T Cells + and CD8 + Human CD4 IL-2 Upregulates CD86 Expression On

Published January 13, 2012, doi:10.4049/jimmunol.1100181 The Journal of Immunology

IL-2 Upregulates CD86 Expression on Human CD4+ and CD8+ T Cells

Ananta Paine,* Hartmut Kirchner,† Stephan Immenschuh,* Mathias Oelke,‡ Rainer Blasczyk,* and Britta Eiz-Vesper*

The glycoprotein CD86 is an important costimulatory molecule that has been shown to be predominantly expressed on APCs, such as dendritic cells, macrophages, and B cells. More recently, CD86 was also detected on T cells in speciﬁc pathological conditions. The mechanisms of how CD86 might be induced and its functional role in T cells are not well understood. In the present study, we showed that treatment with IL-2 markedly upregulated CD86, but not CD80, in human CD4+ and CD8+ T cells. This upregulation occurred in the absence of bystander cells, and isolated naive CD4+ or CD8+ T cells exhibited different time-dependent CD86-expression patterns in response to IL-2. Upregulation of CD86 on activated T cells was reduced by Abs that block IL-2 and IL-2Ra (CD25), indicating a receptor-mediated mechanism. IL-2–dependent CD86 upregulation was blocked by pharmacological inhibitors of the NFAT and mammalian target of rapamycin pathways and was largely reduced by simultaneous exposure to IFN-a. Importantly, a marked increase in CD86 on T cells was also observed in vivo in IL-2–treated patients. In conclusion, IL-2 upregulates CD86 expression on human CD4+ and CD8+ T cells via a receptor-dependent mechanism that involves the NFAT and mammalian target of rapamycin pathways. The Journal of Immunology, 2012, 188: 000–000.

D86, also known as B7.2, is a central costimulatory T cell-mediated immune responses (17–19). Moreover, IL-2 has molecule that is considered to be mainly expressed on been widely used for therapeutic applications in patients with C APCs, such as dendritic cells (DCs), macrophages, and malignant (17, 20–22) and infectious disorders (23–26). Although B cells (1). CD86 is a ligand for CD28 and CTLA-4, both of which most previous reports focused on the impact of IL-2 on T cell are immunologically important receptors on T cells and provide survival and proliferation, only few studies addressed IL-2–in- signals for the activation or inhibition of Ag-specific T cells (2). duced phenotypic changes in T cells, which are particularly rel- Similar to CD86, another member of the B7 family, CD80 (B7.1) evant in cytokine therapy. Because the role of IL-2 in regulating also interacts with CD28 and CTLA-4 (3). Although increased CD86 expression in T cells is not well understood, the goal of the levels of CD86 and CD80 on mature and activated APCs improve current study was to investigate the specific effects of IL-2 on T cell-mediated immune responses toward pathogens and/or ma- CD86 in human T cells. lignant cells, excessive expression of these molecules has been It is shown that IL-2 upregulates the expression of CD86, but not associated with autoimmune responses (1, 4–7). Thus, expression that of CD80, on cell cultures of CD4+ or CD8+ T cells, even in the of these costimulatory molecules is controlled via a complex absence of bystander cells. Although exposure to IL-2 led to network of regulatory pathways. comparable CD86+ CD4+ and CD8+ T cells in PBMCs, separately More recently, CD86 was shown to be expressed on T cells in cultured naive CD4+ or CD8+ T cells showed differences in their specific pathological conditions (8–11). Independent groups dem- CD86-expression patterns in response to IL-2. Studies with phar- onstrated that CD86 was detected on allergen-specific T cells (9), macological inhibitors showed that CD86 upregulation by IL-2 tumor-infiltrating lymphocytes (11), T cells from HIV-infected is mediated via the NFAT and mammalian target of rapamycin patients (8, 12), and hepatitis C virus-specific CD8+ T cells (13). (mTOR) pathways. Moreover, IL-2–dependent CD86 upreg- Collectively, these reports suggested that TCR/costimulation- ulation was largely reduced by IFN-a. Finally, an increased ex- dependent activation of T cells might be a prerequisite for CD86 pression of CD86 was also observed on T cells in vivo in patients expression on T cells (14–16). It is well known that IL-2 plays during IL-2 therapy. a central role for T cell activation in vitro and in vivo (17) and in Materials and Methods Isolation of PBMCs and T lymphocyte subsets *Institute for Transfusion Medicine, Hannover Medical School, D-30625 Hannover, PBMCs were isolated from blood samples of healthy blood donors and Germany; †Department of Hematology-Oncology, Hospital Siloah, D-30449 Hannover, Germany; and ‡Department of Pathology, The Johns Hopkins School of Medicine, patients, as indicated. Prior approval for this study was received Baltimore, MD 21205 from the local ethics committee. PBMCs were isolated by discon- tinuous-gradient centrifugation, washed twice in sterile PBS, and Received for publication January 19, 2011. Accepted for publication December 8, resuspended at a concentration of 1 3 106 cells/ml in M’ medium sup- 2011. plemented with 10% heat-inactivated human AB serum (C.C.pro, This work was supported in part by Deutsche Jose´ Carreras Leuka¨mie Stiftung. Neustadt, Germany). Standard M’ medium was prepared, as described + + Address correspondence and reprint requests to Dr. Ananta Paine, Institute for Trans- before (27), and was used in all cell culture experiments. CD3 ,CD4 , fusion Medicine, Hannover Medical School, Carl-Neuberg-Strasse 1, D-30625 Hannover, and CD8+ T cells from PBMCs of healthy donors were enriched by Germany. E-mail address: [email protected] magnetic cell sorting (MACS), using negative selection kits (Miltenyi Abbreviations used in this article: CsA, cyclosporine A; DC, dendritic cell; mTOR, Biotec, Bergisch Gladbach, Germany), or FACS, using a FACSAria mammalian target of rapamycin; PFA, paraformaldehyde; Rapa, rapamycin. (BD Biosciences, Heidelberg, Germany). Unless specified, the purity of the respective cell populations was 90–99.8%, as determined by flow Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 cytometry.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100181 2 IL-2 UPREGULATES CD86 ON HUMAN CD4+ AND CD8+ T CELLS

Abs, reagents, flow cytometry, and intracellular staining In vitro costimulatory assay The following Abs were used for cell surface marker staining: anti–CD3- In vitro costimulatory assay was performed using naive CD4+ T cells as FITC (UCHT1), anti–CD4-FITC (13B8.2), anti–CD8-PE/FITC (B9.11), responder cells. To obtain CD86+ and CD862 T cells, PBMCs from anti–CD86-PE (HA5.2B7), anti–CD80-FITC (MAB104), and anti–CD122- healthy donors were cultured with 100 IU/ml IL-2 for 2 wk. Subsequently, FITC (2RB), all of which were purchased from Beckman Coulter (Kre- the IL-2–stimulated cells were stained with anti-CD86 and anti-CD3 feld, Germany). Anti–CD3-PerCP (SK7), anti–CD4-PerCP (L200), anti– mAbs, and CD86+ and CD862 T cells were obtained by FACS sorting. CD8-PerCP (G42-8), anti–CD86-allophycocyanin (FUN-1), anti–CD80-PE Purity, as determined by flow cytometry, was up to 99%. Isolated CD86+ (L307.4), anti–CD45RA-FITC/PE–Cy7 (HI100/L48), anti–CD45RO- and CD862 T cells were fixed with 1% paraformaldehyde (PFA) for 10 PE–Cy7/allophycocyanin-H7 (UCHL1), anti–CD70-FITC (Ki-24), anti– min and washed three times before use. Naive CD45RA+CD4+ T cells CD30-FITC (Ber-H83), anti–CD25-PE/PE–Cy7 (M-A251), anti–CD69- were purified from PBMCs of healthy blood donors by FACS sorting us- FITC (FN50), anti–p-STAT1–PE (pY701), anti–p-STAT3–PE (pY705), ing anti-CD4, anti-CD8, anti-CD45RA, and anti-CD45RO mAbs (purity anti–p-STAT5–PE (pY694), anti–CD44-FITC (G44-26), anti–CD38-FITC up to 99%). Isolated CD4+ T cells were stained with CFSE. For CFSE (HB7), anti–CD71-FITC (M-A712), anti–CD98-FITC (UM7F8), anti– staining, 0.5–2 3 106 naive CD4+ T cells were labeled with 1 mM CFSE CD62L-PE (SK11), anti–CCR7-PE–Cy7 (3D12), anti–CD127-PE–Cy7 (Invitrogen). Thereafter, 2.5 3 105 CFSE-stained naive CD4+ T cells from (HIL-7R-M21), anti–FOXP3-PE (259D/C7), anti–CD132-PE (TUGh4), healthy donors (n = 8) were cultured with anti-CD3 Ab-conjugated beads and anti–CD25-FITC (M-A251) were all purchased from BD Biosciences (Dynabeads human CD3; Invitrogen) in combination with 2.5 3 105 PFA- (Heidelberg, Germany). For blocking experiments, purified mouse anti- fixed CD86+ or CD862 T cells/well in a 96-well round-bottom