cells were pulsed for 18–20 h with [³H]thymidine, 0.4 µCi/well (specific activity, 1 Ci/mmol, Amersham Biosciences, Freiburg, Germany), and [³H]thymidine incorporation was measured. Results are presented as the mean [³H]thymidine incorporation of triplicate wells ± SD.

Antigen-nonspecific suppression assays were performed using CD4⁺CD25^– responder T cells, as described previously [⁸ ]. For antigen-specific suppression assays, the T cell clone Z1a was used as a responder T cell [⁸ ]. The antigen dose-response curve of Z1a is sigmoid-to-bell-shaped and the sensitivity of Z1a for antigen can vary in time. The antigen dose-response curve was determined in each individual experiment, and suppression of Z1a responses on different experimental days was determined at peptide concentrations yielding similar T cell proliferation rates in the linear part of the dose-response curve. Responder T cells (10⁴ per well) were cultured in round-bottom, 96-well plates with CD4+ cells, which had been sorted based on expression of CD25 and/or CD134 (ratio of responder cells:sorted cells=1:1), with irradiated thymocytes as APC (5x10⁵ per well) and with the specific antigenic peptide. After 72 h, proliferation was assessed by [³H]thymidine incorporation, as described above.

Statistical evaluation
Statistical significance of differences in the development of AA was evaluated with a Mann-Whitney test using GraphPad Prism 3.02 (GraphPad Software, San Diego, CA, USA).

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Identification of in vivo-activated FoxP3⁺CD4⁺CD25⁺ Tregs in naïve rats
Previously, we showed that in vivo-activated, CD134-expressing CD4⁺CD25⁺ Tregs derived from naïve rats suppress T cell responses without additional stimulation in vitro [⁸ ]. This feature of CD4⁺CD134⁺CD25⁺ cells cannot be demonstrated in a classic, antigen-nonspecific suppression assay, as in this assay, responder and suppressor T cells are activated by anti-TCR mAb, resulting in equally potent suppression by CD4⁺CD134⁺CD25⁺ and CD4⁺CD134^–CD25⁺ cells (Fig. 1A and ref. [⁸ ]). However, in an antigen-specific suppression assay, only CD4⁺CD134⁺CD25⁺ T cells and not CD4⁺CD134^–CD25⁺ T cells were able to suppress the proliferation of T cell clone Z1a to its stimulatory peptide (Fig. 1B and ref. [⁸ ]). In addition, we found that the suppressive capacity of CD4⁺CD134⁺CD25⁺ T cells was inversely correlated with the strength of responder T cell stimulation (Fig. 1B) .

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Figure 1. Coexpression of CD25 and CD134 defines a population of Foxp3-expressing CD4+ T cells, which suppress antigen-specific T cell responses in vitro. (A and B) Naïve, splenic CD4+ T cells were sorted on the basis of CD25 and/or CD134 expression. (A) CD4+CD134+CD25+ T cells (open bar) or CD4+CD134–CD25+ T cells (black bar) were assessed for their capacity to suppress the proliferation of naive CD4+CD25– responder T cells by coculturing these cells in a 1:1 ratio with anti-TCR mAb and irradiated T cell-depleted spleen cells. (B) CD4+CD134–CD25– T cells (hatched bars), CD4+CD134+CD25+ T cells (open bars), or CD4+CD134–CD25+ T cells (black bars) were tested for their capacity to suppress the proliferation of the CD4+ rat T cell clone Z1a, stimulated with 0.05, 0.5, or 5.0 µg/ml Ac-MBP72–85 and irradiated thymus APC. (A and B) Data are from representative experiments (one out of three) and are expressed as means ± SD of T cell [3H]thymidine incorporation (cpm) from triplicate wells. (C and D) Pooled PLN and ILN cells from naïve rats were stained for CD4, CD25, CD134, and intracellular FoxP3 and analyzed by flow cytometry. (C) Representative histograms indicate FoxP3 (black lines) and isotype control (filled histograms) levels in cells gated for CD4+CD25+CD134+ T cells (left) or CD4+CD25+CD134– T cells (right). (D) The percentage of FoxP3+ cells of CD4+CD134–CD25– T cells (hatched bars), CD4+CD134+CD25+ T cells (open bars), or CD4+CD134–CD25+ T cells (black bars) was determined. The results are shown as the mean percentage ± SD of three rats.

