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IL-15 and Cognate Antigen Successfully Expand De Novo-Induced Human Antigen-Specific Regulatory CD4⁺ T Cells That Require Antigen-Specific Activation for Suppression

Department for Blood Transfusion and Transplantation Immunology, University Medical Center, Nijmegen, The Netherlands

	Abstract

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An important prerequisite in using regulatory T cells for immunotherapy is their ex vivo expansion without loss of suppressor function. Human anergic regulatory T cells are expandable by Ag-specific stimulation in the presence of IL-2. IL-15, like IL-2, is a T cell growth factor that, in contrast to IL-2, stimulates survival of T cells. In this study, we examined whether IL-15 could be exploited as a superior growth factor of human CD4⁺ anergic regulatory T cells that were generated by costimulation blockade. Next, IL-15, as compared with IL-2, was investigated with respect to expansion and function of these regulatory T cells. Optimal expansion required cognate allogeneic stimulation in the presence of exogenous IL-15. IL-15 resulted in enhanced survival that was paralleled by an increased number of Bcl-2-expressing cells. Moreover, IL-15 induced a distinct type of anergy characterized by hyperreactivity to IL-15, resulting in improved expansion. This is likely attributed to increased propensity of these cells to up-regulate both - and -chains of the IL-2 and IL-15 receptor. Notably, IL-15-expanded regulatory CD4⁺ T cells suppressed both naive and memory T cells in a superior way. Immunosuppression required alloantigen-specific stimulation and appeared gamma-irradiation resistant and independent of IL-10, TGF, or CTLA-4 interactions. These regulatory T cells were stable suppressors, mediating bystander suppression upon TCR stimulation, but leaving recall responses unaffected in the absence of cognate Ag. Finally, human naturally occurring regulatory CD4⁺CD25⁺ T cells appeared important in generating regulatory T cells by costimulation blockade. In conclusion, IL-15-expanded, de novo-induced human anergic regulatory CD4⁺ T cells are of interest in Ag-specific immunotherapy.

	Introduction

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Immunoregulatory suppressive T cells play a crucial role in the homeostasis of the immune system and in prevention of immune pathology (1, 2). Immunoregulatory T cells were identified in rodents and humans and may broadly be divided into two groups: naturally occurring regulatory cells (i.e., CD4⁺CD25⁺) (3, 4, 5, 6, 7) and various types of de novo-induced suppressive T cells (8, 9, 10, 11, 12). Both kinds of immunosuppressive regulatory T cells share an anergic phenotype (low proliferative capacity in vitro) and need TCR triggering to become suppressive, yet contrasting and incompletely understood suppressor mechanisms have been postulated. Briefly, immunoregulatory T cells might inhibit target T cells directly and/or indirectly by impairment of the APC function, often in a cell-cell contact-dependent manner (2, 13). Some studies indicated an important role for immunosuppressive cytokines such as IL-10 and TGF (9, 14, 15, 16) or interactions via CTLA-4 (15, 17, 18).

Immunosuppressive regulatory T cells play an evident role in allograft tolerance and prevention of autoimmune disease; hence there are substantive therapeutic prospects for these cells in transplantation and autoimmunity (2, 13, 19, 20, 21). Moreover, the therapeutic use of Ag-specific suppressor T cells would be an advantage to the currently used non-Ag-specific immunosuppressive drugs. For future human immunotherapy, sufficient immunosuppressive cell numbers will be needed, and therefore, it is crucial to reverse the anergic state and enhance the growth of these cells, while preserving their (Ag-specific) suppressor function.

In previous work, we have shown the novo induction of human anergic allo-MHC-specific suppressive regulatory T cells via anti-CD86/CD40 mAb blockade (10). Moreover, we showed that exogenous IL-2 recovered the anergic state and enhanced growth whereby the cells retained their anergic state after withdrawal of IL-2 (10). Similar observations were made for human naturally occurring CD4⁺CD25⁺ T cells following TCR stimulation in the presence of exogenous IL-2 or mixtures of IL-2 and either IL-4 or IL-15 (4, 5, 6). In addition, naturally occurring CD4⁺CD25⁺ regulatory T cells preserved their suppressive potential following TCR stimulation in the presence of exogenously added cytokines (4, 7, 22, 23). These cytokine-mediated growth features are of interest in the expansion of alloantigen-specific suppressor T cells for future immunotherapy. Therefore, in this study, we examined the potential of IL-2 and IL-15 in alloantigen-specific expansion of anergic suppressor T cells.

IL-15 and IL-2 share two receptor subunits: the -chain (CD122) (24, 25) and the common -chain (CD132) (26), including a number of biological functions (27, 28). Both cytokines stimulate the proliferation of activated T cells (25), but they distinctly contribute to T cell survival (29, 30). IL-15 is generally considered to be an inhibitor of apoptosis (30, 31), whereas IL-2 is important in activation-induced cell death of CD4⁺ T cells (32, 33). In addition, IL-15, in contrast to IL-2, selectively stimulated proliferation of the CD8⁺ memory T cell subset (34, 35, 36) and redirected apoptosis toward anergy in partially stimulated T cells (37). Recently, IL-15 was proposed to be critical in the proliferation of human regulatory T cells (38, 39).

Considering the opposite effects of IL-2 and IL-15 on T cell survival and selective growth of certain T cell subsets, we hypothesized that IL-15 would be ideally suited for expansion of the novo-induced anergic regulatory CD4⁺ T cells. In this report, we describe superior expansion of de novo-induced anergic regulatory CD4⁺ T cells by stimulation with cognate Ag and exogenously added IL-15. After expansion, these cells remained anergic and immunosuppressive. Immunosuppression required alloantigen-specific TCR triggering, was independent of TGF, IL-10, or CTLA-4, and was sustained in the presence of a concurrent autologous productive T cell response.

In conclusion, these IL-15-expanded, de novo-induced regulatory CD4⁺ T cells meet important criteria needed for donor-specific immunosuppression and hence are of potential interest for clinical application in the prevention of graft rejection.

	Materials and Methods

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Cells

For all experiments, PBMC were isolated by density gradient centrifugation (Lymphoprep; Nycomed Pharma, Oslo, Norway) from buffy coats of normal healthy donors. Cells were frozen and stored in liquid nitrogen until use. After thawing, viability of the cells was determined by trypan blue exclusion.

CD4⁺ T cells were purified from PBMC by a magnetic bead-based negative-selection system purchased from StemCell Technologies (Vancouver, British Columbia, Canada) in combination with the Stem Sep CD4⁺ T cell enrichment mixture (StemCell Technologies). Naturally occurring CD4⁺CD25⁺ T cells were depleted using CD25⁺ microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). The isolation procedures were conducted according to the manufacturer’s instructions and resulted in an enrichment of 90–95% CD4⁺ T cells and in a depletion (<0.2%) of CD25⁺ T cells, respectively.

