Originally published online as doi:10.1189/jlb.1003491 on December 23, 2003

Published online before print December 23, 2003

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(Journal of Leukocyte Biology. 2004;75:785-791.)
© 2004 by Society for Leukocyte Biology

4-1BB-dependent inhibition of immunosuppression by activated CD4⁺CD25⁺ T cells

Beom K. Choi^*, Jun S. Bae^*, Eun M. Choi^*, Woo J. Kang^*, Shimon Sakaguchi, Dass S. Vinay and Byoung S. Kwon^*,,1

^* Immunomodulation Research Center, University of Ulsan, Korea;
Department of Experimental Pathology, Institute for Frontier Medical Sciences, Kyoto University, Japan; and
Louisiana State University Eye Center, Louisiana State University Health Sciences Center School of Medicine, New Orleans

¹ Correspondence: Immunomodulation Research Center, University of Ulsan, 29 Mukeo-Dong, Nam-Ku, Ulsan, Korea, 680-749. E-mail: bskwon{at}mail.ulsan.ac.kr

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
4-1BB (CD137) is a costimulatory molecule involved in the activation and survival of CD4, CD8, and natural killer cells. Although a great deal has been learned as to how 4-1BB-mediated signaling governs the immunity of conventional T cells, the functional role of 4-1BB in the context of CD4⁺CD25⁺ regulatory T cell (Tr) activation is largely unknown. Using 4-1BB-intact and -deficient mice, we investigated the effect of the 4-1BB/4-1BB ligand pathway on the suppressive function of Tr cells. Our data indicate that although 4-1BB is expressed on Tr cells, its contribution to their proliferation is minimal. We also showed that signaling through the 4-1BB receptor inhibited the suppressive function of Tr cells in vitro and in vivo. It is interesting that anti-4-1BB-mediated but not anti-GITR-directed inhibition was more potent when Tr cells were preactivated. Collectively, these data indicate that 4-1BB signaling is critical in Tr cell immunity.

Key Words: regulatory T lymphocytes • GVHD • tolerance • suppression

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Since their discovery, CD25⁺ (CD4⁺CD25⁺) regulatory T (Tr) cells have been found to play important roles in immune function [^{1 2 3} ]. Upon coculture, they immunosuppress CD4⁺CD25^– and CD8⁺ T cell function, presumably by down-regulating interleukin (IL)-2 production [^{2 3 4 5} ] by a distinctive process in that it is rendered more potent by prior activation of the CD25⁺ cells [² ]. Despite rapid progress, the molecular basis of the immunosuppression remains elusive. Some studies have suggested that IL-10 and transforming growth factor-ß (TGF-ß) [⁴ ] contribute to the suppressive potency of the CD25⁺ Tr cells, but subsequent observations of IL-10 and TGF-ß-deficient mice do not fully support this view [² , ⁶ ]. Suppression appears to be contact-dependent and is not mediated by IL-4 or IL-10, as CD25⁺ Tr cells from IL-4- or IL-10-deficient mice are as effective as those from wild-type mice [² ]. Recent data suggested that glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR) plays some part in inhibiting CD25⁺ Tr cell-mediated suppression [⁷ ].

4-1BB, the inducible T cell antigen (Ag) present on CD4⁺, CD8⁺, natural killer, and dendritic cells, provides CD28-independent costimulation of T cell activation [^{8 9 10} ]. 4-1BB-mediated signaling plays a critical role in preventing activation-induced cell death, promoting the rejection of cardiac allografts and skin transplants, enhancing integrin-mediated cell adhesion, increasing T cell cytolytic potential, and eradicating established tumors [^{8 9 10 11 12 13 14} ]. 4-1BB-deficient mice have normal T and B cell numbers but have defects in Ag-specific interferon- expression and cytolytic T lymphocyte (CTL) activity [¹⁵ ].