culture human IL-2 mAb (5334.21), anti-CD25 (anti–IL-2Ra, 22722), and con- plate. Each experiment was performed in triplicates. CFSE-stained naive trol Ab mouse-IgG1 (11711) were purchased from R&D Systems (Min- CD4+ T cells, cultured alone or with only anti-CD3–conjugated beads, neapolis, MN). Pharmacological inhibitor cyclosporine A (CsA) and were used as negative control. Naive CD4+ T cells, cultured with anti- rapamycin (Rapa) were from Sigma-Aldrich (Hamburg, Germany). Ex- CD3/CD28–conjugated beads (Dynabeads human CD3/CD28 T Cell Ex- pression levels of phenotypic markers on T cells, monocytes, and DCs pander; Invitrogen), were used as positive control. After 6 d, cell prolif- were determined by six-color flow cytometry (FACSCanto II, Becton eration was measured as CFSE dye dilution by flow cytometry (29). The Dickinson, Heidelberg, Germany) after staining with fluorescent-tagged results are given as the percentage of cells undergoing proliferation in Abs, according to the manufacturer’s instructions. In general, ∼1 3 105 response to stimulation with anti-CD3–conjugated beads in combination cells suspended in PBS (with 0.5% human AB serum) were incubated with with either CD86+ or CD862 T cells or anti-CD3/CD28–conjugated beads. the respective Abs for 15 min at room temperature or for 30 min on ice. Thereafter, cells were washed with PBS and analyzed by flow cytometry. Patients and treatment regimen Intracellular staining for FOXP3 was performed using the FOXP3 staining Blood samples from cancer patients treated with s.c. IL-2 (three patients kit from BD Biosciences, according to the manufacturer’s instructions. with melanoma and one patient with metastatic renal cell carcinoma) were analyzed for expression of CD86 on T cells before and after IL-2 ad- Cell culture ministration. Patients were treated with IL-2 (Proleukin; Novartis), ac- PBMCs from patients and healthy donors were cultured for up to 21 d in cording to therapeutic regimen that has been described elsewhere (30). The M’ medium, supplemented with 10% human AB serum, in the presence or first day of IL-2 administration was designated as day 1. Prior approval for absence of cytokines. IL-2 (Proleukin; Novartis, Basel, Switzerland) con- this study was received from the local ethics committee. centrations used in these experiments were 50, 100, 200, and 400 IU/ml; 100 IU/ml IFN-a (IFN-a-2a, Roferon-A; Hoffmann-La Roche, Grenzach- Results Wyhlen, Germany) and 100 U/ml IL-7, IL-15 (PeproTech, Rocky Hill, NJ) IL-2 upregulates CD86 on T cells were used for the in vitro experiments. The media were changed every 3–4 d, and cells were analyzed for expression of different cell surface markers We studied the phenotypical changes of T cells in IL-2-treated and costimulatory molecules, as indicated in Results. In experiments with PBMCs from healthy blood donors (Fig. 1). An unexpected upreg- purified T cell subsets, isolated T cells were cultured in the presence of 100 IU/ml IL-2 for 10 d before analyzing CD86 expression by flow cytometry. ulation of CD86, also termed B7.2, on T cells was observed by For IL-2–blocking experiments, isolated CD4+/CD8+ T cells were cultured flow cytometry studies (Fig. 1A). As a control, levels of CD80 with anti-CD3/CD28–conjugated beads (Dynabeads CD3/CD28 T Cell (also known as B7.1), another member of the B7 family, were not Expander; Invitrogen, Karlsruhe, Germany), with or without anti–IL-2 altered in response to IL-2 (Fig. 1B). We also determined the and anti-CD25 mAb (each 10 mg/ml), for 6 d; thereafter, they were ana- levels of CD86 on CD4+ and CD8+ T cell subsets separately. lyzed for CD86 expression by flow cytometry. To determine the effect of pharmacological inhibitors, PBMCs were cultured with IL-2 (100 IU/ml), CD86 levels increased in both T cell subsets and reached a max- with or without 0.5 mg/ml CsA or 100 nM Rapa, for 10 d. imum at day 10 (Fig. 1C,1D). After day 10, a gradual decrease in CD86+ was observed in CD4+, but not in CD8+, T cells (Fig. 1D). Generation of human mature DCs from primary monocytes We also performed IL-2 dose-dependency studies and found Primary monocytes were isolated from PBMCs of healthy donors using a statistically significant correlation between