Intracellular staining for FoxP3 revealed that >95% of CD4⁺CD134⁺CD25⁺ cells were positive for this Treg marker, whereas the CD4⁺CD134^–CD25⁺ subset in peripheral LN and spleen contained a substantial percentage of FoxP3^– cells (Fig. 1C and 1D) . Thus, CD134 expression identifies a pure FoxP3⁺ population of CD4⁺CD25⁺ Tregs, which suppress T cell proliferation without in vitro stimulation.

Identification of CD4⁺ Tregs activated during the course of AA
Next, we analyzed the suppressive capacity of CD4⁺ T cell subsets expressing CD25 and/or CD134 during the course of AA. Rats were immunized with Mt, and on Days 10 (before onset of clinical signs) and 35 (during active disease), cells from PLN (draining the joints) and ILN (draining the site of immunization) were sorted into CD4⁺CD134^–CD25^–, CD4⁺CD134⁺CD25⁺, CD4⁺CD134^–CD25⁺, and CD4⁺CD134⁺CD25^– fractions. The in vivo-induced, suppressive capacity of these CD4⁺ T cell subsets was assessed in an antigen-specific suppression assay using the AA-unrelated MBP-specific rat CD4⁺ T cell clone Z1a as responder. On Days 10 and 35 after Mt immunization, CD4⁺CD134⁺CD25⁺ cells from PLN and ILN suppressed the Z1a T cell response, whereas CD4⁺CD134^–CD25⁺ cells showed no or a much less pronounced suppression (Fig. 2A ).

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Figure 2. CD4+CD25+CD134+ T cells isolated during AA suppress antigen-specific T cell responses in vitro and reduce the severity of AA. (A) On Day 10 or Day 35 after Mt immunization, PLN and ILN cells from 15–25 rats were isolated and pooled per LN type. CD4+ cells were sorted into CD134–CD25– (hatched bars), CD134+CD25+ (open bars), CD134–CD25+ (black bars), and CD134+CD25– (cross-hatched bars) fractions and tested for their capacity to suppress the proliferation of the CD4+ T cell clone Z1a, stimulated with peptide. Ac-MBP72–85 peptide concentrations used for Days 10 and 35 experiments were chosen so that Z1a proliferation rates were similar (Day 10, 0.5 µg/ml; Day 35, 1 µg/ml). Responder T cells and sorted cells were cocultured at a 1:1 ratio. The results are shown as the mean [3H]thymidine incorporation (cpm) of triplicate wells ± SD for PLN subsets and ILN subsets. The indicated percentage of inhibition was calculated as the relative difference between the mean proliferative response of responder T cells in the presence of CD134–CD25– and CD134+CD25+ cells. One of two experiments yielding similar results is shown. (B) On Day 10 after Mt immunization, PLN and ILN were isolated from donor rats (n=30) and pooled, and CD4+ subsets were prepared as described in A. The sorted subsets were then transferred to recipient rats (2x105 sorted cells per recipient), which received a Mt immunization simultaneously. Rats were followed for development of clinical disease, and results are presented as the mean arthritis score ± SEM per group (n=4).

To confirm the suppressive potential of CD4⁺CD134⁺CD25⁺ cells isolated from rats during AA, we tested whether low numbers of these cells could suppress AA symptoms without in vitro stimulation prior to adoptive transfer. T cells were isolated from Mt-immunized donor rats on Day 10, and as few as 2 x 10⁵ cells were transferred to recipient rats, which were immunized with Mt at the time of adoptive T cell transfer and monitored for arthritis development. Of all transferred subsets, only CD4⁺CD134⁺CD25⁺ T cells decreased the AA disease scores significantly (P<0.05, Fig. 2B ). Thus, similar to naïve animals, expression of CD134 on CD4⁺CD25⁺ T cells in an activated immune system is indicative for their capacity to suppress T cell responses without additional stimulation. It is important that these data also indicate that functionally active CD4⁺CD25⁺ Tregs are present during the onset of disease.