Flow cytometry, intracellular cytokine staining, and Abs

Cells were phenotypically analyzed by three- or four-color immunofluorescence. Briefly, cells were washed twice with buffer (PBS containing 0.2% BSA) and labeled with the Abs of interest conjugated with FITC, PE, PE-Texas Red-X, or PE-CY5 (PC5).² Cells were incubated for 20 min in the dark at room temperature, washed twice, and analyzed on a Coulter Epics XL flow cytometer (Beckman Coulter, Fullerton, CA). A total of 5,000–10,000 living cells were collected and analyzed by Coulter Epics Expo 32 (Beckman Coulter). Isotype-matched Abs, usually below background staining, were used to define marker settings. The following conjugated mAbs were used: CD3(UCHT1), CD4(MT310), CD8(DK25), CD25(ACT1), CD27(M-T271), CD45RA(4KB5), CD45RO(UCHL1) (FITC or PE labeled) (DAKO, Glostrup, Denmark); CD122(CF1) (PE labeled) (Immunotech, Marseille, France); CD4(T4), CD8(T8) (PE-Texas Red-X labeled) (Coulter, Miami, FL); CD152(BNI13) PE, CD4(T4), CD8(T8), and CD25(B1.49.9) (PC5 labeled) (Immunotech, Marseille, France); and CD25(M-A251) PE, CD132(AG184) PE, anti-BclII(BclII/100) PE (BD Biosciences, San Jose, CA), and anti-human IL-15R-biotin (R&D Systems, Abingdon, Oxon, U.K.), which was used in combination with streptavidin-PC5 (Beckman Coulter). Anti-human IL-2 PE, IL-4 PE, IL-10 PE, IFN- FITC, and TNF- FITC were purchased from BD Biosciences. Intracellular cytokine staining was analyzed after polyclonal stimulation with PMA and ionomycin, such as described previously (40). Data were analyzed by Coulter Epics Expo 32 software (Beckman Coulter).

To detect intracytoplasmic CTLA-4 (CD152), Bcl-2, or cytokines, the cells of interest were fixed and permeabilized (Fix and Perm reagent A and B; Caltag Laboratories, Vienna, Austria) according to the instructions of the manufacturer and, thereafter, were labeled with the mAb of interest. After 45-min incubation at room temperature, the cells were washed three times and analyzed by flow cytometry. Appropriate isotype control mAbs were used for marker settings.

Cell division kinetics according to CFSE dilution

Cell division was studied by labeling responder T cells with 2 µM CFSE (Molecular Probes, Eugene, OR) just before stimulation. In the cell, esterases cleave the acetyl group, leading to the fluorescent CFSE. At each cell division, the mean CFSE fluorescence halves. Simultaneous Ab labeling of selective T cell subsets enables division kinetics of a particular subset. Samples were measured by flow cytometry. Data were analyzed using ModFit LT (Verity Software House, Topsham, ME) or Coulter Epics Expo 32 software (Beckman Coulter).

Cell cycle analysis by flow cytometry

Cell cycle analysis was performed by analyzing the DNA content with the vital cell dye TOPRO-3 (Molecular Probes). Cells were washed in PBS and permeabilized in cold 70% ethanol for at least 1 h. Next, the cells were washed and stained with TOPRO-3 (0.3 µM) containing 0.1 mg/ml RNase. TOPRO-3 staining was measured on a Coulter Epics Elite flow cytometer (Beckman Coulter). Surface staining by FITC- and/or PE-labeled mAb was performed before permeabilization of the cells as mentioned above. Data were analyzed by ModFit LT (Verity Software House) and/or Coulter Epics Expo 32 software (Beckman Coulter).

HLA typing

Serological HLA-A, HLA-B, HLA-DR, and HLA-DQ phenotyping (broad specificities and splits) was performed using the standard microcytoxicity assay. Additional class I and II (sub)typing was performed by molecular methods. Preparation of genomic DNA was performed using the QIA-Amp blood kit (Qiagen, Hilden, Germany). HLA-A, -B, -DR, and -DQ low-to-intermediate resolution typing was by a PCR-sequence-specific primer technique (Pel-Freeze Clinical Systems, Milwaukee, WI). HLA-DRB and -DQB subtyping was performed using a PCR-sequence-specific primer technique (DRB1*, B3*, B4*, B5*, and DQB subtyping kits; Dynal, Oslo, Norway).

Primary MLC

Primary one-way MLC were performed by culturing 5 x 10⁴ isolated CD4⁺ T cells with 1 x 10⁵ HLA-mismatched gamma-irradiated (30 Gy) stimulator PBMC in 96-well round-bottom plates (Greiner, Frickenhausen, Germany) in 200 µl of culture medium (RPMI 1640 with glutamax supplemented with pyruvate (0.02 mM), 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Life Technologies, Paisley, U.K.) and 5–10% heat-inactivated pooled human serum) at 37°C, 95% humidity, and 5% CO₂. Proliferation was analyzed by [³H]thymidine incorporation at the indicated point of measurement. To this end, 0.037 MBq (1 µCi) of [³H]thymidine (ICN Pharmaceuticals, Irvine, CA) (specific activity, 7.4 x 10¹⁰ Bq/mmol (2.0 Ci/mmol)) was present during the last 18 h. [³H]Thymidine incorporation was analyzed by a gas scintillation counter (Matrix 96 beta counter; Canberra Packard, Meriden, CT). The ³H incorporation is expressed as counts per 5 min (mean ± SD) of at least quadruplicate measurements.

Induction of T cell anergy and secondary MLC

To induce anergic CD4⁺ T cells, primary MLC were performed for 7 days in the presence of two antagonistic mAbs directed against either CD40 or CD86 (ch5D12 and chFUN-1, respectively; 0.5–1 µg/ml; kindly provided by Tanox Pharma (Amsterdam, The Netherlands)). Anergy was examined in the secondary MLC as follows: the cells from the primary MLC were harvested, washed, and allowed to recuperate for 2–3 days. Thereafter, the cells were washed again, and the nonviable cells were removed by density gradient centrifugation (Lymphoprep; Nycomed Pharma). Next, 2 x 10⁴ recovered viable CD4⁺ T cells were restimulated with 1 x 10⁵ gamma-irradiated (30 Gy) stimulator PBMC in 96-well round-bottom plates, referred to as secondary MLC. The stimulator PBMC used in the second and first step were derived from the same donor PBMC pool to assure allospecific responsiveness. Completely MHC-mismatched stimulator PBMC did not result in recall responses. The proliferative response of the secondary MLC was examined at the indicated time points. T cell anergy was defined as hyporesponsiveness upon antigenic restimulation that is reversed by addition of exogenous IL-2 (12.5 U/ml; Proleukin; Eurocetus, Amsterdam, The Netherlands) (10).

Tertiary MLC

To analyze whether anergic T cells that were reversed by IL-2 or IL-15 preserved their anergic state, tertiary MLC were performed according to a three-step culture system. In step 1, primary MLC were performed in the presence or absence of costimulation blockade, and in step 2, these cells were allogeneically restimulated in the absence or presence of exogenous IL-2 (12.5 U/ml; Proleukin) or IL-15 (10 ng/ml; BioSource International, Nivelles, Belgium) as mentioned earlier. IL-15 titration curves revealed that concentrations of 10 ng/ml resulted in optimal proliferative responses. In step 3, the tertiary MLC, 2 x 10⁴ viable T cells (obtained after density gradient centrifugation) that were derived after expansion in the secondary MLC in the presence of IL-2 or IL-15 were restimulated by 1 x 10⁵ irradiated (30 Gy) allogeneic PBMC. At each (re)stimulation step, allogeneic stimulator PBMC derived from the same donor were used.

Cocultures to study immunosuppression

The regulatory capacity of IL-2- or IL-15-reversed anergic CD4⁺ T cells was studied in coculture MLC. To this end, anergic CD4⁺ T cells or control primed CD4⁺ T cells that were expanded with alloantigen and exogenously added IL-2 or IL-15 were added to a newly set-up primary or secondary MLC. Cocultures were performed in 96-well round-bottom plates; 5 x 10⁴ previously expanded anergic CD4⁺ T cells or control primed CD4⁺ T cells were added to a newly set-up MLC consisting of the 5 x 10⁴ original responder PBMC (from which the anergic cells originate) and 5 x 10⁴ original gamma-irradiated (30 Gy) stimulator PBMC (used to generate the anergic cells). In some experiments, the responder cell fraction was labeled with CFSE. In case of ³H incorporation studies, the tests were performed in quadruplicate. Relative suppression was calculated according to the following equation: percentage of suppression = (1 - (³H incorporation coculture/³H incorporation control MLC) x 100%).