Although 4-1BB is constitutively expressed on CD25⁺ Tr cells [¹⁶ , ¹⁷ ], the consequence of this expression is largely unknown. In the present study, we analyzed its effect in systems involving CD4⁺ T cell immunity. Using wild-type and 4-1BB-deficient mice, we showed that 4-1BB signaling is required to neutralize the suppressive function of CD25⁺ Tr cells in vitro and in vivo and that this neutralizing action is much more potent when the CD25⁺ Tr cells are activated. Thus, signaling through the 4-1BB receptor is critical for CD25⁺ Tr cell immunity. The ability of 4-1BB-dependent regulatory processes to counter the suppressive effect of CD25⁺ Tr cells in vitro and in vivo also points to a novel role for the 4-1BB receptor.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Mice
Female mice (5–6 weeks) were used in all experiments. Wild-type C57BL/6 mice were purchased from Harlan (Indianapolis, IN) and B6.C-H2^bm12/KhEg (bm12) mice, from The Jackson Laboratory (Bar Harbor, ME). 4-1BB-deficient C57BL/6 mice [¹⁵ ] were bred and maintained under specific, pathogen-free conditions in the animal facilities of the University of Ulsan (Korea).

Reagents and antibodies (Ab)
Anti-CD3 monoclonal Ab (mAb; 145.2C11), biotin-labeled CD25 (7D4), biotin–major histocompatibility complex (MHC) II (I-A^b, AF6-120.1), Fc blocker (2.4G2), fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (H129.19) mAb, and isotype-control Ab were purchased from BD PharMingen (San Diego, CA). Phycoerythrin (PE)-conjugated anti-CD25 (PC61.5) and biotin–CD8a (53-6.7) were purchased from E-Biosciences (San Diego, CA). Anti-CD4, anti-CD8, as well as streptavidin-conjugated microbeads, were obtained from Miltenyi Biotec (Auburn, CA). Recombinant human (rh)IL-2 was purchased from PeproTech (Rocky Hill, NJ). Dr. Robert S. Mittler (Emory University, Atlanta, GA) kindly provided agonistic anti-4-1BB mAbs (3H3 and 3E1), and production of agonistic anti-GITR mAb, DTA-1 has been described [⁷ ].

Cell isolation
Cell populations were isolated with a VarioMACS^TM magnetic cell sorter (Miltenyi Biotec), according to the manufacturer’s protocols. Briefly, red blood cell-depleted splenocytes were combined with lymph node cell suspensions in phosphate-buffered saline (PBS), supplemented with 0.5% bovine serum albumin, and incubated with Fc receptor-blocking mAb 2.4G2 for 10 min at 4°C. CD8⁺ T and MHC II⁺ cells were depleted by staining with biotinylated anti-CD8 and anti-MHC II mAb and streptavidin microbead. The CD25⁺ Tr cells were enriched by incubating the CD8^–MHC II^– fraction with a biotinylated anti-CD25 mAb and microbeads. CD25^– T cells were isolated from the CD8^–MHC II^–CD25^– fraction with microbead-conjugated anti-CD4 mAb. The CD25⁺ and CD25^– populations were >90% and >96% pure, respectively. To activate the purified CD25⁺ Tr cells, they were plated at 2 x 10⁶/well in six-well plates with 0.5 µg/ml anti-CD3 mAb and 20 U/ml rhIL-2 for 3 days. Activated CD25^– T cells were prepared by adding 0.5 µg/ml anti-CD3 mAb to the culture and incubating for 24 h. The activated cells were extensively washed with PBS and used immediately.

Cell proliferation
CD25⁺ Tr cells (1x10⁵ cells/well) and CD25^– T cells (2x10⁵ cells/well) were incubated with X-irradiated (20 Gy) splenocytes (5% with respect to total cells/well) for 3 days in the presence 0.5 µg/ml anti-CD3 mAb alone or in combination with 5 µg/ml 3H3 (anti-4-1BB mAb) or DTA-1 (anti-GITR mAb). rhIL-2 (10–20 U/ml) and a different number of CD25⁺ Tr cells were used in some experiments. The cells were labeled with 1 µCi/well [³H]-thymidine for the final 8 h, harvested, and counted in a liquid scintillation counter (Packard, Albertville, MN).