Identification of effector CD4⁺ T cells during AA
We next studied the proliferative response to disease-related antigen of CD4⁺ T cell subsets expressing CD25 and/or CD134 in different LN stations during the course of AA. Autoaggressive effector T cells induced during AA have been shown previously to proliferate to Mt HSP60_176–190, which contains a disease-associated epitope [¹⁴ ]. On Day 10, a strong, proliferative response to Mt HSP60_176–190 was observed in the CD134⁺CD25^– fraction of ILN, which drain the site of immunization directly (Fig. 3 ). In PLN, responses to Mt HSP60_176–190 in the different T cell subsets resembled the situation in ILN, although proliferation levels were generally much lower. In contrast, the highest response to Mt HSP60_176–190 in the PLN on Day 35 of AA was observed in the CD134^–CD25⁺ subset. At this time-point, proliferation to Mt HSP60_176–190 in the ILN was decreased compared with Day 10 but was still most pronounced in the CD134⁺CD25^– fraction. Thus, the course of AA is characterized by changes in the phenotype and localization of T cells reacting to disease-related antigen. Furthermore, we demonstrated that effector CD4⁺ T cells express CD25 during the later stages of AA, precluding the use of CD25 as a single and exclusive marker for Tregs in an activated immune system.

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Figure 3. Antigen responsiveness of CD4+ T cells expressing CD25 and/or CD134 during AA. On Day 10 or Day 35 after Mt immunization, PLN and ILN cells from 15–25 rats were prepared as described in Figure 2A . CD4+ cells were sorted into CD134–CD25– (hatched bars), CD134+CD25+ (open bars), CD134–CD25+ (black bars), and CD134+CD25– (cross-hatched bars) fractions and tested for their proliferative response to Mt HSP60176–190 peptide. The results are shown as the mean [3H]thymidine incorporation (cpm) of triplicate wells ± SD for PLN subsets and ILN subsets. None of the fractions isolated at Day 10 or Day 35 proliferated in response to the irrelevant Ac-MBP72–85 peptide (control), and all fractions showed a similar response to recombinant IL-2 (data not shown). One of two experiments yielding similar results is shown.

Activation status of effector cell and Treg subsets during AA
To further study the activation status of effector cell and Treg subsets induced during AA (as characterized above), we analyzed the cell size and expression of the activation marker MHC class II [²⁰ ] of the different CD4⁺ T cell subsets on Days 10 and 35 after Mt immunization. On Day 10, CD134⁺CD25^– effector T cells and CD134⁺CD25⁺ Tregs had an activated phenotype, characterized by their larger cell size and increased expression of MHC class II compared with the nonactivated CD134^–CD25^– cells (Fig. 4A ). The phenotype of the CD4⁺ subsets in ILN and PLN was comparable at this time-point.

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Figure 4. Effector cells and Tregs induced during AA display an activated phenotype. (A) On Day 10 or Day 35 after Mt immunization, PLN and ILN cells were stained for CD4, CD25, and CD134. Cells were additionally stained for MHC class II (RT-1.BL) and were analyzed by flow cytometry. Dot-plots show the MHC class II expression on live CD4+ cells, gated for CD25 and/or CD134 expression as indicated. Quadrants were set based on the fluorescence intensity of cells stained with the isotype control and were used to calculate the percentage of MHC class II+ cells, as indicated in the upper-right quadrants. Histograms show the forward-scatter (FSC) profile of each subset. The numbers in these plots indicate the mean fluorescence intensity (MFI). (B and C) PLN cells from rats 35 days after Mt immunization were stained for CD4, CD25, CD134, and intracellular FoxP3 and analyzed by flow cytometry. (B) Representative histograms indicate FoxP3 (black lines) and isotype control (filled histogram) levels in cells gated for CD4+CD25+CD134+ T cells (left) or CD4+CD25+CD134– T cells (right). Numbers in plots indicate the percentage of FoxP3+ cells. (C) The representative dot-plot shows the relationship between FoxP3 and FSC levels on CD4+CD25+CD134– T cells from PLN. (D) Histograms indicate the forward-scatter levels of CD4+CD25+CD134– T cells in different organs from rats 35 days after Mt immunization.

