CD8 T cells produce IL-2, which is required for CD4 CD25 T cell regulation of effector CD8 T cell development for contact hypersensitivity responses

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1 CD8 T cells produce IL-2, which is required for CD4 CD25 T cell regulation of effector CD8 T cell development for contact hypersensitivity responses Danielle D. Kish,*,1 Anton V. Gorbachev,* and Robert L. Fairchild*, *Department of Immunology, Cleveland Clinic Foundation, Ohio; and Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio Abstract: Interleukin (IL)-2 functions to promote, as well as down-regulate, expansion of antigen-reactive CD4 and CD8 T cells, but the role of IL-2 in hapten-specific CD8 T cell priming for contact hypersensitivity (CHS) responses remains untested. Using enzyme-linked immunospot to enumerate numbers of hapten-specific CD4 and CD8 T cells producing IL-2 in hapten-sensitized mice, the number of IL-2-producing CD8 T cells was tenfold that of CD4 T cells. Hapten-primed CD4 T cells produced low amounts of IL-2 during culture with hapten-presenting Langerhans cells, whereas production by hapten-primed CD8 T cells was fivefold greater. CD8 T cells did not express CD25 during hapten priming, but treatment with anti-il-2 or anti-cd25 monoclonal antibodies during hapten sensitization increased hapten-specific effector CD8 T cells as well as the magnitude and duration of the CHS response. These results indicate that CD8 T cells are the primary source of IL-2 and that this IL-2 is required for the function of a population of CD4 CD25 T cells to restrict the development of the hapten-reactive effector CD8 T cells that mediate CHS responses. J. Leukoc. Biol. 78: ; Key Words: delayed-type hypersensitivity T regulatory cells skin T cell priming INTRODUCTION Factors promoting the clonal expansion of antigen-reactive T cells during priming have multiple roles in regulating the extent of this expansion. For the expansion of antigen-reactive CD8 T cells, interleukin (IL)-2 is an early factor promoting clonal expansion and differentiation to effector, cytolytic T cell function [1 3]. Alternatively, IL-15 is an important growth factor in the development and maintenance of memory CD8 T cells [3 5]. IL-2 is also an important factor down-regulating clonal proliferation of T cells. One mechanism of IL-2-mediated restriction of CD8 T cell expansion is through the induction of activation-induced cell death [6]. In addition, IL-2 sustains a cellular regulatory mechanism of T cell immunity. Recently, populations of IL-2-dependent CD4 CD25 T cells, which inhibit T cell activation to self- and allo-antigens, have been described [7 11]. Overt autoimmunity is observed in IL-2- or CD25-deficient animals, which is preventable by transfer of wild-type (WT) CD4 CD25 T cells [7 12]. Contact hypersensitivity (CHS) is a T cell-mediated, inflammatory response of the epidermis to cutaneous sensitization and subsequent challenge with a hapten. Following hapten application or sensitization, epidermal dendritic cells, Langerhans cells (LC), are stimulated to traffic to the skin-draining lymph nodes, where hapten-major histocompatibility complexes (MHC) are presented to and prime specific T cell populations [13 15]. Challenge of sensitized individuals with the hapten directs primed T cell infiltration into the site of challenge and the activation of the T cells to produce cytokines that mediate the characteristic tissue edema of the response. In contrast to CD4 T cell-mediated, classical, delayed-type hypersensitivity responses to protein antigens, hapten-reactive CD8 T cells are the primary effector cells of CHS responses, which in mice treated with CD8-depleting antibodies and in class I MHC-deficient mice, are low-to-absent, whereas responses in mice treated with CD4-depleting antibodies and in class II MHC-deficient mice, are increased in magnitude and duration [16 18]. In vitro stimulation of purified, haptenprimed CD8 T cells from lymph nodes of sensitized mice induces interferon- (IFN- ) production, whereas stimulation of CD4 T cells induces type 2 cytokines, including IL-4, IL-5, and IL-10 but little IFN- [18, 19]. Challenge of haptensensitized mice normally results in ear-swelling responses, which peak at h after challenge and then rapidly decline to background levels. The increased magnitude and extended duration of CHS responses observed in the absence of CD4 T cells suggest their role as regulators of this response. The CD4 T cells mediating regulation of CHS and whether this regulation is directed at the point of T cell priming during sensitization or at elicitation of the response remain unknown. In the current study, the role of IL-2 in the clonal expansion and development of hapten-specific CD4 and CD8 T cell 1 Correspondence: NB3-30, Department of Immunology, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH Received February 4, 2005; revised April 15, 2005; accepted May 20, 2005; doi: /jlb /05/ Society for Leukocyte Biology Journal of Leukocyte Biology Volume 78, September