the dose of IL-2 and MACS magnetic separation system with CD14 MicroBeads (Miltenyi CD86+ expression on CD4+ and CD8+ T cells (Fig. 1E). Taken 6 Biotec). Purified monocytic cells (1 3 10 cells/ml) were cultured in together, these results demonstrated that IL-2 upregulates ex- M’ medium supplemented with 2% heat-inactivated human AB serum (C. pression of CD86 on human CD4+ and CD8+ T cells. C.pro), 500 U/ml IL-4, and 800 U/ml GM-CSF for 5 d. Media change and addition of fresh media, IL-4, and GM-CSF was done after 2 d. On day 5, IL-2-induced CD86+ T cells are of effector/memory phenotype DCs were matured by treating them with 1 mg/ml LPS (Escherichia coli + O111: B4; Calbiochem, La Jolla, CA) for an additional 2 d. We further characterized IL-2–treated CD86 T cells by flow cytometry with Abs against various cell surface markers. As mRNA quantification by real-time RT-PCR summarized in Table I, CD86 positivity is mainly observed on 2 + Total RNA was extracted using an RNeasy kit (Qiagen), and cDNA was CD45RA CD45RO effector/memory T cells. In addition, we synthesized using 1 mg RNA using the High Capacity cDNA Reverse found that CD86+ T cells expressed higher levels of the activation Transcription Kit (Applied Biosystems, Darmstadt, Germany), as de- markers CD25, CD38, CD69, CD71, and CD98 compared with scribed previously (28). Inventoried mixes purchased from Applied Bio- CD862 T cells (Table I). However, the expression of these acti- systems were used for quantification of mRNA levels for CD86 (TaqMan Gene Expression Assay, Hs01567025_m1). Amplification was performed vation markers was mainly transient. Interestingly, higher levels of using TaqMan Gene Expression Master Mix (Applied Biosystems) on the costimulatory molecules CD70 and CD30 were also observed a StepOnePlus Real-Time PCR System (Applied Biosystems). Thermal on a fraction of in vitro IL-2–induced CD86+ T cells but not on cycling was performed at 95˚C for 10 min, followed by 40 cycles at 95˚C CD862 T cells (Table I). We also analyzed expression level of for 15 s and 60˚C for 1 min. The constitutively expressed gene, GAPDH, + 2 was used as a control for normalization of cDNA levels. The DDCT FOXP3, CD127, CD62L, CCR7, and CD44 on CD86 and CD86 method was used to semiquantify mRNA levels, according to the manu- T cells, and fractions of both subsets expressed FOXP3, CD127, facturer’s protocol (Applied Biosystems). CD62L, CCR7, and CD44 (Table I). The Journal of Immunology 3

FIGURE 1. IL-2 upregulates CD86 expression on T cells which are of effector/memory phenotype. A, PBMCs from healthy blood donors were cultured with 100 IU of IL-2. Mature DCs were generated from isolated monocytes after culturing them with IL-4 and GM-CSF for 8 d and subsequently treating them with 1 mg/ml LPS for 2 d. Thereafter, cells were analyzed by flow cytometry for expression of CD86 on DCs and T cells (left panel) (results from one representative experiment are shown, n = 4). Mean fluorescence intensity (MFI) detected for CD86 expressed on T cells and mature DCs are presented as bar graphs (right panel). B, Expression of CD80 on T cells before and after 10 d of IL-2 exposure was analyzed by flow cytometry. Results from one representative of four independent experiments are shown (n =4).C, PBMCs from healthy donors (n = 16) were cultured in the presence of 100 IU of IL-2 for 10 d, after which CD86 expression on T cells was measured by flow cytometry. ***p , 0.0001, two-tailed paired t test. D, Kinetics of CD86 expression on T cells by IL-2: PBMCs from four healthy donors were cultured in the presence of 100 IU of IL-2 for 21 d, and CD86 expression on T cells was subsequently monitored by flow cytometry. Results are expressed as percentage of CD86+ cells among the CD4+ and CD8+ T cells (d, donor 1; s, donor 2; ▼, donor 3; 4, donor 4). (E) Dose-response curve for IL-2–induced CD86 expression on T cells: PBMCs derived from six healthy blood donors were cultured in the presence of increasing concentrations of IL-2 (50–400 IU/ml). After 10 d of culturing, CD86 expression on CD3+ T cells was measured by flow cytometry. Results are expressed as the percentage of CD86+ T cells among CD4+ and CD8+ Tcells. **p , 0.001, ***p , 0.0001, ANOVA.