On Day 35, CD134⁺CD25⁺ Tregs still showed a strongly activated phenotype in PLN, whereas the activation status in ILN was decreased. CD134⁺CD25^– effector T cells in the PLN, which were not reactive to Mt HSP60_176–190, appeared more activated than in the ILN at this time-point (Fig. 4A) . It is interesting that the CD134^–CD25⁺ cell population of PLN, containing Mt HSP60_176–190-reactive T cells, showed the highest activation level but appeared phenotypically dichotomic. The fraction contained large, activated cells, which were mostly MHC class II⁺, and the smaller cells were MHC class II^–. In the ILN, the CD134^–CD25⁺ fraction was more uniform and showed an intermediate activation level. To further investigate the nature of cells residing in the heterogeneous PLN CD134^–CD25⁺ cell population at Day 35 of AA, cells were stained for the Treg marker FoxP3. In comparison with the CD134⁺CD25⁺ Treg population, which was still almost entirely FoxP3⁺ (93.4%±1.8%, n=3 rats) at this late stage of AA, the CD134^–CD25⁺ population contained a much lower proportion (58.0%±3.9%, n=3 rats) of FoxP3⁺ cells (Fig. 4B) as compared with naïve rats (see Fig. 1 ). In addition, the majority of FoxP3-expressing cells of the Day 35 CD134^–CD25⁺ PLN population was small, whereas the largest cells lacked FoxP3 expression (Fig. 4C) . Most likely, therefore, the smaller FoxP3⁺ cells represent nonactivated Tregs, whereas the larger FoxP3^– cells represent effector/memory cells, which caused the observed proliferation of the CD134^–CD25⁺ population to disease-related antigen (Fig. 3) .

We next used the split pattern in forward-scatter levels within the CD134^–CD25⁺ T cell population at Day 35 as an indication of the presence of CD25-expressing effector/memory T cells in different lymphoid organs (Fig. 4D) . It is interesting that a clear split pattern was observed, not only in the PLN but also in blood, and this pattern was less prominent in ILN and SLN or MLN, which do not drain arthritic joints. This suggests that effector/memory T cells migrate via blood to or from inflamed LN.

Treg and effector cell numbers in draining LN during AA
Not only effector T cells but also Tregs can increase in number in response to antigenic stimulation [^{21 22 23} ]. We sought to determine how the numbers of cells characterized as effector cells or Tregs (Figs. 1 2 3 4) varied at different LN stations during the course of AA. The PLN, ILN, and as a control, the SLN were examined prior to (Day 0) and on Days 10 and 35 after Mt immunization by cell count and analytical flow cytometry. Total cell numbers of all CD4⁺ T cell subsets in the ILN increased during the initiation of AA and decreased later during clinical AA (Fig. 5A ). In contrast, the highest cell numbers in the PLN were found on Day 35 of clinical disease. In the control SLN, the numbers of cells in all subsets remained largely unchanged during the course of AA. To study the relative increase in cell number of the different subsets, we expressed these data as fold increase over the cell number on Day 0 (Fig. 5B) . It is interesting that CD134⁺CD25^– and CD134⁺CD25⁺ T cells showed the largest increase in numbers compared with Day 0 in ILN and PLN. Although the timing of expansion and contraction of these CD4⁺ T cell populations differed between PLN and ILN, both populations followed similar kinetics. The relative change in numbers of CD134^–CD25⁺ cells was low and comparable with that of CD134^–CD25^– cells. As the balance between autoaggressive and Tregs may determine the course of autoimmune responses, we next calculated the ratio of CD134⁺CD25^– effector cell and CD134⁺CD25⁺ Treg numbers during AA (Fig. 5C) . In ILN and PLN, this ratio had shifted in favor of CD134⁺CD25^– effector T cells on Day 10, as compared with Day 0. On Day 35, the effector cell:Treg ratio in ILN was returning to the Day 0 situation, whereas in PLN, the ratio was still similar to Day 10. In conclusion, dynamic changes occur in the effector cell and Treg compartments during AA. During onset of disease, Tregs increase in number, but these cells are outnumbered by an even stronger increase in the number of effector T cells. The effector cell/Treg balance is restored during active disease in the injection site-draining LN but not in the arthritic joint-draining LN.

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Figure 5. Kinetics of Tregs and effector T cells in draining LN during AA. On Days 0, 10, and 35 after Mt immunization, cells from ILN, PLN, and SLN were isolated, counted, stained for CD4, CD25, and CD134, and analyzed by flow cytometry. Bars indicate the mean total numbers of lymphocytes in the indicated fractions of three rats ± SD. Note that the scale of the y-axis is different for each subpopulation. (B) The fold increase in cell number of the different subsets in ILN and PLN on Days 10 and 35 over the cell number on Day 0 was calculated based on total cell numbers determined in A. (C) The ratio of the number of CD134+CD25– T cells:CD134+CD25+ T cells in ILN and PLN on Days 10 and 35 was calculated based on cell numbers determined in A.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In the present study, we show that costaining of CD25 and CD134 on CD4⁺ T cells allows the discrimination of effector cells and Tregs activated during experimental arthritis. We used this approach to monitor the dynamics of effector cells and Tregs in secondary lymphoid organs, which play a role in the induction phase or active disease phase of AA.