2 populations was investigated. These studies implicate IL-2 and CD4 CD25 T cells as critical factors regulating the development of hapten-specific CD4 and CD8 T cell populations to sensitization as well as the CHS response. These studies demonstrate for the first time the negative regulatory role of IL-2 in the development of effector CD8 T cell responses to mediate responses to antigens deposited on the skin. MATERIALS AND METHODS Mice BALB/c (H-2 d ) and C57BL6 (H-2 b ) mice were obtained through Dr. Clarence Reeder (National Cancer Institute, Frederick, MD). Adult female mice of 8 10 weeks old were used throughout these studies. Antibodies and cytokines Purified monoclonal antibodies (mab) YTS and GK1.5 (anti-mouse CD4) and YTS 169 and TIB-150 (anti-mouse CD8) were purchased from Ligocyte (Bozeman, MT). Culture supernatants of the immunoglobulin G (IgG)- producing hybridomas S4B6 (anti-mouse IL-2) and PC (anti-mouse CD25) were used to purify mab by protein G chromatography. Polyclonal rat IgG was purchased from Sigma-Aldrich (St. Louis, MO). Fluorescein isothiocyanate (FITC) and phycoerythrin (PE)-labeled mab specific for CD4, CD8, CD25, CD11a, and rat IgG and capture and detection mab for IL-2, IL-4, and IFN- were purchased from PharMingen (San Diego, CA). Hapten sensitization and elicitation of CHS Mice were sensitized to 2,4-dinitrofluorobenzene (DNFB) by painting the shaved abdomen with 25 l 0.25% DNFB (Sigma-Aldrich) and with 10 l on each footpad on days 0 and 1. On day 5, hapten-sensitized and negative control, unsensitized mice were challenged with 10 l 0.2% DNFB on both sides of each ear. The increase in ear swelling was measured at 24 h intervals after challenge using an engineer s micrometer (Mitutoyo, Elk Grove Village, IL) and expressed in units of 10 4 in, as previously reported [18]. The ear-swelling response is presented as the mean increase of each group of four sensitized or nonsensitized mice (i.e., eight ears) SEM. Each experiment shown includes its own positive and negative control groups. In vivo and in vitro depletion of CD4 and CD8 T cells For in vivo depletion of CD4 T cells, mice were injected with 100 g each anti-cd4 mab, YTS 191 and GK1.5, intraperitoneally (i.p.) on 3 consecutive days before hapten sensitization on days 0 and 1 as described previously [18, 20]. In vivo depletion of CD8 T cells was performed by injecting mice with 100 g each anti-cd8 mab, YTS 169 and TIB-150. In each experiment, treated, sentinel mice were used to evaluate the efficiency of CD4 or CD8 T cell depletion by antibody staining and flow cytometry analysis of spleen and lymph node cells (LNC) and were always 95% when compared with cells from control rat IgG-treated mice. For in vitro depletion of CD4 or CD8 T cells, LNC from hapten-sensitized mice were incubated with specific antibodycoated magnetic beads (Dynabeads, Dynal A.S., Oslo, Norway). Treatment with anti-il-2 and anti-cd25 antibodies Mice were injected with 250 g anti-il-2 or anti-cd25 mab (S4B6 or PC61, respectively) on days 0, 1, 2, and 3. As a control, mice were treated with the same amount of rat IgG on the same days. Enzyme-linked immunospot (ELISPOT) assays for enumeration of hapten-specific T cells producing IFN- and IL-4 ELISPOT assays were performed as described previously [18, 21]. Briefly, ELISPOT plates (Whatman Polyfiltronics, Clifton, NJ) were coated with 4 g/ml IFN- -, IL-4-, or IL-2-specific mab and incubated overnight at 4 C. The plates were blocked with 1% bovine serum albumin in phosphate-buffered saline (PBS) and washed four times with PBS. LNC from unsensitized or DNFB-sensitized mice treated with control IgG, anti-cd25, or anti-il-2 mab were prepared on day 5 postsensitization and used as responder cells. Syngeneic spleen cells from naïve mice were labeled with 100 g/ml 2,4- dinitrobenzene sulfonic acid (DNBS) and treated with 50 g/ml mitomycin C before use as stimulator cells, which were plated at cells/well with or responder cells/well in serum-free HL-1 medium (BioWhittaker, Walkersville, MD), supplemented with 1 mm L-glutamine and 1 mm antibiotic, as described previously. Responder cells plated with unlabeled splenocytes were used in each assay as a negative (hapten-specificity) control. After 24 h of cell culture at 37 C in 5% CO 2, cells were removed from the plate by extensive washing with PBS/0.05% Tween-20 (PBS-T). Biotinylated anti- IFN-, anti-il-4, or anti-il-2 mab (2 g/ml) was added, and the plate was incubated overnight at 4 C. The following day, the plate was washed three times with PBS-T, and conjugated streptavidin-alkaline phosphatase was added to each well. After2hatroom temperature, the plates were washed with PBS-T, and nitroblue tetrazolium/5-bromo-4-cholor-30-indolyl substrate (Bio- Rad Laboratories, Hercules, CA) was added for the detection of IFN-, IL-4, or IL-2. The resulting spots were counted with an ImmunoSpot Series I analyzer (Cellular