IL-2 upregulates CD86 expression on naive CD8+ T cells but due to different expression levels of IL-2R subunits on these not on naive CD4+ T cells cells. Next, we examined the levels of CD86 on naive or effector/memory CD86 expression on CD4+ and CD8+ T cells does not require T cells. Naive and effector/memory CD4+ and CD8+ T cells were the presence of bystander cells separated by cell sorting, based on the presence of the phenotypic markers CD45RA and CD45RO. Our experiments showed that Because CD86 is predominantly expressed on professional APCs naive CD4+ T cells did not express CD86, even after 10 d of IL-2 (31), the presence of this protein on T cells might be a conse- treatment, whereas CD8+ naive T cells exhibited CD86 upreg- quence of membrane transfer from CD86-expressing bystander ulation similar to its effector/memory counterparts (Fig. 2A). In- cells (32, 33) or the uptake of CD86-containing exosomes (34). creased levels of CD86 in response to IL-2 on CD4+ and CD8+ Alternatively, the T cell phenotype might be affected by bystander T cells were clearly visible in effector and memory T cells. Thus, cells through cell–cell interactions or secreted cytokines. To ex- our experiments indicated that naive CD4+ and CD8+ T cells amine how CD86 might be upregulated on IL-2–activated T cells, have a different pattern of IL-2–dependent CD86 upregulation. we puriﬁed CD4+ and CD8+ T cell subsets from PBMCs and We further determined the expression of the three IL-2R chains cultured them with IL-2 in the absence of other cells. As shown in (IL-2Ra [CD25], IL-2Rb [CD122], and IL-2Rg [CD132]) in na- Fig. 3A, upregulation of CD86 was also observed on isolated ive and memory CD4+ and CD8+ T cells. Our experiments showed CD4+ and CD8+ T cells. In our in vitro experiments, .90% of that expression levels of these three subunits were higher in T cells became CD86+ when cultured with 100 IU/ml IL-2 for memory subsets of CD4+ and CD8+ T cells (Fig. 2B). Further- 10 d (Fig. 3B), suggesting that CD86 expression on T cells is due more, levels of IL-2Ra,IL-2Rb,andIL-2Rg in naive CD8+ to increased synthesis rather than to protein transfer from by- T cells were higher compared with their naive CD4+ counterparts stander cells. (Fig. 2B). Thus, the differences in IL-2–dependent CD86 upreg- To further investigate whether CD86 expression on CD4+ and ulationinnaiveandmemoryCD4+ and CD8+ T cells might be CD8+ T cell subsets might be interdependent of each other, we 4 IL-2 UPREGULATES CD86 ON HUMAN CD4+ AND CD8+ T CELLS

Table I. Phenotypic characteristics of CD86+ and CD862 T cells tages of CD4+CD86+ T cells were comparable (∼45% and 55% of CD86+CD4+ in donor 2 and 3, respectively) (Fig. 3B). Markers CD862 T Cells CD86+ T Cells Expression of CD86 mRNA by real-time RT-PCR was analyzed in + CD45RA 2/+ 2 puriﬁed CD3 T cells before or after exposure to IL-2. We observed CD45RO 2/+ + that IL-2 caused a marked increase in CD86 mRNA synthesis in CD25 2/+ +a/2 T cells (Fig. 3C). These results further indicated that CD86 ex- a CD69 2 + /2 pression on T cells is due to increased synthesis of CD86. In con- CD70 22/+ + 22clusion, these results demonstrated that CD86 expression on CD4 CD30 /+ + FOXP3 2/+ 2/+ and CD8 T cells is not dependent on the presence of bystander cells. 2 2 CD127 /+ /+ Exposure to IL-2 upregulates CD86 expression on CD62L 2/+ 2/+ 2 2 CD38 2 + CD25 CD86 T cells CD44 2/+ 2/+ We also investigated whether CD86+ T cells would occur as CD71 2/+ 2/+ + 2 a result of the expansion of initially present CD86 T cells or as CD98 /+ + 2 + CCR7 2/+ 2/+ a result of CD86 T cells acquiring a CD86 phenotype in re-