Flow cytometry
Naïve CD25^– T cells and CD25⁺ Tr cells were stained with PE-conjugated anti-CD25 and FITC-labeled anti-CD4 mAb after blocking with Fc receptor-blocking mAb 2.4G2 for 10 min at 4°C. Expression of 4-1BB on CD25⁺ Tr cells was measured by staining with FITC-conjugated 3E1 mAb for 30 min at 4°C and analysis on a FACScan^TM (BD Biosciences, San Jose, CA).

Induction of graft-versus-host disease (GVHD)
Recipient mice (bm12) were sublethally irradiated with 6.0 Gy (¹³⁷Cs) total body irradiation. After 6 h, the mice were intravenously infused with freshly purified CD25^– T cells (2x10⁵) or CD25^– and CD25⁺ Tr cells (2x10⁵) from wild-type C57BL/6 mice. In a separate experiment, CD25^– cells were prepared from 4-1BB-deficient mice and naïve CD25⁺ Tr cells from wild-type mice and activated as described above. The naive CD25^– T cells (3x10⁵) were injected into bm12 recipients together with naïve or activated CD25⁺ Tr cells (3x10⁵). Where indicated, the recipient mice were injected intraperitoneally with 200 µg 3H3 or DTA-1 mAb. The mice were monitored daily for GVHD lethality.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The 4-1BB does not influence CD25⁺ Tr cell proliferation
CD25⁺ Tr cells constitutively express 4-1BB at a low level and the same increases upon activation [¹⁶ ]. To assess the significance of this expression, we purified CD25⁺ Tr cells from wild-type C57BL/6 mice as described previously (Fig. 1A ). Flow cytometric analysis confirmed constitutive- as well as activation (with anti-CD3/IL-2)-enhanced 4-1BB expression on the surface of naïve CD25⁺ Tr cells (Fig. 1B) . Having found that the CD25⁺ Tr cells express 4-1BB, we tested whether this expression was responsible for activation signals, as it is in conventional T cells [¹⁸ ]. When naïve CD25⁺ Tr cells from wild-type mice were stimulated with anti-CD3 mAb, the addition of agonistic anti-4-1BB mAb (3H3) resulted in negligible enhancement of proliferation, whereas proliferation of CD25^– T cells was stimulated by the addition of 3H3 mAb (Fig. 1C) . If the CD25⁺ Tr cells were contaminated with a few effector cells, and these cells would be expanded to a certain extent during the proactivation step, then the anti-4-1BB stimulation may be stimulating those activated effectors to produce IL-2 and thereby break suppression. To remove this possibility and to test the effect of 4-1BB stimulation on the activated CD25⁺ Tr cells, CD25⁺ Tr cells were activated with anti-CD3 mAb plus IL-2 for 3 days and CD25^– cells with anti-CD3 mAb alone for 24 h, and proliferation assay was performed. Activated CD25⁺ Tr cells showed no significant enhancement of proliferation by 3H3 mAb (Fig. 1D) . Thus, signaling via the 4-1BB in CD25⁺ Tr cells does not result in cell proliferation as it does in conventional CD4⁺ or CD8⁺ T cells [¹⁹ ].