Technology Ltd., Cleveland, OH), which was designed to detect enzyme-linked immunosorbent assay (ELISA) spots with predetermined criteria for spot size, shape, and colorimetric density. The data are shown as the mean number SEM of cytokine-producing cells in triplicate cultures after subtraction of spots from control wells containing T cells with unlabeled stimulator cells (always less than five spots/well) for pooled LNC from two mice or for LNC from two individual mice as indicated. Stimulation of hapten-primed CD4 and CD8 T cells to produce cytokines in culture As stimulator cells, hapten-presenting LC (hplc) were purified from LNC suspensions of DNFB-sensitized mice on day 1 following hapten sensitization using positive selection with anti-cd11c, mab-coated magnetic beads (Miltenyi Biotec, Auburn, CA). As responder cells, CD4 -orcd8 -enriched cell populations were prepared from LNC from unsensitized or DNFB-sensitized mice on day 4 following hapten sensitization. Aliquots of T cell populations were cultured in a 96-well plate with hplc in RPMI medium supplemented with 10% heat-inactivated fetal calf serum (FCS) and 5 mm L-glutamine at 37 C in 5% CO 2. After 36 h of culture, supernatants were collected and tested for concentrations of IL-2, IL-4, and IFN- by sandwich ELISA (R&D Systems, Minneapolis, MN), according to the manufacturer s protocol. Results are reported as concentration of cytokine produced in pg/ml culture supernatant SEM. Flow cytometry analyses LNC were prepared from naïve mice or from hapten-sensitized mice on day 3 or 4 postsensitization. The cells were washed twice with staining buffer (Dulbecco s PBS with 2% FCS/0.2% NaN 3 ), and cell aliquots were incubated on ice in 150 l rat serum (Rockland, Gilbertsville, PA), diluted 1:1000 in the staining buffer. After 30 min, the cells were washed twice and stained with fluorochrome-labeled anti-mouse mab (in these studies, CD25-, CD44-, CD11a-, CD4-, and CD8- or rat IgG-specific mab were used) at 10 g/ml. After 30 min on ice, the cells were washed five times, resuspended in staining buffer, and analyzed by two-color flow cytometry using a FACScan and CellQuest software (Becton Dickinson, San Jose, CA). Sample data were collected on 20,000-gated cells. RESULTS IL-2 production by hapten-specific CD8 and CD4 T cells during sensitization for CHS The cellular source of IL-2 during sensitization for CHS was tested directly using ELISPOT assays. LNC suspensions were prepared from sensitized and naïve mice 5 days after sensitization with DNFB, and enriched CD4 and CD8 T cell 726 Journal of Leukocyte Biology Volume 78, September

3 populations were tested for the number of IL-2-producing cells during culture with unlabeled and dinitrophenyl (DNP)-labeled stimulator cells. When isolated from the lymph nodes of naïve mice, the numbers of CD4 (open bars) or CD8 (solid bars) T cells producing IL-2 during culture with DNP-labeled stimulator cells were low-to-absent (Fig. 1A). In LNC from sensitized mice, low numbers of hapten-specific CD4 T cells producing IL-2 were observed. In contrast, the number of hapten-specific CD8 T cells producing IL-2 was almost tenfold higher. Few spots (less than five) were observed in any cell population during culture with unlabeled cells. As an alternative approach to investigating the source of IL-2 production during hapten sensitization, LNC suspensions were prepared from sensitized and nonsensitized/naïve mice and tested for the ability to produce IL-2 in vitro. Enriched populations of CD4 and CD8 T cells were prepared and cultured with purified hplc from sensitized mice. After 36 h, the supernatants were collected and tested by ELISA to assess T cell production of IL-2, IL-4, and IFN- (Fig. 1B). Culture with hplc failed to stimulate detectable production of IL-4 or IFN- by naïve CD4 or CD8 T cells and low amounts of IL-2 production by naïve CD8 but not CD4 T cells. As previously shown, hapten-primed CD8 but not CD4 T cells produced IFN- during stimulation in culture, and haptenprimed CD4 but not CD8 T cells produced IL-4 [18, 19]. Consistent with the ELISPOT results, hapten-primed CD4 T cells produced low amounts of IL-2, whereas hapten-primed CD8 T cells produced fivefold more during culture with the hplc. Cytokine production observed by all test T cell populations required stimulation with hplc, as no detectable cytokine production was observed when the enriched T cell populations were cultured in the absence of the hplc (data not shown). CD8 T cells do not express CD25 during hapten sensitization To begin to examine the potential role of IL-2 in the expansion of hapten-specific CD4 and CD8 T cells during sensitization for CHS, the expression of CD25 was compared on T cells from groups of five naïve and DNFB-sensitized mice on day 4 after sensitization. Expression of CD25 was observed on CD4 T cells from naïve ( % of total CD4 T cells) and DNFB-sensitized ( %) mice. In contrast, CD8 T cell expression of CD25 was low-to-absent in naïve ( %) mice and did not increase during sensitization ( %). Representative flow cytometry data from one mouse in each group are shown in Figure 2. Analysis of CD8 T cells on days 2 and 3 after hapten sensitization also indicated no increase in CD25-expressing cells (data not shown). CD8 T cells do not require IL-2 for expansion during CHS Previous studies from this laboratory have indicated that expression of the activation marker CD11a is up-regulated on hapten-specific CD4 and CD8 T cells during priming for CHS [19]. The absence of CD25 on CD8 T cells during sensitization suggested that IL-2 was not a critical growth factor in the development of these T cells and that antibodies to IL-2, given during sensitization, would not affect this development. To assess the requirement for IL-2 in the expansion of CD4 and CD8 T cells during sensitization, groups of four mice were treated with control or anti-il-2 mab during sensitization with DNFB on days 0 and 1. On day 3, LNC suspensions were stained to analyze the expression of CD11a on CD4 and CD8 T cell populations. A low percentage of CD4 and CD8 T cells expressed high levels of CD11a in unsensitized, naïve mice (Fig. 3 and Table 1). As previously observed, sensitization resulted in marked increases in the CD4 /CD11a high and CD8 /CD11a high cell populations. Treatment with anti-il-2 mab decreased the size of the CD4 / CD11a high cell population. Alternatively, anti-il-2 mab did not decrease the CD8 /CD11a high cells but resulted in further expansion of these cells. Collectively, the results in Figures 1 3 indicate that more hapten-specific CD8 than CD4 T cells produces IL-2 during sensitization for CHS and suggest that this cytokine is not required for expansion of these CD8 T cells but instead, may negatively regulate this expansion. Treatment with anti-cd25 mab does not deplete CD4 T cells As CD4 and not CD8 T cells were observed to express CD25, a potential mechanism of rat anti-cd25, mab-mediated enhancement of the CHS response was through deletion of CD4 /CD25 regulatory T cells. To examine this, LNC from groups of three mice treated with anti-cd25 or control mab on days 0 through 3 during sensitization were stained for expression of rat IgG or CD4 on CD25 cells on day 4 following sensitization. Consistent with observations in Figure 2, LNC from mice treated with control mab had a considerable number of CD4 CD25 cells ( %), and no CD4 cells stained positively for rat IgG ( %). However, LNC from mice that were treated with anti-cd25 mab had fewer CD4 cells staining positive for CD25 ( %) but also had CD4 T cells staining positively for rat IgG ( %). Representative flow cytometry data from a single mouse in each group are shown in Figure 4. In LNC from mice treated with anti-cd25 mab, the percentage of CD4 cells staining positively for rat IgG added to the percentage of CD4 CD25 cells was nearly identical to the percent of CD4 CD25 cells from control, untreated mice. These observations suggest that within the time-frame of these experiments, the PC61 mab binds to CD25 and blocks the activity of the receptor without depleting the receptor-expressing cells. Elevated and prolonged CHS responses in hapten-sensitized mice treated with anti-il-2 or anti-cd25 mab To examine the role of IL-2 in effector CD8 T cell priming for CHS, the effect of treatment with anti-il-2 or anti-cd25 mab on the CHS response was tested. Groups of mice were sensitized with DNFB on days 0 and 1 and were treated daily with control rat IgG, anti-il-2 mab, or anti-cd25 mab on days 0 through 3. All sensitized mice and a group of unsensitized mice were challenged on the ears with DNFB on day 5, and the change in ear thickness was measured in 24 h increments Kish et al. Regulation of hapten-specific CD8 T cell priming 727

4 Fig. 1. Antigen-specific CD8 T cells are the primary source of IL-2 during sensitization for CHS. (A) C57BL/6 mice were sensitized with 0.25% DNFB on days 0 and 1. On day 5 after sensitization, LNC suspensions were prepared from sensitized and naïve mice. CD4 (open bars) and CD8 (solid bars) T cell-enriched populations were cultured with DNBS-labeled or unlabeled syngeneic splenocytes on anti-il-2 mab-coated ELISPOT plates. After 24 h, cells were removed, and the ELISPOT assay was developed to detect IL-2-producing T cells. The mean number SEM of IL-2-producing CD4 or CD8 T cells per cells in triplicate cultures for pooled LNC from two mice is shown after subtraction of spots from control wells containing T cells with unlabeled stimulator cells (less than five spots per well). Results are representative of two individual experiments. *, P 0.005, compared with the number of spots in sensitized CD4 and naïve CD8 T cell cultures using Student s t-test. (B) BALB/c mice were sensitized with 0.25% DNFB on days 0 and 1. On day 4, LNC suspensions were prepared from groups of four sensitized and naïve mice and pooled. CD4 and CD8 T cell-enriched populations were cultured with hplc. After 36 h, culture supernatants were tested by ELISA to determine IL-2, IL-4, and IFN- production. The mean concentration SEM of IL-2, IL-4, and IFN- produced by naïve and hapten-primed CD4 and CD8 T cells in triplicate cultures is shown. Detectable cytokine production was not observed when the enriched T cell populations were cultured in the absence of the hplc (data not shown). *, P 0.005, compared with cytokine production in all other groups for each specific cytokine using Student s t-test. ND, Not detected. 728 Journal of Leukocyte Biology Volume 78, September