2 + sponse to IL-2 exposure. To this end, we selectively purified The phenotype of the CD86 and CD86 T cells was compared by flow cyto- 2 2 metric analysis with fluorescent-labeled mAbs against the markers specified above. CD25 CD86 T cells by flow cytometry-based cell sorting and Patient and in vitro data are summarized in the table above (n = 4–10). cultured them with IL-2. Our experiments showed that, even in aTransient expression. 2 2 . +, Notable/high level of expression; 2, low expression/undetectable. isolated CD25 CD86 T cells (purity 99.9%), 69% of those cells acquired a CD86+ phenotype (after 10 d, Fig. 3D). As demonstrated in Fig. 3D, .63% cells were CD86+CD252, performed additional experiments with isolated CD4+ and CD8+ whereas ∼5% were CD86+CD25+ (day 10). T cells. In donor 1, treatment with IL-2 caused CD86 upregulation of 54% in CD4+ T cells and 93% in CD8+ T cells, inde- Upregulation of CD86 on activated T cells is mediated via pendent of whether they were cocultured or cultured separately secreted IL-2 (Fig. 3B). Moreover, in two donors, exposure to IL-2 resulted in Activation of T cells was shown to cause an increase in CD86 on a higher percentage of CD8+CD86+ T cells when CD8+ T cells T cells (14–16). Because this upregulation might be due to an were cultured in the presence of the CD4+ counterpart compared autocrine mechanism via secreted IL-2, we performed studies with with cell culture alone (donors 2 and 3). In these cases, percen- anti–IL-2– and anti–IL-2R–blocking Abs in T cells. More spe-

FIGURE 2. IL-2 upregulates CD86 expression on naive CD8+ T cells but not on naive CD4+ Tcells.CD45RA+ (naive) and CD45RO+ (effector/ memory) CD4+ and CD8+ T cells were isolated from PBMCs by cell sorter and were cultured with 100 IU/ml of IL-2 for 10 d. Thereafter, cells were stained with different fluorescent-labeled Abs against different phenotypic markers. One representative example is shown (n = 3). Results are expressed as the percentage of CD86+ cells among CD4+ (A)orCD8+ T cells (B). C, Expression levels of different receptor chains of IL-2R (i.e., CD25 [IL-2Ra], CD122 [IL-2Rb], and CD132 [IL-2Rg]) on naive and memory CD4+ and CD8+ T cells were analyzed by flow cytometry, and the average of respective mean fluorescence intensity (MFI) values obtained from four different independent experiments are presented as bar graphs (n =4). The Journal of Immunology 5

FIGURE 3. Upregulation of CD86 on T cells is not dependent on the presence of bystander cells or initial presence of CD86+ T cells. A, Isolated CD3+ T cells were cultured for 10 d with or without IL-2 (100 IU/ml), and CD86 expression on T cells and the presence of other cells were analyzed by flow cytometry. Cells were gated on live cells. One representative example is shown (n =3).B, CD4+ and CD8+ T cells were isolated from PBMCs of three healthy blood donors by FACS or MACS. Purified CD4+ T cells and CD8+ T cells were cultured either alone or together in the presence of 100 IU/ml of IL-2 for 10 d. Thereafter, CD86 expression on CD4+ T cells and CD8+ T cells was analyzed by flow cytometry. Results from three independent experiments are shown and are expressed as the percentage of CD86+ cells among CD4+ or CD8+ T cells. C, Expression of CD86 mRNA in T cells prior (NC) and after incubation with IL-2 (day 21) was analyzed by real-time RT-PCR. Human primary monocytes were used as positive controls (n =2).D, CD3+CD252CD862 T cells were purified by flow cytometry-based cell sorting and cultured for 10 d with IL-2 (100 IU/ml). Expression levels of CD86 and CD25 were monitored by flow cytometry before and after IL-2 exposure. Representative results from one of three independent experiments with similar results are shown (n =3). cifically, isolated