We found that during AA, T cells with autoaggressive potential, as defined by a strong, proliferative response to Mt HSP60_176–190 [¹⁴ ], resided in CD4⁺ T cell subsets expressing CD25 or CD134. In contrast, CD4⁺ T cells expressing CD25 and CD134 showed a strong, regulatory phenotype throughout the course of disease, as demonstrated by suppression of responder T cells in vitro and suppression of experimental arthritis. The strength of the regulatory capacity of CD4⁺CD25⁺CD134⁺ T cells was emphasized by their capacity to down-regulate the antigen-specific response of a rat CD4⁺ T cell clone in vitro without the need for additional stimulating factors and by the capacity of these cells to suppress AA after injecting only 2 x 10⁵ cells, which were not preactivated in vitro into fully immunocompetent recipients. Thus, similar to what we showed previously in naive rats [⁸ ], we here show that also in an activated immune system, the CD134-expressing subpopulation of CD4⁺CD25⁺ T cells represents in vivo-activated Tregs. The CD4⁺CD134⁺CD25⁺ population may have developed as a result of activation of the naturally occurring, thymus-derived CD4⁺CD25⁺ Treg population. Alternatively, these cells may have been induced in the periphery by disease-associated antigen stimulation [²⁴ , ²⁵ ]. It is important that despite the presence and intact suppressive capacity of Tregs during AA, the disease still ensued. Similarly, functionally active Tregs were shown to be present in humans during active RA [²⁶ , ²⁷ ]. The failure of Tregs to suppress disease may occur, as the Tregs are outnumbered by even more vigorously expanding effector T cells, or these effector T cells are less sensitive in vivo to the suppressive activity of Tregs.

We demonstrated that CD4⁺CD25⁺CD134^– T cells do not inhibit the development of AA upon transfer to Mt-immunized rats. Although others found that transferred CD25⁺ Tregs could interfere in experimental autoimmune diseases [^{28 29 30 31} ], lymphopenic animals have been used as recipients in these studies. In a lymphopenic environment, however, the in vivo regulatory capacity of T cells merely results from preferential expansion, irrespective of specificity and intrinsic regulatory capacity [³² , ³³ ]. In nonlymphopenic or CD25-depleted animals, transfer of total CD4⁺CD25⁺ T cells has only led to suppression of autoimmune disease when the transferred cells were preactivated in vitro [^{34 35 36} ]. Our findings also indicate that Tregs need to be activated before they can exert their suppressive function.

Our results indicate that CD25 cannot be used as a sole marker to identify CD4⁺ Tregs during an ongoing autoimmune response. Most clearly, this is shown by the strong proliferation to disease-associated antigen of CD4⁺CD25⁺ T cells resident in PLN during active disease (Day 35). In this CD134^–CD25⁺ population, we found a split pattern with regard to forward-scatter, MHC class II, and FoxP3 expression. The larger MHC class II⁺, FoxP3^– T cells may represent Mt HSP60_176–190-reactive, autoaggressive T cells, which have developed from recently activated CD134⁺CD25^– T cells into effector/memory T cells. Indeed, subsets of memory cells have been described to express CD25 [³⁷ , ³⁸ ]. The smaller FoxP3⁺ cells share phenotypic features with CD134^–CD25⁺ T cells present on Day 10 after Mt immunization and in naïve rats and most likely represent nonactivated, naturally occurring Tregs.

In RA, a high percentage of synovial fluid-derived CD4⁺CD25⁺ Tregs expresses high levels of CD134 [³⁹ ]. Others showed that CD4⁺CD25⁺CD134⁺ T cells with regulatory capacity could be discriminated from allo-specific effector T cells, which only expressed CD134 in a murine acute graft-versus-host disease model [⁴⁰ ]. Also in type 1 diabetes patients, increased levels of CD25⁺CD134⁺ T cells reactive to diabetes-associated antigens were demonstrated [⁴¹ ]. However, the functional phenotype of these autoreactive T cells, autoaggressive or suppressive, was not investigated. Recently, sustained expression of CD27, another member of the TNFR family, was shown to distinguish activated Tregs from effector T cells in inflamed synovia of arthritis patients [⁴² ]. These and our own findings indicate that differential regulation of expression of certain TNFR family members, such as CD27 and CD134 on Tregs versus effector cells, can serve as an important tool to discriminate between these T cell populations in an activated immune system. Whether CD134 plays a role in the suppressive function of Tregs is not clear. In general, CD134 is important for costimulation and survival [⁴³ , ⁴⁴ ] and plays a role in adhesion to CD134L-expressing vascular endothelial cells [⁴⁵ , ⁴⁶ ]. We speculate that the sustained expression of CD134 on Tregs facilitates their function, long-term survival, and entrance of inflammatory sites.