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Figure 1. CD25+ Tr cells show resistance to 4-1BB-mediated signaling. (A) The purity of CD25– and CD25+ cells assessed by staining with FITC-conjugated anti-CD4 mAb and PE-conjugated anti-CD25 mAb. (B) 4-1BB expression on naïve and activated CD25+ Tr cells. Cells were cultured in RPMI-1640 medium, supplemented with 0.5 µg/ml anti-CD3 mAb and 20 U/ml IL-2 for 3 days. They were then stained with FITC-conjugated anti-4-1BB mAb (3E1) and were analyzed with a FACScanTM. (C) Freshly purified CD25– T and CD25+ Tr cells were plated in 96-well culture plates at 1 x 105/well. Cells were activated with 0.5 µg/ml anti-CD3 mAb in the absence or presence of 5 µg/ml anti-CD28 or 3H3 (anti-4-1BB mAb) for 3 days. (D) CD25– T and CD25+ Tr cells were activated as described in Materials and Methods. After washing cells with PBS, they were plated at 1 x 105/well and stimulated with 0.5 µg/ml anti-CD3 mAb in the absence or presence of 5 µg/ml anti-CD28 or 3H3 (anti-4-1BB mAb) for 3 days. All samples were labeled with 1 µCi [3H]-thymidine for the last 8 h.

Signal through the 4-1BB receptor is required to neutralize the suppressive effect of CD25⁺ Tr cells in vitro
Previous results (Fig. 1B) showed that 4-1BB was expressed at a high level on rIL-2-activated CD25⁺ Tr cells. Thus, we hypothesized that activation of CD25⁺ Tr cells might be required to exert 4-1BB-mediated signaling. We tested whether 4-1BB-mediated signaling requires IL-2 to promote proliferation in coculture experiments. CD25^– T cells were cultured with CD25⁺ Tr cells in the presence of agonistic anti-4-1BB (3H3) mAb with and without IL-2. To establish the role of 4-1BB, CD25^– T cells from 4-1BB-deficient mice were cultured with CD25⁺ Tr cells from wild-type mice to direct the 4-1BB effects to the suppressors rather than the responders. CD25^– T cell proliferation was clearly inhibited by the Tr cells, and the 3H3 mAb permitted the proliferation of the CD25^– T cells in the presence of exogenous IL-2 (Fig. 2A ). This result suggests that 4-1BB signaling, together with exogenous IL-2, can antagonize the suppression by CD25⁺ Tr cells in a manner similar to the effect of GITR ligation, which efficiently reverses suppressive function of naïve CD25⁺ Tr cells [¹⁶ ]. Similar results were obtained when CD25^– T cells from 4-1BB-deficient mice were cocultured with CD25⁺ Tr cells from wild-type mice (Fig. 2B) . To determine whether 4-1BB stimulation could neutralize the suppressive activity of activated CD25⁺ Tr cells, we prepared "activated" CD25⁺ Tr cells by culturing them with anti-CD3 mAb and IL-2 for 3 days. When naïve CD25^– T cells were cocultured with activated CD25⁺ Tr cells, agonistic anti-4-1BB mAb (3H3) efficiently induced proliferation even in the presence of CD25^– T cells from 4-1BB-deficient mice (Fig. 2E and 2F) but not in the presence of naive CD25⁺ Tr cells as previously reported (Fig. 2C and 2D) .

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Figure 2. Effect of 4-1BB on the suppressive activity of CD25+ Tr cells. CD25+ Tr cells were purified from wild-type C57BL/6 mice, and CD25– T cells, from wild-type and 4-1BB-deficient C57BL/6 mice. To exclude the effect of anti-4-1BB mAb on activated CD25– T cells, 1 x 105 wild-type CD25+ Tr cells were mixed with 2 x 105 CD25– T cells from wild-type or 4-1BB-deficient mice and were stimulated with 0.5 µg/ml anti-CD3 mAb in the presence of 5 µg/ml rat immunoglobulin G (IgG), 3H3, DTA-1 mAb, and/or 10 U/ml rhIL-2. (A and B) Naive 4-1BB-intact and -deficient CD25– T cells were cocultured with naïve CD25+ Tr cells in the presence of 5 µg/ml control IgG, 3H3 mAb, and/or 10 U/ml IL-2. (C and D) Naïve CD25+ Tr cells were mixed with freshly isolated CD25– T cells from 4-1BB-intact and -deficient mice in the presence of 5 µg/ml control IgG, 3H3, or DTA-1 mAb. (E and F) Activated CD25+ Tr cells were prepared as described previously and cocultured with naive CD25– T cells from 4-1BB-intact and -deficient mice in the presence 5 µg/ml control IgG, 3H3, or DTA-1 mAb. Proliferation was measured on the third day by labeling with 1 µCi [3H]-thymidine for the last 8 h.