5 Fig. 2. CD4 T cells and not CD8 T cells express CD25 during sensitization for CHS. Groups of five BALB/c mice were sensitized with 0.25% DNFB on days and 0 and 1. On day 4 postsensitization, LNC suspensions from naïve and sensitized mice were stained with FITC-anti-CD25 mab and PE-anti-CD4 or -CD8 mab. Cells from representative animals are shown, and the percentages of CD4 or CD8 cells expressing CD25 in the CD4 or CD8 T cell populations are indicated in the upper right-hand quadrants. The mean percentage for each group of five animals SEM is indicated in the text. postchallenge (Fig. 5, A and B). Treatment with anti-il-2 or anti-cd25 mab resulted in an elevated and prolonged CHS response to hapten challenge when compared with the responses observed in animals treated with control IgG during sensitization. The CHS responses in mice treated with anti-il-2 or anti-cd25 mab were similar to responses in mice depleted of CD4 T cells just prior to hapten sensitization (Fig. 5C), which have been shown to negatively regulate the magnitude and duration of the CHS response [18, 19]. Enhanced development of T cells by anti-il-2 or anti-cd25 mab during sensitization The effect of anti-il-2 mab and anti-cd25 mab on the development of hapten-specific CD4 and effector CD8 T cell populations during priming for CHS was tested directly. The numbers of CD8 IFN- -producing cells and CD4 IL-4-producing cells were compared in the lymph nodes of animals treated with control IgG, anti-il-2, or anti-cd25 mab during sensitization. On day 5 postsensitization, LNC suspensions were separated into CD4 - or CD8 - enriched cell populations using magnetic beads and tested in ELISPOT assays to enumerate the number of haptenspecific cells producing cytokines. Hapten-specific CD8 T cells producing IFN- were detected in lymph nodes of sensitized (70 spots/ cells) but not naïve mice (Fig. 6A). Consistent with the elevated ear-swelling responses, LNC from mice treated with anti-il-2 (110 spots/ cells) or anti-cd25 mab (183 spots/ cells) contained more hapten-specific CD8 T cells producing IFN- than the control IgG-treated group. Consistently, the numbers of hapten-specific CD4 T cells producing IL-4 in the lymph nodes of DNFB-sensitized mice were not increased significantly by treatment with anti-il-2 or anti-cd25 mab during sensitization (Fig. 6B). Absence of anti-cd25 mab-mediated enhancement of effector CD8 T cell development in mice depleted of CD4 cells To examine a potential synergistic effect between CD25 antagonism and CD4 cell depletion on effector CD8 T cell development, mice were depleted of CD4 cells followed by treatment with control IgG or anti-cd25 mab during hapten sensitization. On day 5 postsensitization, LNC from each group were tested by ELISPOT assay to enumerate hapten-specific cells producing IFN-. Consistent with the increased CHS response, depletion of CD4 T cells prior to hapten sensitization increased the numbers of IFN- -producing cells when compared with control-treated, sensitized mice. However, the number of hapten-specific cells producing IFN- did not increase further when the mice depleted of CD4 cells were treated additionally with anti-cd25 mab (Fig. 7). Thus, anti-cd25 mab-mediated enhancement of effector CD8 T cell development for CHS was dependent on CD4 T cells. Kish et al. Regulation of hapten-specific CD8 T cell priming 729

6 Fig. 3. Expression of CD11a on CD4 or CD8 T cells from mice treated with anti-il-2 mab during sensitization for CHS. Groups of four BALB/c mice were sensitized with 0.25% DNFB on days 0 and 1 and were treated with control rat IgG or with anti-il-2 mab on days 0, 1, 2, and 3. On day 4, LNC suspensions from naïve and sensitized mice were stained with FITC-anti-CD11a mab and PE-anti-CD4 or anti-cd8 mab. Cells from a representative animal are shown, and the percentages of CD4 or CD8 cells expressing CD11a in the gated CD4 or CD8 T cell populations are indicated in the upper right-hand quadrants. The mean percentage for each group SEM is indicated in Table 1. Increase in number of hapten-specific CD4 T cells producing IL-4 in animals depleted of CD8 cells prior to anti-cd25 mab treatment The results to this point have indicated that the hapten-reactive CD8 T cell compartment produced more IL-2 during priming than the CD4 T cells and that the negative regulation of effector CD8 T cell development was dependent on IL-2. Therefore, the effect of anti-cd25 