Ag specificity of the regulatory phenomenon was examined in cocultures performed with third-party stimulator PBMC that were either completely HLA mismatched or partially HLA matched (with an isolated class II mismatch) in relation to the original stimulator cells used. Neutralizing Abs (20 µg/ml) against IL-10 (MAB217) and TGF (MAB1835) (both R&D Systems) or blocking anti-CTLA-4 mAb (20 µg/ml; Innogenetics, Ghent, Belgium) were added during the coculture to study the role of these cytokines or surface marker in immunosuppression. Irrelevant isotype-matched Abs were used to control for specificity. These Abs never abrogated suppression.

To study whether expanded regulatory CD4⁺ T cells interact with autologous T cell responses, fresh autologous donor PBMC from which the regulatory cells originate were used in a recall Ag stimulation assay. To this end, 1 x 10⁵ autologous gamma-irradiated (30 Gy) stimulator PBMC and 1 x 10⁵ autologous responder PBMC and 5 x 10⁴ expanded regulators or primed control CD4⁺ T cells were cocultured in 96-well round-bottom plates in the presence of 10 µg/ml Candida albicans extract (ARTHU Biologicals, Lelystad, The Netherlands) or tetanus toxoid (RIVM, Bilthoven, The Netherlands). Proliferation was measured at the indicated time points. In some experiments, gamma-irradiated (30 Gy) original stimulator cells (i.e., used to generate the anergic regulatory T cells) or MHC-II-mismatched allogeneic stimulator cells were added (at the indicated ratio) to the autologous stimulator fraction. The proliferative response of quadruplicate samples was analyzed by ³H incorporation.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Increased reversal of the hyporesponsive state of anergic CD4⁺ T cells by exogenously added IL-15

In isolated allogeneic CD4⁺ T cells, anergy was induced in primary MLC by the addition of two antagonistic mAbs directed against the costimulatory ligands CD40 or CD86. The presence of these antagonistic mAbs resulted in inhibition of the primary MLC (Fig. 1A).

View larger version (22K): [in this window] [in a new window]   FIGURE 1. Increased reversal of the hyporesponsive state of anergic CD4+ T cells by exogenously added IL-15. A, Primary MLC were performed with isolated CD4+ responder T cells (5 x 104) and irradiated allogeneic PBMC (1 x 105) as stimulator cells in the presence of two antagonistic mAbs directed against CD40 and CD86, respectively. Cell division according to 3H incorporation (y-axis) is shown at days 5 and 6 of the culture. As a control, the response to autologous gamma-irradiated PBMC and the response of allogeneic stimulator PBMC only are shown. B, At day 7 of the primary MLC, the cells were collected, washed, and rested for 2–3 days. Next, the dead cells were removed by density gradient centrifugation. The recovered viable CD4+ T cells (2 x 104) that were derived from primary mAb-treated MLC or control primary MLC were antigenically restimulated by gamma-irradiated, fresh original allogeneic PBMC (1 x 105) in the absence or presence of exogenously added IL-2 (12.5 U/ml) or IL-15 (10 ng/ml). Note scale differences. C, The recovered viable CD4+ T cells (2 x 104) that were derived from primary mAb-treated MLC or control primary MLC were stimulated by exogenously added IL-2 (12.5 U/ml) or IL-15 (10 ng/ml) only, in the absence of stimulator PBMC. Note scale differences. Proliferation (3H incorporation; y-axis) is shown during the course of restimulation (days; x-axis). Figures show representative results of at least four individual experiments. Results are shown as mean ± SD of quadruplicate measurements.

IL-15 results in increased expansion and elevated Bcl-2 expression in anergic CD4⁺ T cells

To better understand the increased responsiveness following allogeneic restimulation with exogenous IL-15 in terms of cell death and cell survival, cell cycle levels were studied by DNA content analysis (Fig. 2A). Compared with IL-2, addition of exogenous IL-15 during allogeneic restimulation of anergic CD4⁺ T cells resulted in enhanced survival, because the numbers of dividing cells in S-G₂-M phase were increased (19 vs 28%), whereas the numbers of cells in the hypoploid (apoptotic) phase of the cell cycle were reduced (17 vs 4%; Fig. 2A). Antiapoptotic molecules such as Bcl-2 are known to promote cell survival (41, 42). To determine whether Bcl-2 might play a role in the increased survival of anergic CD4⁺ T cells during allogeneic restimulation in the presence of exogenous IL-15 or IL-2, the presence of intracellullar Bcl-2 was analyzed by flow cytometry. Exogenous IL-15, as compared with IL-2, resulted in a higher number of anergic CD4⁺ T cells expressing Bcl-2 (48.1 vs 12.4%; Fig. 2B). Similar observations were made for allogeneically primed control CD4⁺ T cells; therefore, IL-15 does not selectively or specifically act on anergic cells.

View larger version (40K): [in this window] [in a new window]   FIGURE 2. IL-15 results in enhanced survival of anergic CD4+ T cells that is paralleled by an increase in Bcl-2. A, IL-15 results in an increased survival of anergic CD4+ T cells. Cell cycle progression of anergic (and control) CD4+ T cells was studied by flow-cytometric analysis of the DNA content according to TOPRO-3 labeling. Anergic and control CD4+ T cells were derived from primary MLC that were performed in the presence (or absence) of anti-CD40 plus anti-CD86 antagonistic mAbs. These cells were allogeneically restimulated (secondary MLC) in the presence of IL-2 or IL-15. At day 5 of the cultures, the cells were harvested, surface stained, and analyzed for DNA content. The histograms show the DNA content of anergic and control CD4+ T cells in the presence of either IL-2 or IL-15 (conditions are indicated at the top). Numbers indicate the percentage of CD4+ cells in <G1 (left), G1 (middle), and S/G2/M (right) cell cycle phase, representing the dead cells, resting cells, and dividing cells, respectively. B, IL-15 results in an increased expression of Bcl-2 in anergic CD4+ T cells. The effect of exogenously added IL-2 or IL-15 during allogeneic restimulation on the presence of Bcl-2 in CD4+ anergic (and control) T cells was analyzed by flow cytometry. Dot plots (Bcl-2 expression on the x-axis; forward scatter on the y-axis) show the relative number (percentage) of CD4+ T cells expressing intracytoplasmic Bcl-2 at day 5 of cultures. The conditions during the cultures are indicated. One representative experiment is shown (n = 5). C, Compared with IL-2, IL-15 results in an increased expansion. CD4+ T cells were counted at start of secondary cultures and after 5 days of culture in the presence of stimulator cells and the indicated cytokines. The cellular expansion of six individual experiments was calculated. Mean ± SD of the six experiments are included.

Thus, IL-15 results in increased survival of anergic CD4⁺ T cells, which is paralleled by an increased number of cells expressing Bcl-2.

Both IL-15- and IL-2-expanded anergic CD4⁺ T cells retain their anergic state, but IL-15 results in advanced growth and a distinct type of anergy

Previously, we reported that hyporesponsive anergic T cells that were recovered by allogeneic restimulation in the presence of exogenously added IL-2 retained their anergic state (10). This finding was again observed for isolated CD4⁺ T cells in our current study (Fig. 3A). Now, we addressed the question of whether this effect was also observed when IL-15 was used during allogeneic restimulation of anergic CD4⁺ T cells. To this end, in the first expansion cycle, anergic CD4⁺ T cells or control primed CD4⁺ T cells were allogeneically restimulated in the presence of exogenously added IL-2 or IL-15. The viable recovered CD4⁺ T cells were allogeneically restimulated in a tertiary MLC in the absence or presence of exogenous IL-2 or IL-15. Fig. 3A shows that both IL-2- and IL-15-expanded allogeneic anergic T cells retained their anergic state, i.e., compared with expanded allogeneically primed control CD4⁺ T cells, the cells were hyporesponsive, whereas addition of exogenous IL-2 or IL-15 reversed their hyporesponsive state. Notably, anergic CD4⁺ T cells that were first expanded in the presence of IL-15 showed an increased proliferative response (hyperreactivity) and expansion upon allogeneic restimulation and exogenously added IL-15 as compared with the use of IL-2 (Fig. 3, B and C). In contrast, allogeneic restimulation in the presence of IL-2 in the first expansion phase prevented the IL-15-mediated hyperreactivity during the second expansion phase (Fig. 3, B and C). Thus, during the first expansion cycle, the presence of IL-15 is essential to ensure hyperreactivity upon re-encounter with alloantigen and IL-15.