T cells were stimulated with anti-CD3/anti- a on the IL-2–dependent CD86 upregulation in T cells, we cul- CD28 Ab-coated cell-sized beads in the presence or absence of tured PBMCs with IL-2 (100 IU/ml) alone or in combination with anti–IL-2 and anti–IL-2Ra (anti-CD25)-blocking Abs. Although IFN-a (IFN-a–2a, 100 IU/ml). IFN-a alone was used as a nega- incubation of T cells with anti-CD3/anti-CD28–coated beads resulted in a significant increase in CD86 expression on T cells (Fig. 4), we noted that addition of anti–IL-2– and anti–IL-2R– blocking Abs inhibited the upregulation of CD86 expression on activated T cells (Fig. 4). Therefore, the results suggested that CD86 upregulation on activated T cells might be primarily due to an autocrine mechanism that is mediated via secreted IL-2. CsA and Rapa block IL-2–dependent CD86 upregulation on T cells CsA and Rapa are immunosuppressants, which are commonly used in the context of transplantation, and are known to prevent T cell- mediated graft rejections. Importantly, both compounds interfere with IL-2 secretion or IL-2–dependent signaling in T cells. To examine whether these compounds might have an impact on CD86 upregulation by IL-2 on T cells, we cultured T cells with IL-2 in the presence or absence of CsA or Rapa. Interestingly, both CsA and Rapa markedly reduced the number of CD86+ T cells in the presence of IL-2 (Fig. 5A). CsA and Rapa prevented or reduced CD86 expression in CD45RO2 naive, as well as CD45RO+ effector or memory, T cells (Fig. 5B). These results indicated that upregulation of CD86 on human T cells by IL-2 is regulated, at least in part, via the NFAT and mTOR pathways. IFN-a has an inhibitory effect on IL-2–dependent upregulation FIGURE 4. CD86 upregulation on activated T cells is due to secreted + + of CD86 on T cells IL-2. Isolated CD4 /CD8 T cells were activated with CD3/CD28 beads (T cell expander) in the presence of isotype-control Ab (IgG1, 20 mg/ml) IFN-a is frequently used in combination with IL-2 for the treat- or anti-IL-2 (10 mg/ml) and anti-CD25 mAb (10 mg/ml) for 6 d; thereafter, ment of cancer patients (21, 35). To determine the effects of IFN- CD86 expression was analyzed by flow cytometry (n = 4). 6 IL-2 UPREGULATES CD86 ON HUMAN CD4+ AND CD8+ T CELLS

STAT5 is involved in IL-2–mediated upregulation of CD86 on T cells. IL-2–dependent CD86 upregulation on T cells does not provide costimulatory signals We conducted costimulation assays to evaluate whether IL-2– induced CD86 upregulation on T cells might have functional consequences and affect the activation of T cells. For this pur- pose, CD3+CD86+ and CD3+CD862 T cells were sorted, fixed, and cocultured with CFSE-labeled naive CD4+ T cells in combination with anti-CD3–conjugated beads. In our assays, CD86 expressed on IL-2–treated T cells showed no apparent costimulatory effect (Fig. 7). CD86 levels on T cells are upregulated in vivo by IL-2 To investigate the impact of IL-2 in vivo, we collected PBMCs from IL-2–treated patients and studied the levels of CD86 on T cells. Increased levels of CD86 on T cells from IL-2–treated patients appeared to correlate with our in vitro experiments (Tables II, III). CD86 expression increased from 1.8% on day 0 to 41.2% on day 7 in patient 1 (Tables II, III). Similarly, .32% of the second patient’s T cells expressed CD86 during treatment (Tables II, III). In patients 3 and 4, after 7 d of therapy, up to 48.7% of T cells were CD86+ (Tables II, III). In patients 1 and 2, the upregulation of CD86 expression was more evident in CD8+ T cells than in CD4+ T cells (Tables II, III). After 7 d of stimulation, up to 42% of CD8+ T cells were CD86+ compared with up to 22% of CD4+ CD86+ T cells. In