mab treatment on the development of haptenreactive CD4 T cells producing IL-4 in the absence of CD8 T TABLE 1. Treatment with Anti-IL-2 mab during Hapten Sensitization Increases CD11a high /CD8 T Cells in Skin-Draining Lymph Nodes a Naive Sensitized Anti-IL-2 mab treated during sensitization CD4 4.24% 0.97% 18.57% 0.82% b 13.09% 0.83% c CD8 4.87% 0.77% 12.46% 2.14% b 26.16% 7.91% c a Percentages of CD4 and CD8 T cells expressing high levels of CD11a. Data are expressed as mean percent of gated T cell population SEM for a group of four animals. b P 0.05 when compared with percent-positive cells from naïve animals. c P 0.05 when compared with percent-positive cells from sensitized-only animals. cells was tested. Groups of mice were treated with control rat IgG or anti-cd8 mab on days 3, 2, and 1 and then sensitized with DNFB on days 0 and 1. One group of CD8 T cell-depleted mice was also treated with anti-cd25 mab during sensitization. On day 5, single-cell suspensions of skin-draining lymph nodes were prepared and tested in ELISPOT assays to enumerate the numbers of hapten-specific T cells producing IL-4 in each of the treatment groups (Fig. 8). Depletion of CD8 T cells resulted in a 2.5-fold increase in the number of IL-4-producing cells when compared with LNC from sensitized animals with the CD8 T cell compartment intact ( spots/ cells versus spots/ cells). This increase is likely to reflect the absence of total CD8 T cells in the lymph nodes. Treatment with anti-cd25 mab in sensitized mice without CD8 T cells resulted in a striking increase in hapten-specific T cells producing IL-4 ( spots/ cells). Thus, in animals depleted of CD8 T cells, development of IL-4-producing CD4 T cells is enhanced by treatment with anti-cd25 mab. DISCUSSION The expansion of antigen-specific CD8 T cells during development for many immune responses is dependent on helper 730 Journal of Leukocyte Biology Volume 78, September

7 Fig. 4. Treatment with anti-cd25 mab does not delete CD4 T cells expressing CD25. BALB/c mice were sensitized to 0.25% DNFB on days 0 and 1 and treated with 250 g anti-cd25 mab i.p. on days 0, 1, 2, and 3. On day 4, LNC suspensions were prepared and stained to detect the presence of CD25 and rat IgG on CD4 T cells. Cells from representative animals are shown, and the percentages of CD4 cells staining positively for CD25 and rat IgG in the CD4 T cell populations are indicated in the upper right-hand quadrants. The mean percentage for each group of three mice SEM is indicated in the text. signals provided by CD4 T cells [22 25]. These helper signals may include IL-2, which promotes antigen-reactive CD8 T cell expansion during the early stages of antigen priming but later mediates apoptosis to maintain homeostasis within the priming site [1, 26]. During antiviral responses, IL-2 may also play a critical role in directing the development of CD8 T cells expressing CD25 to effector, cytolytic cells [3]. Sensitization with hapten induces disparate populations of cytokineproducing T cells: IFN- -producing CD8 T cells, which are the primary effector cells of the response to hapten challenge, and populations of CD4 T cells, which produce type 2 cytokines including IL-4 and IL-5 [18, 19]. In contrast to antiviral responses, CD8 T cells did not express CD25 during development for CHS responses. The absence of CD25 expression on hapten-primed CD8 T cells may indicate that these T cells do not develop to express cytolytic function during elicitation of CHS. However, cytolysis of keratinocytes and hapten-labeled spleen cell targets by hapten-primed CD8 T cells in vitro has been observed in some laboratories but not in others [27 29]. The goal of the current study was to investigate the influence of IL-2 in the expansion of hapten-reactive T cell populations during sensitization for CHS. It is surprising that the number of hapten-specific CD8 T cells producing IL-2 was almost tenfold greater than IL-2-producing CD4 T cells, and haptenprimed CD8 T cells produced much more IL-2 during in vitro stimulation with hplc than did hapten-primed CD4 T cells. These results indicate that CD8 T cells are the major source of IL-2 during CD4 and CD8 T cell priming to DNFB sensitization. The IL-2 production and effector functions of hapten-specific CD4 and CD8 T cells during priming for CHS responses are distinct from these aspects of CD4 and CD8 T cell function in many other immune responses including antiviral and classical delayed-type hypersensitivity responses. Kish et al. Regulation of hapten-specific CD8 T cell priming 731