View larger version (40K): [in this window] [in a new window]   FIGURE 3. Both IL-2- and IL-15-expanded anergic CD4+ T cells retain their anergic state, but IL-15 results in advanced growth and a distinct type of anergy. Anergic or primed control CD4+ T cells were expanded by allogeneic restimulation, in a secondary MLC, in the presence of exogenously added IL-2 or IL-15 (Ag + IL-2 expanded (exp.) or Ag + IL-15 exp. cells, respectively). At day 5 of the culture, the cells were collected and allowed to rest for 2 days. A, The recovered viable CD4+ T cells (2 x 104) were restimulated with gamma-irradiated allogeneic stimulator PBMC (1 x 105), in a so-called tertiary MLC, in the absence or presence of exogenously added IL-2 or IL-15. Conditions during restimulation are given on top of the figures. B, Repeated allogeneic stimulation in the presence of IL-15 results in increased expansion. Anergic T cells were derived from a secondary MLC, which was performed in the presence of either IL-2 or IL-15 (at the right in the first column). These cells were allogeneically restimulated in a tertiary MLC, again in the presence of either IL-2 or IL-15 (at the right in the second column). The tertiary proliferative response (3H incorporation) is shown at day 3 of the cultures. Means ± SD of one representative experiment are shown (n = 3). C, The number of viable cells was microscopically determined at days 5 and 6 of the cultures (x-axis). Anergic cells were first expanded by stimulation with alloantigen and either IL-15 or IL-2 and restimulated by Ag only or Ag and IL-2 or IL-15 (legends). Cell numbers were counted in triplicate; SD were <10%. D, IL-15 and IL-2 receptor -chain expression on Ag and IL-15- or IL-2-expanded anergic T cells. Anergic T cells expanded with Ag and either IL-15 or IL-2 were derived from secondary MLC. These cells were rested and allogeneically restimulated (tertiary MLC). Surface expression of the IL-15 and IL-2 receptor -chain (x-axis) at the start (dashed histogram) and 48 h after stimulation (gray-shaded histogram) was analyzed by flow cytometry. Number of events, scaled to 100% of the peak value, is shown on the y-axis. The percentage of positive cells and mean fluorescence intensity of the activated cells are shown in the upper and lower right corner of each histogram, respectively. CD25 (IL-2R chain), CD122 (IL-15/IL-2R chain), and CD132 (IL-15/IL-2R chain). One of two similar experiments is shown.

The improved expansion of IL-15-expanded cells when stimulated by alloantigen and IL-15 is likely the result of the abovementioned increase in IL-15 and IL-2 receptor expression, especially the IL-15R chain.

Together, these data indicate that repeated allogeneic restimulation in the presence of exogenous IL-15 results in advanced growth of anergic CD4⁺ T cells and a functionally distinct anergic state.

Superior suppression of naive and memory T cell division by IL-15-expanded anergic CD4⁺ T cells

Next, we addressed the question of whether IL-15- or IL-2-expanded anergic CD4⁺ T cells were indeed able to suppress naive and memory T cell responses. This was studied in coculture experiments, whereby IL-2- or IL-15-expanded anergic or primed control CD4⁺ T cells were added to cultures consisting of fresh allogeneic stimulator gamma-irradiated (30 Gy) PBMC and either fresh naive or memory responder T cells, respectively. Both stimulator and responder PBMC were derived from the donor cell pool that was originally used to generate the anergic or control primed CD4⁺ T cells. The responder PBMC were labeled with CFSE, and cell division of CD4⁺ and CD8⁺ T cells was analyzed by flow cytometry. Both IL-15- and IL-2-expanded anergic CD4⁺ T cells suppressed the division of naive CD4⁺ T cells (naive CD8⁺ T cells hardly divide in primary MLC) (Fig. 4A). Especially, IL-15-expanded anergic T cells were powerful suppressors of naive T cell proliferation (Fig. 4A; 20 vs 31% dividing cells in the presence of IL-15- or IL-2-expanded anergic cells, respectively). This phenomenon was also observed at effector cell:regulatory cell ratios of 1:0.5 and 1:0.25 (data not shown). Despite the suppression of T cell division, activation of the responder population was evident as blast formation (increase in forward scatter) and concomitant CD25 expression took place (Fig. 4A). This indicates that expanded anergic regulatory CD4⁺ T cells did not mediate suppression by killing the responder population. Importantly, immunosuppression by IL-2- or IL-15-expanded anergic CD4⁺ T cells appeared dose dependent (Fig. 4C) and gamma-irradiation resistant (D). The latter suggests that suppression is not the result of copious IL-2 consumption by the regulatory T cells. IL-15-expanded anergic CD4⁺ T cells, as compared with their IL-2-expanded counterpart, were also better suppressors of CD4⁺ and CD8⁺ memory T cell division (Fig. 4B; 44 vs 64% dividing CD4⁺ memory T cells, 71 vs 85% dividing CD8⁺ memory T cells in the presence of IL-15- or IL-2-expanded anergic cells, respectively). Generally, memory T cells were suppressed to a lesser degree than naive T cells. Thus, although both IL-2- and IL-15-expanded anergic CD4⁺ T cells are immunosuppressive, IL-15-expanded regulatory CD4⁺ T cells clearly are more potent suppressors.

View larger version (77K): [in this window] [in a new window]   FIGURE 4. Superior suppression of naive and memory T cell division by IL-15-expanded anergic regulatory CD4+ T cells. IL-2- or IL-15-expanded anergic or primed control CD4+ T cells were added to a primary MLC (A) or a secondary MLC (B) to study their immunosuppressive capacity toward naive and memory T cells, respectively. The responder cells used in both the primary and secondary MLC were labeled with the fluorescent dye CFSE. Cell division of anti-CD4-, anti-CD8-, and anti-CD25-labeled responder cells was measured by flow cytometry. A, A total of 5 x 104 naive CFSE-labeled responder PBMC were stimulated with 5 x 104 gamma-irradiated (30 Gy) stimulator PBMC (primary MLC) in the presence of 5 x 104 IL-2- or IL-15-expanded anergic CD4+ T cells or primed control CD4+ T cells. The dot plots show forward scatter on y-axis and CFSE intensity on the x-axis at day 5 of the culture. The activated CD4+ dividing cells were gated and analyzed for CFSE intensity and CD25 expression as shown in the histograms below. Percentages shown in the histograms indicate the relative number of dividing cells and CD25-expressing cells, respectively. The gray cells at the left of the dot plots are the non-CFSE-labeled cells. B, A total of 2 x 104 previously allogeneically primed T cells were CFSE labeled and stimulated with 5 x 104 allospecific gamma-irradiated (30 Gy) stimulator PBMC (secondary MLC) in the presence of 2 x 104 IL-2- or IL-15-expanded anergic CD4+ T cells or primed control CD4+ T cells. The dot plots show forward scatter on y-axis and CFSE intensity on the x-axis at day 2 of the cultures. The dividing cells were gated and analyzed for CFSE intensity in anti-CD4- and anti-CD8-stained T cell subset (shown in the histograms below). Percentages indicate the relative number of dividing CD4+ and CD8+ T cells. The gray-colored cells at the left of the dot plots are the non-CFSE-labeled cells. C, Suppression by alloantigen and IL-2- or IL-15-expanded regulatory CD4+ T cells is dose dependent. Proliferation (y-axis; 3H incorporation) of naive T cells (5 x 104 PBMC) was studied in primary MLC in the absence or presence of increasing numbers of regulatory T cells (x-axis). D, Immunosuppression of naive T cells by the expanded anergic regulatory CD4+ T cells is gamma-radiation resistant. Cocultures of primary MLC and either live or gamma-irradiated (30 Gy), expanded (IL-2 or IL-15) anergic regulatory cells or primed control cells were performed. The percentage of dividing cells in the presence of anergic or primed control cells was analyzed according to CFSE dilution. From these percentages, the relative suppression (x-axis) per condition (y-axis) was calculated. Data for live cells are derived from the dot plots mentioned under A (data for irradiated cells are not shown). One representative experiment is shown (n = 3).