Signaling through 4-1BB attenuates the suppression of activated CD25⁺ Tr cells
To further confirm the observed, desuppressive effect of 3H3 mAb, naïve CD25^– T cells were cocultured with different ratios of CD25⁺ T cells with control IgG, 3H3, or DTA-1 mAb. As previously reported [¹⁶ ], DTA-1 mAb only reversed the suppression when the activated CD25⁺ Tr cell number is low. In contrast, the 3H3 mAb abrogated the suppression of activated CD25⁺ Tr cells at all ratios tested (Fig. 3A ). To test the dose-dependent effect of 3H3 mAb, naive CD25^– T cells and activated CD25⁺ Tr cells (2:1 ratio) were cocultured with the indicated doses of 3H3 or DTA-1 mAb. Agonistic anti-4-1BB mAb (3H3) abrogated activated CD25⁺ Tr cell-mediated suppression in a dose-dependent manner when added to cultures containing CD25⁺ Tr cells and CD25^– T cells (Fig. 3B) .

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Figure 3. Stimulation through the 4-1BB receptor reverses the suppression of activated CD25+ Tr cells. (A) Naïve CD25– Tr cells (2x105) from 4-1BB-deficient mice were cocultured with the indicated number of activated CD25+ Tr cells and treated with 5 µg/ml control IgG, 3H3, or DTA-1 mAb. (B) Naïve CD25– Tr cells (2x105) from 4-1BB-deficient mice and 2 x 105 of activated CD25+ Tr cells were mixed with the indicated concentration of control IgG, 3H3, or DTA-1 mAb. All samples were stimulated with 0.5 µg/ml anti-CD3 mAb, and proliferation was measured on the third day.

It is possible to deduce from these results that CD25⁺ Tr cells express a high level of GITR even in the resting status [⁷ ], but only a subpopulation of naïve CD25⁺ Tr cells expressed 4-1BB (Fig. 1B) . In this condition, 4-1BB stimulation could not reverse the suppression of naïve CD25⁺ Tr cells as a result of the low level of 4-1BB, suggesting that to elicit effective desuppression, 4-1BB must be expressed at higher levels. Thus, we conclude that the suppression of activated but not "resting" CD25⁺ Tr cells is more effectively neutralized by 4-1BB than GITR stimulation.

4-1BB stimulation renders CD25^– T cells resistant to suppression but does not fully alter the suppressive function of CD25⁺ Tr cells
4-1BB stimulation is known to enhance the proliferation, function, and survival of CD4⁺ and CD8⁺ T cells [¹¹ , ²⁰ ]. We therefore tested whether CD25^– T cells stimulated by 3H3 mAb are resistant to suppression by CD25⁺ Tr cells. Activated CD25^– T cells were prepared from wild-type and 4-1BB-deficient mice as a negative control and were cultured with anti-CD3 mAb for 24 h. This activation step led to the increased expansion of 4-1BB-positive cells (>70%; data not shown). Stimulation through 4-1BB rendered activated CD25^– T cells more resistant to the suppression by CD25⁺ Tr cells but not activated CD25^– T cells from 4-1BB-deficient mice (Fig. 4A and 4B ). It is interesting that activated CD25^– T cells were poorly suppressed by "naïve" CD25⁺ Tr cells but not by activated CD25⁺ Tr cells (Fig. 4C) . In a separate experiment, we tested whether 4-1BB stimulation permanently altered the suppressive activity of the CD25⁺ Tr cells, which were activated with anti-CD3 mAb and IL-2 for 3 days in the presence or absence of 3H3 mAb. Activated cells were extensively washed and cocultured with naïve CD25^– T cells. We found that they recovered their ability to suppress upon removal of the 3H3 mAb (Fig. 4D) , suggesting that sustained stimulation through 4-1BB is required for the increased resistance of activated CD25^– T cells.