8 Fig. 5. Treatment with anti-il-2 or anti-cd25 mab during sensitization increases the magnitude and duration of CHS responses and the development of antigen-specific T cells producing IFN- and IL-4. Groups of four BALB/c mice were sensitized with 0.25% DNFB on days 0 and 1 and challenged with 0.2% DNFB on day 5. One group of mice was treated with 250 g control rat IgG ( ) i.p. on days 0, 1, 2, and 3 during DNFB sensitization, and this group is shown as the positive control (A C), and each experimental group was tested at the same time. The increase in ear swelling in sensitized and naïve, nonsensitized ( ) mice was measured at 24 h intervals postchallenge and is expressed in 10 4 in SEM. (A) Mice were treated with 250 g anti-il-2 mab (e) i.p. on days 0, 1, 2, and 3 during DNFB sensitization. *, P 0.02, using Student s t-test. (B) Mice were treated with 250 g anti-cd25 mab (e) i.p. on days 0, 1, 2, and 3 during DNFB sensitization. *, P (C) Mice were given 200 g anti-cd4 mab (e) i.p. on days 3, 2, and 1 and then sensitized with 0.25% DNFB on days 0 and 1 and challenged on the ears with 0.2% DNFB. *, P The expression of CD25 on CD4 but not CD8 T cells during hapten priming led us to predict that treatment with antagonizing antibodies to IL-2 or to CD25 would inhibit the development of hapten-specific, type 2, cytokine-producing CD4 T cells but not the IFN- -producing effector CD8 T cells. Treatment with either antibody enhanced the development of hapten-specific CD8 T cells producing IFN- and hapten-specific CD4 T cells producing IL-4. Furthermore, treatment with either antibody during hapten sensitization resulted in CHS responses of higher magnitude and longer duration, which correlated with the enhanced development of the effector CD8 T cell compartment. Recent studies from this Fig. 6. Treatment with anti-il-2 or anti-cd25 mab during sensitization increases the numbers of hapten-specific CD8 T cells and CD4 T cells producing cytokines in DNFB-sensitized mice. BALB/c mice were sensitized with 0.25% DNFB on days 0 and 1. Mice were treated during sensitization with control rat IgG, anti-il-2, or anti-cd25 mab as above. On day 5 after sensitization, CD8 T cell-enriched populations were prepared from lymph nodes and cultured with DNBS-labeled or unlabeled syngeneic splenocytes on anti-ifn- mab-coated ELISPOT plates. CD4 T cell-enriched populations were cultured on anti-il-4 mab-coated plates. After 24 h, cells were removed, and the ELISPOT assay was developed to enumerate hapten-specific and IFN- - and IL-4-producing T cells. The mean number SEM of (A) IFN- -producing CD8 T cells and (B) IL-4-producing CD4 T cells in triplicate cultures for pooled LNC from two mice is shown after subtraction of spots from control wells containing T cells with unlabeled stimulator cells. Results are representative of two individual experiments. *, P 0.05, using Student s t-test, in comparison with LNC from sensitized, IgG-treated, control mice. ND, Not detected. 732 Journal of Leukocyte Biology Volume 78, September

9 Fig. 7. Anti-CD25 mab treatment does not increase hapten-specific CD8 T cell development in the absence of CD4 T cells. BALB/c mice were given 200 g control, rat IgG, or anti-cd4 mab i.p. on days 3, 2, and 1 and were sensitized with 0.25% DNFB on days 0 and 1. Groups of the mice were treated during sensitization with control rat IgG or anti-cd25 mab as above. On day 5 after sensitization, CD8 T cell-enriched populations were prepared and tested for numbers of hapten-specific, IFN- -producing cells. Results are representative of two individual experiments. *, P 0.04, in comparison with sensitized, IgG-treated, control mice. ND, Not detected. laboratory have shown that the increased numbers of haptenspecific CD8 T cells in CD4-depleted animals are a result of increased expansion of these T cells in the lymph nodes rather than an increase in frequency in the absence of CD4 T cells [30]. The enhancement of hapten-specific CD4 and CD8 T cell development in anti-il-2 mab-treated mice indicated that IL-2 is not a critical growth factor for these T cell populations. The enhanced development of hapten-specific CD8 T cells and elicited CHS responses in mice treated with anti-il-2 or anti- CD25 mab suggested that the antibodies neutralized a regulatory component of the response and were reminiscent of the absence of regulation observed in mice depleted of CD4 T cells during hapten sensitization. Our previous observations that hapten-specific CD4 T cells develop to IL-4- and IL-5- producing cells [18] led us to suspect that type 2 cytokines produced by the CD4 T cells were regulating CD8 T cell development and the CHS response. However, enhanced CD8 T cell responses observed in anti-il-2 or anti-cd25 mabtreated mice were coincident with enhanced IL-4-producing CD4 T cell development. These results suggest that the CD4 T cells regulating CD8 T cell development and CHS are not likely to be the hapten-specific CD4 T cells producing IL-4 and that a common CD4-mediated mechanism regulates the development of hapten-specific CD4 and CD8 T cell populations during sensitization. The results further suggest that IL-2 is not a growth factor for the hapten-specific CD4 or CD8 T cell populations but plays a critical role in downregulating the development of these T cell populations and the CHS response. The current results indicate that a primary point of regulation