Anergic regulatory CD4⁺ T cells and control primed CD4⁺ T cells were expanded by allospecific restimulation in the presence of exogenous IL-15. At day 6, these expanded cells were harvested, washed, and allowed to rest for 48 h. The recovered viable T cells (>95% CD4⁺) were analyzed by flow cytometry for surface expression of CD25 (IL-2 receptor chain), CD122 (IL-2/IL-15 receptor chain), the differentiation markers CD45RO, CD45RA, and CD27, and intracellular expression of CTLA-4. The majority of expanded anergic regulatory CD4⁺ T cells expressed CD45RO (90%); part of these cells expressed CD27 (50%), CTLA-4 (30%), CD25 (30%), and CD122 (19%); and a minority expressed CD45RA (10%) (Fig. 5A). Upon comparison of expanded anergic CD4⁺ T cells vs primed control CD4⁺ cells, the latter showed an increased percentage of CD25⁺, CD45RO⁺, and CTLA-4⁺ cells, similar numbers of CD122⁺ cells, and a reduced number of CD27⁺ and CD45RA⁺ cells (Fig. 5). The use of IL-2 or IL-15 resulted in a similar phenotype (data not shown).

View larger version (45K): [in this window] [in a new window]   FIGURE 5. Phenotype and intracellular cytokine staining pattern of IL-15-expanded regulatory CD4+ cells. Anergic regulatory CD4+ T cells and primed control CD4+ T cells were allogeneically restimulated in a secondary MLC in the presence of IL-15. After 5 days, the cells were collected and rested for 2 days. The viable CD4+ T cells (>95% CD4+ T cells) were analyzed for surface expression (A) and intracellular cytokine staining (B) by flow cytometry. A, Histograms show surface marker expression of the indicated CD marker and intracytoplasmatic staining of CTLA-4. Fluorescence intensity and relative number of events are shown on the x- and y-axes, respectively. The percentage of positive cells are indicated at the right upper corner of each histogram. B, Dot plots show intracellular cytokine fluorescence intensity (x-axis) and forward scatter (y-axis) after activation. The percentage of positive cells and mean fluorescence intensity are indicated at the lower left corner. Fluorochrome-conjugated isotype mAbs were used for marker settings. One representative experiment of four is shown.

Immunosuppression by IL-15-expanded anergic CD4⁺ T cells requires Ag-specific TCR triggering and is independent of IL-10, TGF, or CTLA-4

Immunosuppressive therapy should ideally be Ag specific. We hypothesized (in line with Waldmann and Cobbold (19)) that recognition of the cognate MHC-Ag on the APC by the suppressive immunoregulatory CD4⁺ T cells as generated in our model system is a prerequisite to activate the suppressor function of the regulatory T cells. In this scenario, omission of the cognate allo-MHC-II (i.e., fully MHC-mismatched APC) precludes activation of regulatory anergic CD4⁺ T cells. Fig. 6A shows that addition of IL-15-expanded anergic regulatory CD4⁺ T cells suppressed the proliferative response of naive alloreactive cells only in the presence of fully (83% suppression) or partially HLA-DR-matched (59% suppression) stimulator APC (i.e., cognate Ag), but not in the case of MHC-II-mismatched stimulators (8% suppression). To further characterize the mechanism of suppression by IL-15-expanded anergic regulatory CD4⁺ T cells, the role of formerly postulated immunosuppressive mechanisms (i.e., CTLA-4 or the cytokines TGF and IL-10) was examined. Previously, we have demonstrated that regulatory cells that were generated by costimulation blockade did not produce detectable levels of IL-10 or TGF as measured by ELISA (10). In this study, we show that IL-15-expanded anergic regulatory CD4⁺ T cells did not mediate suppression via either CTLA-4, TGF, or IL-10, because neither blockade of CTLA-4 nor neutralization of TGF or IL-10 by mAbs reversed suppression (Fig. 6B).

View larger version (23K): [in this window] [in a new window]   FIGURE 6. Suppression by IL-15-expanded, de novo-induced anergic regulatory CD4+ T cells requires Ag-specific stimulation and is independent of IL-10, TGF, or interactions via CTLA-4. A, IL-15-expanded, de novo-induced regulatory CD4+ T cells require specific Ag to become suppressive. A total of 5 x 104 gamma-irradiated, IL-15-expanded anergic regulatory CD4+ T cells or IL-15-expanded primed control CD4+ cells were cocultured with 5 x 104 autologous responder PBMC and 5 x 104 gamma-irradiated (30 Gy) stimulator PBMC. The stimulator PBMC were either allospecific (from the source originally used to generate the regulatory cells), partially matched (isolated HLA class-II match), or completely HLA class-II mismatched with respect to the allospecific stimulator PBMC used. Proliferation at day 5 of the culture was examined by 3H incorporation (x-axis). B, IL-15-expanded regulatory CD4+ T cells or primed control CD4+ cells were gamma-irradiated and cocultured in a primary MLC (consisting of fresh autologous responder PBMC and stimulator PBMC, from the source originally used to generate the regulatory CD4+ T cells) in the presence of neutralizing mAb against TGF (20 µg/ml) and IL-10 (20 µg/ml), and anti-CTLA-4 mAb (20 µg/ml). Abs were titrated up to 75 µg/ml showing identical results (data not shown). 3H incorporation (mean ± SD) is shown at day 6 of the cultures. One representative experiment of three is shown.

IL-15-expanded regulatory CD4⁺ T cells are stable suppressors that mediate bystander suppression upon TCR stimulation but do not affect recall responses in the absence of cognate Ag

Next, we studied whether IL-15-expanded, alloantigen-specific regulatory CD4⁺ T cells suppress T cells specific for other Ags. Allospecific IL-15-expanded autologous regulatory CD4⁺ T cells were added to autologous PBMC in the presence of soluble recall Ags (C. albicans or tetanus toxoid). Recall responses against C. albicans or tetanus toxoid were not suppressed by these regulatory CD4⁺ T cells (Fig. 7A). Notably, addition of 20% or more allospecific stimulator APC (carrying cognate alloantigen-Ag) to the above cocultures restored the immunosuppressive function of the regulatory cells (55% suppression). In contrast, addition of HLA class II-mismatched APC, instead of the original stimulator cells, did not restore the suppressor function of the regulatory T cells (Fig. 7B). This again confirms the need for cognate Ag to activate suppression by these IL-15-expanded regulatory cells, as was also shown in Fig. 6A. In addition, these results indicate that TCR stimulation of IL-15-expanded regulatory T cells likely results in bystander suppression, leading to down-regulation of proliferation by Candida-specific T cells.