As only activated Tregs can suppress other T cell responses, specific monitoring of those Tregs, which received an activation signal in vivo, provides exclusive information about the involvement of Tregs in (auto)immune responses. We assessed the dynamics of Tregs and effector T cells activated in the course of experimental arthritis by analyzing the T cell expression patterns of CD25 and CD134. It is interesting that the cellular dynamics in ILN and PLN, which are involved in different stages of AA, were different. The highest total cellularity and T cell activation status in ILN, which drain the Mt immunization site and play an important role in the induction phase of AA, were observed on Day 10 and were decreased on Day 35 after immunization. In contrast, in PLN, which drain the arthritic joints, sustained T cell activation, and increasing T cell numbers were seen on Day 35 during active disease. As expected, the number of CD4⁺CD134⁺CD25^– effector T cells was markedly elevated in ILN and PLN during the course of AA as compared with naïve rats. It is important that the CD134⁺CD25⁺-activated Treg population expanded and contracted with similar kinetics as the effector T cell population, although with lower amplitude. Consequently, the ratio of the number of CD134⁺CD25^– effector T cells versus CD134⁺CD25⁺ Tregs increased during AA and peaked on Day 10 in ILN and on Day 35 in PLN. This ratio may be indicative for the immune status of LN, as nonstimulated, inflamed, or returning back to steady-state. Our data about the dynamics of effector cells and Tregs during antigen stimulation are consistent with a recent study describing the synchronous expansion and contraction of conventional cells and Tregs after CFA immunization [²³ ]. Similar kinetics but with much lower amplitude were seen in the CD134^–CD25^– nonactivated T cell population. The increase in number of nonactivated T cells in LN draining the inflammatory site during AA can be explained by the generalized block or "shutdown" in lymphocyte egress from inflamed LN, which has been described as a process that helps increase the precursor frequency in lymphoid organs [⁴⁷ , ⁴⁸ ]. This process could also have caused the increase in number of CD134^–CD25⁺ T cells on Day 10 in ILN. Alternatively, these nonactivated Tregs may have been actively recruited into inflamed LN as a result of localized production of chemokines or were retained specifically in the LN upon antigen recognition. During active disease (Day 35), CD134^–CD25⁺ effector/memory T cells, strongly reacting to disease-associated antigen, were found in peripheral blood and PLN, which may be explained by the suggested capacity of effector/memory T cells to migrate to inflamed LN [⁴⁹ ]. Future research should be directed toward understanding whether the observed changes in total cell numbers in each fraction are a result of proliferation, migration, and/or retention of T cells in LN involved in AA.

In conclusion, we developed and verified a method to simultaneously monitor effector cells and Tregs activated during an autoimmune response. We show that the combined analysis of CD25 and CD134 expression on CD4+ T cells reliably identifies effector T cells reacting to disease-associated antigen and in vivo-activated Tregs during AA. Spatiotemporal differences in the dynamics of Treg and effector T cell populations were found in secondary lymphoid organs involved in AA. These results form the basis of future research directed at assessing how the balance of Tregs and effector T cells affects the outcome of autoimmune responses.

 
Part of the work was carried out within UNYPHAR, a research network between Yamanouchi Europe B.V. and the Universities of Groningen, Leiden and Utrecht. We thank Drs. L. S. Taams and M. A. Nolte for criticial comments about the manuscript.

 
¹ These authors contributed equally to this work.

² Current address: Genmab B.V., P.O. Box 85199, 3508 AD, Utrecht, The Netherlands.

³ Current address: TNO Quality of Life, Zeist, The Netherlands.

⁴ Current address: Department of Radiology, Erasmus MC, Rotterdam, The Netherlands.

Received June 27, 2007; revised September 10, 2007; accepted September 14, 2007.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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