in the CHS response is the CD4 CD25 T cell-mediated restriction of effector CD8 T cell development to sensitization, and this limits the magnitude and duration of the swelling/ inflammation elicited by hapten challenge. In other immune responses, regulation mediated by CD4 CD25 T cells may be expressed during elicitation of the response by suppressing the function of the effector T cells. For example, recent studies by Belkaid and co-workers [31] have indicated the accumulation of CD4 CD25 T cells at infection sites of experimental leishmaniasis and the down-regulation of ongoing immune responses to eliminate the parasite. Whether the CD4 CD25 regulatory T cells that restrict hapten-reactive CD8 T cell development also function in down-modulating ongoing earswelling responses at the antigen challenge site is untested. In summary, the current report indicates that IL-2 is an important factor in the regulation of effector CD8 T cell priming and the CHS response. The role of IL-2 is not mediated directly at the level of hapten-specific CD4 and CD8 T cell development during sensitization as a growth factor. Rather, IL-2 is required to maintain the presence and activity of CD4 CD25 T cells that restrict the development of effector CD8 T cells and in turn, the magnitude and duration of the CHS response. The results of this study have further exposed Kish et al. Regulation of hapten-specific CD8 T cell priming 733

10 Fig. 8. Anti-CD25 mab treatment increases hapten-specific T cell production of IL-4 in the absence of CD8 T cells. BALB/c mice were given 200 g control rat IgG or anti-cd8 mab i.p. on days 3, 2, and 1 and were sensitized with 0.25% DNFB on days 0 and 1. Groups of CD8-depleted mice were treated during sensitization with control rat IgG or anti-cd25 mab as above. On day 5 following sensitization, LNC suspensions were prepared and tested for numbers of hapten-specific, IL-4-producing cells. Results are representative of two individual experiments. *, P 0.05, in comparison with sensitized IgG-treated control mice. ND, Not detected. the complexity of the cells induced to mediate and regulate CHS responses. Hapten sensitization primes populations of hapten-specific, IFN- -producing CD8 T cells, which are the effector cells of the CHS response as well as hapten-specific, IL-4-producing CD4 T cells, whose function in the response remains unclear. Although the function of the CD4 CD25 regulatory T cells is dependent on IL-2, the hapten specificity and other requirements to elicit the regulatory function of these cells are undefined currently. Clarification of these requirements should increase understanding of these regulatory cells and the ability to enhance or inhibit CD8 T cell-mediated responses to antigens deposited in the skin. ACKNOWLEDGMENTS This work was supported by the National Institutes of Health Grant AI The authors thank the staff of the Cleveland Clinic Foundation Biological Resources Unit for excellent animal care. REFERENCES 1. Blattman, J. N., Grayson, J. M., Wherry, E. J., Kaech, S. M., Smith, K. A., Ahmed, R. (2003) Therapeutic use of IL-2 to enhance antiviral T-cell responses in vivo. Nat. Med. 9, Cousens, L. P., Orange, J. S., Biron, C. A. (1995) Endogenous IL-2 contributes to T cell expansion and IFN- production during lymphocytic choriomeningitis virus infection. J. Immunol. 155, Manjunath, N., Shankar, P., Wan, J., Weninger, W., Crowley, M. A., Hieshima, K., Springer, T. A., Fan, X., Shen, H., Lieberman, J., von Andrian, Y. H. (2001) Effector differentiation is not prerequisite for generation of memory cytotoxic T lymphocytes. J. Clin. Invest. 108, Ku, C. C., Murakami, M., Sakamoto, A., Kappler, J., Marrack, P. (2000) Control of homeostasis of CD8 memory T cells by opposing cytokines. Science 288, Zhang, X., Sun, S., Hwang, I., Tough, D. F., Sprent, J. (1998) Potent and selective stimulation of memory-phenotype CD8 T cells in vivo by IL-15. Immunity 8, Lenardo, M. J. (1991) Interleukin-2 programs mouse T lymphocytes for apoptosis. Nature 353, Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, M., Toda, M. (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, Shevach, E. M. (2001) Certified professionals: CD4 CD25 suppressor T cells. J. Exp. Med. 193, F41 F Wood, K. J., Sakaguchi, S. (2003) Regulatory T cells in transplantation tolerance. Nat. Rev. Immunol. 3, Furtado, G. C., de Lafaille, M. A. C., Kutchukhidze, N., Lafaille, J. J. (2002) Interleukin 2 signaling is required for CD4 regulatory T cell function. J. Exp. Med. 196, Murakami, M., Sakamoto, A., Bender, J., Kappler, J., Marrack, P. (2002) CD25 CD4 T cells contribute to the control of memory CD8 T cells. Proc. Natl. Acad. Sci. USA 99, Wolf, M., Schimpl, A., Hunig, T. (2001) Control of T cell hyperactivation in IL-2-deficient mice by CD4 CD25 and CD4 CD25 T cells: evidence for two distinct regulatory mechanisms. Eur. J. Immunol. 31, Journal of Leukocyte Biology Volume 78, September

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