View larger version (27K): [in this window] [in a new window]   FIGURE 7. IL-15-expanded regulatory CD4+ T cells are stable suppressors mediating bystander suppression upon TCR stimulation but do not affect recall responses in the absence of cognate Ag. A, Self MHC-restricted recall responses against C. albicans and tetanus toxoid were conducted by culturing 1 x 105 gamma-irradiated (30 Gy) autologous stimulator and 1 x 105 live autologous responder PBMC in the presence of C. albicans extract or tetanus toxoid (10 µg/ml). To these cultures, either no cells (autologous cells only) or 5 x 104 gamma-irradiated (30 Gy) autologous IL-15-expanded anergic regulatory cells or primed control cells were added (see legend). B, Autologous PBMC were cultured without (no Ag) or with C. albicans, in the presence of IL-15-expanded regulatory T cells and increasing numbers of gamma-irradiated (30 Gy) cognate allogeneic stimulator PBMC (i.e., from the source originally used to generate the anergic regulatory T cells; left) or MHC-II mismatched allogeneic stimulator PBMC (right) were added. The relative number of added allogeneic APC is shown on the x-axis. Proliferation (mean ± SD) is shown on the y-axis at day 5 of the cultures. The legend indicates the culture conditions. Similar results were obtained when tetanus toxoid was used as a source of soluble Ag (data not shown).

Naturally occurring CD4⁺CD25⁺ T cells are necessary for the generation of anergic regulatory CD4⁺ T cells by costimulation blockade

Recently, Blazar and coworkers (43) showed the importance of naturally occurring CD4⁺CD25⁺ T cells in the generation of regulatory T cells via costimulation blockade in mice. Similarly, we analyzed the role of human naturally occurring CD4⁺CD25⁺ T cells. Isolated CD4⁺ T cells were depleted from naturally occurring CD4⁺CD25⁺ T cells (Fig. 8A) and allogeneically stimulated in the absence or presence of mAbs directed against CD40 and CD86 (B). Depletion of CD4⁺CD25⁺ T cells did not affect the inhibitory potential of the mAbs (Fig. 8B). Conversely, the anergic state (Fig. 8C) and suppressive potential (D) were abrogated when CD4⁺CD25⁺ were depleted during costimulation blockade.

View larger version (32K): [in this window] [in a new window]   FIGURE 8. Naturally occurring CD4+CD25+ T cells are necessary for the generation of anergic regulatory CD4+ T cells by costimulation blockade of CD40 and CD86. A, Isolated CD4+ T cells (CD4 Total; upper dot plot) were depleted from CD4+CD25+ naturally occurring T cells (CD4 CD25-; lower dot plot) by magnetic microbeads. CD25 (m-A251) and CD4 fluorescence are shown on the x- and y-axes, respectively. B, Primary MLC using either CD4 Total or CD4 CD25- as responder cells were performed in the absence or presence of anti-CD40 and anti-CD86 mAb. Proliferation (3H incorporation; x-axis) is shown at day 6 of the primary MLC. C, T cells derived from primary MLC, as performed in B, were allogeneically restimulated in the absence (upper two graphs) or presence of exogenously added IL-2 (lower two graphs). Proliferation of CD4 Total (left) and CD4 CD25- (right) are shown in time (x-axis) (n = 3). D, Anti-CD40 and anti-CD86 mAb-blocked CD4 Total or CD4 CD25- cells (5 x 104) derived from primary MLC were examined for their suppressive potential in MLC cocultures consisting of 5 x 104 naive responder PBMC and 5 x 104 stimulator PBMC. Relative suppression (y-axis) was calculated as mentioned in Materials and Methods. Two independent suppression assays are shown (x-axis).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Both IL-2 and IL-15 stimulate proliferation of activated CD4⁺ and CD8⁺ T cells (50, 51), but differ with respect to T cell survival. IL-2 plays an important role in T cell death (33, 52), whereas IL-15 is generally considered as an inhibitor of apoptosis (30, 31). This inhibition is at least partially mediated via up-regulation of the antiapoptotic protein Bcl-2 (Ref. 53 and this report). In addition, in murine models, IL-15, but not IL-2, supported survival of activated CD4⁺ T cells (29), selectively propagated growth of CD8⁺ memory T cells (34, 35, 36), and redirected T cell apoptosis toward anergy in partially stimulated T cells (37). Similarly, human CD4⁺ memory effector T cells and T regulatory type 1 (Tr1) cell clones especially proliferated in response to IL-15 (39, 54). Especially the latter study (39) and our current study underline the importance of IL-15 in the maintenance of regulatory CD4⁺ T cells (38). However, it should be noted that it is not likely that IL-15 plays a prominent role in the induction of regulatory cells (39) such as has been recently described for IL-10 plus IFN- (55) and TGF (56).

Notably, allogeneic restimulation of anergic regulatory CD4⁺ T cells in the presence of either exogenously added IL-2 or IL-15 resulted in a distinct type of T cell anergy. The first expansion cycle with alloantigen and IL-15 resulted in anergic cells that are hyperresponsive to subsequent allogeneic restimulation and exogenously added IL-15. In contrast, the use of alloantigen and IL-2 in the first expansion phase prevented this IL-15-induced hyperreactivity. Thus, optimal expansion of anergic regulatory CD4⁺ T cells by exogenous IL-15 required the presence of this cytokine from the start of the expansion phase.

Expansion of human de novo-induced regulatory CD4⁺ T cells by either IL-15 or IL-2 required alloantigen-specific TCR stimulation, because hardly any proliferation occurred to IL-15 or IL-2 alone. This latter finding is in contrast with a recent observation showing that expansion of human Tr1 cell clones is independent of TCR signaling and requires exogenous cytokines only (39). However, the need for Ag in cytokine-mediated expansion of naturally occurring CD4⁺CD25⁺ regulatory T cells was also shown in two murine studies (47, 48). The necessity of donor Ag was stressed by the finding that only Ag-specific stimulation of the regulatory T cells resulted in Ag-specific immunosuppressive T cells, whereas polyclonal stimulation did not (48).

In the current study, allospecific anergic T cells were generated by stimulating human peripheral CD4⁺ T cells with MHC-mismatched stimulator cells (primary MLC) in the presence of mAb directed against CD40 and CD86. Within the human peripheral T cell pool, only a fraction of cells (5–10%) will respond to a particular allogeneic mismatch. In our experiments, it is these particular alloreactive T cells that are made anergic by costimulation blockade, whereas T cells with other specificities will be unaffected and remain present or die and disappear. This results in a polyclonal population of anergic allospecific and nonanergic T cells with other specificity. The presence of the latter was shown by stimulating this polyclonal population with a third-party alloantigen, which resulted in a primary proliferative response (data not shown). This strongly indicates that, next to allospecific anergic T cells, nonanergized T cells with distinct Ag specificity are present. In the experiments that we describe in this report, the anergic T cell population is Ag-specifically restimulated in the presence of IL-15 (or IL-2), which specifically results in growth of the anergic allospecific T cells. This will enrich the allospecific anergic T cells, at the expense of T cells with other specificity, which still might be present.

An important feature of suppressor regulatory T cells is their ability to inhibit the proliferation of conventional responder T cell populations in vitro. In our experiments, IL-15- (or IL-2)-expanded, de novo-induced immunoregulatory suppressor T cells inhibited proliferation of responder T cells, but in this responder population, blast formation and concomitant expression of CD25 still occurred. In a similar way, naturally occurring regulatory CD4⁺CD25⁺ T cells were shown to induce cell cycle arrest in conventional responder T cells, but initial T cell activation was not affected, because early activation Ags were up-regulated (22). Taken together, this indicates that suppression is not mediated by elimination of conventional responder T cells, but by controlling the proliferative response of this population in an active way.