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Figure 4. Effect of signaling through 4-1BB on in vitro suppression of activated CD25– T cells by CD25+ Tr cells. (A and B) Activated CD25– T cells were prepared from 4-1BB-intact and -deficient mice as described previously. Activated CD25– T cells and naïve CD25+ Tr cells were mixed and stimulated with 0.5 µg/ml anti-CD3 mAb in the presence of 5 µg/ml control IgG, 3H3, or DTA-1 mAb for 3 days. (C) Activated CD25– T cells and CD25+ Tr cells were prepared from wild-type C57BL/6 mice, mixed, and stimulated with anti-CD3 mAb in the presence of 5 µg/ml control IgG, 3H3, or DTA-1 mAb for 3 days. (D) CD25+ Tr cells were incubated in plates with 0.5 µg/ml anti-CD3 mAb and 20 U/ml IL-2 for 3 days in the presence or absence of 3H3 (5 µg/ml) mAb. The cells were harvested, washed with PBS, and were then serially diluted and cocultured with freshly isolated CD25– T cells for 3 days. Samples were labeled with 1 µCi [3H]-thymidine for the last 8 h, and the extent of cellular proliferation was enumerated.

Signals through 4-1BB abrogate prolonged survival in the face of GVHD induction by transfer of CD25⁺ Tr cells
We also tested whether 4-1BB stimulation can inhibit the suppressive activity of CD25⁺ Tr cells in vivo. We used a system in which purified T cell subsets are introduced into MHC-disparate, sublethally irradiated recipients [²¹ ]. We transferred freshly purified CD25^– T cells or CD25^– and CD25⁺ Tr cells into sublethally irradiated bm12 recipients together with control IgG, DTA-1, or 3H3 mAb. Transfer of CD25⁺ Tr cells delayed death from GVHD, and injection of DTA-1 or 3H3 mAb abolished this delay (Fig. 5A ). Thus, stimulation through GITR or 4-1BB neutralized the suppressive activity of CD25⁺ Tr cells in vivo. However, 20–25% of the mice treated with 3H3 mAb survived, perhaps because of differences in the extent of activation of the CD25⁺ Tr cells in the initial immune response. We therefore prepared activated CD25⁺ Tr cells from wild-type C57BL/6 mice by stimulating them with anti-CD3 mAb and exogenous IL-2 and did the same with naïve CD25^– T cells from 4-1BB-deficient mice to rule out the effect of 3H3 mAb on CD25^– T cells, which from wild-type and 4-1BB-deficient mice, behaved only slightly differently in this GVHD system when 3 x 10⁵ cells were transferred into bm12 mice as described previously [²² ]. Therefore, in the present study, we transferred the naïve or activated CD25⁺ Tr cells with naïve CD25^– T cells into sublethally irradiated bm12 mice along with 3H3 mAb, which caused accelerated death when activated rather than naïve CD25⁺ Tr cells, which were transferred (Fig. 5B) . Taken together, we concluded that stimulation through the 4-1BB receptor has a critical role in the neutralization of activated CD25⁺ Tr cells.

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Figure 5. The effect of signaling through the 4-1BB receptor on the protective function of CD25+ Tr cells during GVHD. (A) Recipient mice (bm12) were sublethally irradiated with 6.0 Gy total body irradiation. Six hours later, 2 x 105 freshly purified CD25– and/or CD25+ T cells from wild-type C57BL/6 mice were infused into the bm12 recipients by tail-vein injection together with DTA-1 or 3H3 mAb. (B) To exclude any effect of 4-1BB on the CD25– T cells, in a separate experiment, 3 x 105 freshly purified CD25– T cells from 4-1BB-deficient C57BL/6 mice were injected into bm12 recipients together with 3 x 105 naïve or activated CD25+ Tr cells. The mice were monitored daily for death caused by GVHD lethality.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In an exception to the activation-dependent expression pattern of 4-1BB on conventional T cells [⁸ ], a recent study reported that Tr cells express this receptor in a constitutive manner [¹⁶ ]. The significance of this expression in the regulation of Tr cells, however, has not been explained in detail.