Immunosuppression by expanded suppressor CD4⁺ T cells, such as generated in our model, required alloantigen-specific TCR triggering. This ensures that suppression takes place only when cognate Ag is present, and moreover, it prevents random suppression by the regulatory cells. It is likely that, for this reason, regulatory T cells did not affect autologous recall T cell responses in the absence of specific alloantigen. However, the presence of allospecific APC in autologous recall cocultures recovered the suppressor activity by the regulatory T cells. This means that either the allogeneic response or the autologous recall response was suppressed; in the latter case, we cannot exclude that bystander suppression took place. Be that as it may, using <20% allogeneic APC, we did not observe suppression. Given that regulatory T cells preferentially reside in the tolerated graft (57) and the unlikelihood that each lymphoid organ contains >20% allogeneic cells, we speculate that generalized nonspecific immunosuppression by regulatory cells, such as generated in our model system, is improbable upon in vivo use.

Some studies indicated an important role for soluble molecules such as IL-10 and TGF (9, 14, 15, 16) or interactions via CTLA-4 (15, 17, 18) as the mechanism of immunosuppression. Previously, we demonstrated in our protocol that de novo-generated anergic regulatory T cells did not produce detectable amounts of IL-10 or TGF as measured by ELISA (10). In this study, immunosuppression by IL-15-expanded, de novo-induced regulatory T cells appeared not to be mediated via TGF, IL-10, or CTLA-4, because neutralizing mAb did not reverse suppression. Also, immunosuppression by IL-15- (as well as IL-2)-expanded, de novo-induced regulatory CD4⁺ T cells appeared gamma-irradiation resistant. This indicates that proliferation and concordant IL-2 consumption by these regulatory T cells is not likely to be the mechanism of suppression. We are aware that irradiated T cells might bind IL-2 and other cytokines and thereby deplete them from the cell cultures at the expense of alloreactive T cell responsiveness. However, in this study, we showed that Ag-specific, activated, IL-15-expanded regulatory CD4⁺ T cells remained suppressive when simultaneously productive autologous recall T cell responses (i.e., cytokine production of IL-2, IFN-, and TNF- with concomitant proliferation) were taking place. Together, this indicates that suppression by these regulatory cells does not merely depend on cytokine deprivation from the culture medium. Recently, it has been claimed that suppression by human naturally occurring CD4⁺CD25⁺ regulatory T cells was (partially) reversed by exogenous addition of T cell growth factors (5, 7, 11). However, addition of growth factors might inadvertently result in proliferation of the naturally occurring CD4⁺CD25⁺ T cells and thus not reflect abrogation of suppression per se.

The IL-15-expanded, costimulation blockade-induced human regulatory CD4⁺ T cells, such as described in this study, are anergic and suppressive upon cognate TCR triggering, and they do not appear to mediate their suppressor function via the typical Th3- or Tr1-associated cytokines TGF or IL-10. Among human de novo-induced CD4⁺ regulatory T cells, the Tr1 and Th3 cells are the best known. Tr1 cells were generated in vitro by culturing CD4⁺ T cells in the presence of Ag and IL-10; these Tr1 cells predominantly secrete IL-10, have low proliferative potential, and suppress Ag-specific T cell responses (14). Th3 cells are CD4⁺ T cells that are characteristically generated following oral administration of Ags (58), and especially produce TGF with various amounts of IL-4 and IL-10 and suppress Ag-specific T cell proliferation (9, 58). Whether Th3 cells are unresponsive to TCR triggering is unknown (16). The CD4⁺ regulatory cells, such as described in our current study, are not related to Th3 or Tr1 cells, and appear to be another subset of regulatory cells.

Mouse CD25⁺CD4⁺ naturally occurring immunoregulatory T cells facilitate the induction of T cell anergy (59) and are necessary to induce a tolerant state via costimulatory blockade in mice (43). Also, human naturally occurring CD4⁺CD25⁺ regulatory T cells contribute to de novo generation of suppressor T cells (60), according to the process of infectious tolerance (61, 62). In our human experimental model, depletion of naturally occurring CD4⁺CD25⁺ T cells before the induction of regulatory anergic T cells by costimulation blockade, prevented the generation of de novo-induced suppressor CD4⁺ T cells. Thus, the presence of naturally CD4⁺CD25⁺ T cells (usually 4–10% of the CD4⁺ T cells) appears to be a prerequisite in de novo induction of human suppressive CD4⁺ regulatory T cells by costimulation blockade in the primary MLC. Apparently, these naturally occurring CD4⁺CD25⁺ cells facilitate a proper environment during costimulation blockade, resulting in de novo anergic regulatory CD4⁺ T cells.

The property to revert to an anergic or suppressive condition once the hyporesponsive state is abrogated (e.g., by IL-15 or IL-2) is a shared feature of both de novo-induced regulatory T cells (Ref. 10 and this report) and naturally occurring CD4⁺CD25⁺ T cells (23). Especially IL-15 appeared of particular interest for large-scale ex vivo generation of donor-specific regulatory T cells to be used as a clinical immunosuppressive tool in humans.

	Acknowledgments

 
We are indebted to A. Pennings and G. Vierwinden from the Hematology Department for expert assistance with cell cycle analysis by flow cytometry. Furthermore, we kindly acknowledge J. Coenen for critical review of this manuscript.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. Hans Koenen, Department for Blood Transfusion and Transplantation Immunology (ABTI/OV603), University Medical Center, Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail address: H.Koenen{at}abti.umcn.nl

² Abbreviations used in this paper: PC5, PE-CY5; Tr1, T regulatory type 1.

Received for publication March 24, 2003. Accepted for publication October 10, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Maloy, K. J., F. Powrie. 2001. Regulatory T cells in the control of immune pathology. Nat. Immunol. 2:816.[Medline]
2. Shevach, E. M.. 2002. CD4⁺CD25⁺ suppressor T cells: more questions than answers. Nat. Rev. Immunol. 2:389.[Medline]
3. Sakaguchi, S., N. Sakaguchi, M. Asano, M. Itoh, M. Toda. 1995. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor -chains (CD25): breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155:1151.[Abstract]
4. Levings, M. K., R. Sangregorio, M. G. Roncarolo. 2001. Human CD25⁺CD4⁺ T regulatory cells suppress naive and memory T cell proliferation and can be expanded in vitro without loss of function. J. Exp. Med. 193:1295.[Abstract/Free Full Text]
5. Dieckmann, D., H. Plottner, S. Berchtold, T. Berger, G. Schuler. 2001. Ex vivo isolation and characterization of CD4⁺CD25⁺ T cells with regulatory properties from human blood. J. Exp. Med. 193:1303.[Abstract/Free Full Text]
6. Jonuleit, H., E. Schmitt, M. Stassen, A. Tuettenberg, J. Knop, A. H. Enk. 2001. Identification and functional characterization of human CD4⁺CD25⁺ T cells with regulatory properties isolated from peripheral blood. J. Exp. Med. 193:1285.[Abstract/Free Full Text]
7. Ng, W. F., P. J. Duggan, F. Ponchel, G. Matarese, G. Lombardi, A. D. Edwards, J. D. Isaacs, R. I. Lechler. 2001. Human CD4⁺CD25⁺ cells: a naturally occurring population of regulatory T cells. Blood 98:2736.[Abstract/Free Full Text]
8. Groux, H., M. Bigler, J. E. de Vries, M. G. Roncarolo. 1996. Interleukin-10 induces a long-term antigen-specific anergic state in human CD4⁺ T cells. J. Exp. Med. 184:19.[Abstract/Free Full Text]
9. Chen, Y., V. K. Kuchroo, J. Inobe, D. A. Hafler, H. L. Weiner. 1994. Regulatory T cell clones induced by oral tolerance: suppression of autoimmune encephalomyelitis. Science 265:1237.[Abstract/Free Full Text]
10. Koenen, H. J., I. Joosten. 2000. Blockade of CD86 and CD40 induces alloantigen-specific immunoregulatory T cells that remain anergic even after reversal of hyporesponsiveness. Blood 95:3153.[Abstract/Free Full Text]
11. Jonuleit, H., E. Schmitt, G. Schuler, J. Knop, A. H. Enk. 2000. Induction of interleukin 10-producing, nonproliferating CD4⁺ T cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells. J. Exp. Med. 192:1213.[Abstract/Free Full Text]
12. Jonuleit, H., E. Schmitt, K. Steinbrink, A. H. Enk. 2001. Dendritic cells as a tool to induce anergic and regulatory T cells. Trends Immunol. 22:394.[Medline]
13. Karim, M., A. Bushell, K. Wood. 2002. Regulatory T cells in transplantation. Curr. Opin. Immunol. 14:584.[Medline]
14. Groux, H., A. O’Garra, M. Bigler, M. Rouleau, S. Antonenko, J. E. de Vries, M. G. Roncarolo. 1997. A CD4⁺ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 389:737.[Medline]
15. Kingsley, C. I., M. Karim, A. R. Bushell, K. J. Wood. 2002. CD25⁺CD4⁺ regulatory T cells prevent graft rejection: CTLA-4- and IL-10-dependent immunoregulation of alloresponses. J. Immunol. 168:1080.[Abstract/Free Full Text]
16. Roncarolo, M. G., R. Bacchetta, C. Bordignon, S. Narula, M. K. Levings. 2001. Type 1 T regulatory cells. Immunol. Rev. 182:68.[Medline]
17. Read, S., V. Malmstrom, F. Powrie. 2000. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25⁺CD4⁺ regulatory cells that control intestinal inflammation. J. Exp. Med. 192:295.[Abstract/Free Full Text]
18. Takahashi, T., T. Tagami, S. Yamazaki, T. Uede, J. Shimizu, N. Sakaguchi, T. W. Mak, S. Sakaguchi. 2000. Immunologic self-tolerance maintained by CD25⁺CD4⁺ regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J. Exp. Med. 192:303.[Abstract/Free Full Text]
19. Waldmann, H., S. Cobbold. 2001. Regulating the immune response to transplants: a role for CD4⁺ regulatory cells?. Immunity 14:399.[Medline]
20. Adler, S., L. Turka. 2002. Immunotherapy as a means to induce transplantation tolerance. Curr. Opin. Immunol. 14:660.[Medline]
21. Sakaguchi, S.. 2000. Regulatory T cells: key controllers of immunologic self-tolerance. Cell 101:455.[Medline]
22. Thornton, A. M., E. M. Shevach. 2000. Suppressor effector function of CD4⁺CD25⁺ immunoregulatory T cells is antigen nonspecific. J. Immunol. 164:183.[Abstract/Free Full Text]
23. Sakaguchi, S., N. Sakaguchi, J. Shimizu, S. Yamazaki, T. Sakihama, M. Itoh, Y. Kuniyasu, T. Nomura, M. Toda, T. Takahashi. 2001. Immunologic tolerance maintained by CD25⁺CD4⁺ regulatory T cells: their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance. Immunol. Rev. 182:18.[Medline]
24. Bamford, R. N., A. J. Grant, J. D. Burton, C. Peters, G. Kurys, C. K. Goldman, J. Brennan, E. Roessler, T. A. Waldmann. 1994. The interleukin (IL) 2 receptor chain is shared by IL-2 and a cytokine, provisionally designated IL-T, that stimulates T-cell proliferation and the induction of lymphokine-activated killer cells. Proc. Natl. Acad. Sci. USA 91:4940.[Abstract/Free Full Text]
25. Grabstein, K. H., J. Eisenman, K. Shanebeck, C. Rauch, S. Srinivasan, V. Fung, C. Beers, J. Richardson, M. A. Schoenborn, M. Ahdieh, et al 1994. Cloning of a T cell growth factor that interacts with the chain of the interleukin-2 receptor. Science 264:965.[Abstract/Free Full Text]
26. Lin, J. X., T. S. Migone, M. Tsang, M. Friedmann, J. A. Weatherbee, L. Zhou, A. Yamauchi, E. T. Bloom, J. Mietz, S. John, et al 1995. The role of shared receptor motifs and common Stat proteins in the generation of cytokine pleiotropy and redundancy by IL-2, IL-4, IL-7, IL-13, and IL-15. Immunity 2:331.[Medline]
27. Fehniger, T. A., M. A. Caligiuri. 2001. Interleukin 15: biology and relevance to human disease. Blood 97:14.[Free Full Text]
28. Waldmann, T. A., S. Dubois, Y. Tagaya. 2001. Contrasting roles of IL-2 and IL-15 in the life and death of lymphocytes: implications for immunotherapy. Immunity 14:105.[Medline]
29. Dooms, H., M. Desmedt, S. Vancaeneghem, P. Rottiers, V. Goossens, W. Fiers, J. Grooten. 1998. Quiescence-inducing and antiapoptotic activities of IL-15 enhance secondary CD4⁺ T cell responsiveness to antigen. J. Immunol. 161:2141.[Abstract/Free Full Text]
30. Marks-Konczalik, J., S. Dubois, J. M. Losi, H. Sabzevari, N. Yamada, L. Feigenbaum, T. A. Waldmann, Y. Tagaya. 2000. IL-2-induced activation-induced cell death is inhibited in IL-15 transgenic mice. Proc. Natl. Acad. Sci. USA 97:11445.[Abstract/Free Full Text]
31. Bulfone-Paus, S., D. Ungureanu, T. Pohl, G. Lindner, R. Paus, R. Ruckert, H. Krause, U. Kunzendorf. 1997. Interleukin-15 protects from lethal apoptosis in vivo. Nat. Med. 3:1124.[Medline]
32. Refaeli, Y., L. Van Parijs, C. A. London, J. Tschopp, A. K. Abbas. 1998. Biochemical mechanisms of IL-2-regulated Fas-mediated T cell apoptosis. Immunity 8:615.[Medline]
33. Van Parijs, L., Y. Refaeli, J. D. Lord, B. H. Nelson, A. K. Abbas, D. Baltimore. 1999. Uncoupling IL-2 signals that regulate T cell proliferation, survival, and Fas-mediated activation-induced cell death. Immunity 11:281.[Medline]
34. Zhang, X., S. Sun, I. Hwang, D. F. Tough, J. Sprent. 1998. Potent and selective stimulation of memory-phenotype CD8⁺ T cells in vivo by IL-15. Immunity 8:591.[Medline]
35. Ku, C. C., M. Murakami, A. Sakamoto, J. Kappler, P. Marrack. 2000. Control of homeostasis of CD8⁺ memory T cells by opposing cytokines. Science 288:675.[Abstract/Free Full Text]
36. Marrack, P., J. Bender, D. Hildeman, M. Jordan, T. Mitchell, M. Murakami, A. Sakamoto, B. C. Schaefer, B. Swanson, J. Kappler. 2000. Homeostasis of TCR⁺ T cells. Nat. Immunol. 1:107.[Medline]
37. Dooms, H., T. Van Belle, M. Desmedt, P. Rottiers, J. Grooten. 2000. Interleukin-15 redirects the outcome of a tolerizing T-cell stimulus from apoptosis to anergy. Blood 96:1006.[Abstract/Free Full Text]
38. Roncarolo, M. G., M. K. Levings, C. Traversari. 2001. Differentiation of T regulatory cells by immature dendritic cells. J. Exp. Med. 193:F5.
39. Bacchetta, R., C. Sartirana, M. K. Levings, C. Bordignon, S. Narula, M. G. Roncarolo. 2002. Growth and expansion of human T regulatory type 1 cells are independent from TCR activation but require exogenous cytokines. Eur. J. Immunol. 32:2237.[Medline] <