In this study, we present evidence that 4-1BB signaling is critical for modulation of the suppressor function of activated but not resting CD25⁺ Tr cells. By contrast, GITR stimulation regulates the activity of naïve but not activated CD25⁺ Tr cells [⁷ , ¹⁶ ]. As resting CD25⁺ Tr cells express GITR on their surface at much higher levels than they do 4-1BB [¹⁶ ], it is possible that 4-1BB must be fully expressed to efficiently antagonize suppression of CD25⁺ Tr cell function.

We also confirmed that agonistic anti-4-1BB mAb not only neutralizes the function of activated CD25⁺ Tr cells in vitro and in vivo but also renders 4-1BB-expressing CD25^– T cells more resistant to suppression by CD25⁺ Tr cells. In this process, the suppressor potential of the CD25⁺ Tr cells increases, and the CD25^– T cells become resistant to suppression. As a result, it may be that only activated CD25⁺ Tr cells are able to efficiently suppress the activated CD25^– T cells. In that case, 4-1BB, 4-1BBL, and GITR ligand-expressing cells, which are known to be dendritic cells (unpublished), would be important for initiation, maintenance, and fine-tuning of an optimal immune response.

Although our present experiments suggest that 4-1BB is critical for countering the suppression of CD25⁺ Tr cell function, it is not clear how this is achieved. Further, although several other molecules are involved in the modulation of CD25⁺ Tr cell function, including CTLA-4 [²³ ], tumor necrosis factor-related activation-induced cytokine/receptor activator of nuclear factor-B [²⁴ ], inducible costimulator (ICOS)/ICOS ligand [²⁵ ], CD40/CD40 ligand [²⁶ ], and B7/CD28 [²⁷ ], it remains to be determined how they regulate the suppression of CD25⁺ Tr cells. A possible role for CTLA-4 and programmed death-1 ligand (PD-L1) in T cell–T cell regulation has recently been suggested [⁴ , ²⁸ ], but the surface molecules involved have not been clearly defined [⁶ ]. Shimizu et al. [⁷ ] report that GITR differs from CD28 or CTLA-4 in the way it attenuates suppression and does not down-regulate CTLA-4 and TGF-ß expression. To test whether 4-1BB-mediated signaling of Tr cells affects PD-L1 and CTLA-4 molecules, we performed a flow cytometric analysis: Ligation of 4-1BB had no appreciable effect on these molecules (data not shown). We also tested whether 4-1BB molecules affect the function and development of CD25⁺ Tr cells and found that 4-1BB-deficient mice showed no deficiency of Tr cells in lymphoid and nonlymphoid organs (data not shown). Moreover, the level of suppression obtained with CD25⁺ Tr cells from 4-1BB-deficient mice was comparable with that achieved with CD25⁺ Tr cells from wild-type mice (data not shown).

In spite of intensive study of regulatory T cells, much uncertainty remains regarding their mode of action [²⁹ ]. It is important for the development of new therapeutic approaches to transplantation, autoimmune diseases, and infections to understand modulation of regulatory T cells at the cellular and molecular levels [^{30 31 32 33} ]. Our results provide novel insight into how costimulatory molecules on the surface of CD25⁺ Tr cells modulate the immune response.

 
The SRC Fund to the IRC at the University of Ulsan from KOSEF and the Ministry of Science and Technology (Korea) and U.S. Public Health Services Grants RO1EY013325 and P30EY002377 from the National Eye Institute, National Institutes of Health (Bethesda, MD), supported this work.

Received October 20, 2003; accepted October 22, 2003.

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