patients 3 and 4, initially the expression of CD86 FIGURE 5. CsA and Rapa block IL-2–dependent upregulation of CD86 was more evident on CD4+ T cells; however, on day 7, percentage expression on T cells, and IFN-a also has an inhibitory effect. A and B, of CD86+ cells was equal or slightly higher in the CD8+ T cell PBMCs were cultured with IL-2 (100 IU/ml) alone or with 0.5 mg/ml + CsA or 100 nM Rapa for 10 d. Thereafter, expression of CD86 (A) and subset compared with the CD4 counterpart. Taken together, these CD45RO (B) was analyzed on CD3+ T cells by flow cytometry (n = 3). results indicated that IL-2 upregulates expression of CD86 (B7.2) + + C, PBMCs were cultured in vitro with 100 IU/ml IL-2 and/or 100 IU/ml on human CD4 and CD8 T cells in vivo. IFN-a for 10 d. CD86 expression on T cells was analyzed by flow cytometry. Results are from four different experiments (n = 4). Discussion The costimulatory molecules CD80 and CD86 are predominantly tive control and caused no induction of CD86 in T cells, whereas expressed on professional APCs (i.e., DCs, macrophages, and ac- IL-2 alone markedly increased the frequency of CD3+CD86+ tivated B cells) (2). Both molecules, which belong to the B7 family T cells significantly. As shown in Fig. 5C, when cultured with 100 of proteins, either interact with the activating receptor CD28 or IU/ml of IL-2 alone for 10 d, 35–45% of T cells expressed CD86. with the inhibitory receptor CTLA-4 on T cells and modulate the For a comparison, only ∼2% of cells became CD86+ when cul- activation of these cells. However, expression of CD80 and tured with IFN-a alone. However, when T cells were cultured with CD86 on nonprofessional APCs is not well studied. Because both cytokines together, the percentage of CD86+ T cells was CD86 was also recently detected on allergen-specific T cells (9), lower compared with cells cultured with IL-2 alone (Fig. 5C). tumor-infiltrating lymphocytes (11), and T cells from HIV- These findings held true for both CD4+ and CD8+ T cells (data not infected (12) and hepatitis C virus-infected individuals (13), we shown). Thus, IFN-a appears to have an inhibitory effect on the examined the role of IL-2 in the upregulation of CD86 on T cells IL-2–mediated upregulation of CD86 on T cells. in this study. We noted that exposure to IL-2 alone markedly increased expression of CD86 on T cells. Furthermore, IL-2 CD86+ T cells exhibit higher levels of p-STAT5 upregulated CD86 expression on T cells in a time- and dose- STAT5 was shown to mediate IL-2 signaling in T cells. We dependent manner, and it was restricted to T cells with an effector/ 2 monitored levels of STAT5 in CD86+ and CD86 T cells by in- memory phenotype. tracellular staining and flow cytometry in response to IL-2 treat- + + ment. Levels of p-STAT5 were significantly higher in CD86+ Upregulation of CD86 by IL-2 in CD4 and CD8 T cell T cells compared with their CD862 counterparts (Fig. 6A). We subsets further determined the levels of p-STAT3 in CD86+ and CD862 IL-2–dependent upregulation of CD86 in CD4+ and CD8+ T cell T cell subsets. In contrast to p-STAT5, only minor differences in subsets exhibited different time-dependent expression patterns the p-STAT3 levels were observed (Fig. 6B). (Fig. 1). Although a decreased percentage of CD86 positivity on To further examine whether other members of the g-chain CD4+ T cells was noted after 10 d, CD8+ T cells appeared to cytokines, IL-7 and IL-15, might also affect CD86 expression on have sustained CD86 expression for up to 21 d. Our study also T cells, PBMCs from healthy donors were cultured with IL-7, showed that isolated naive and effector/memory CD4+ and CD8+ IL-15, or IL-2 for up to 10 d, and CD86 on T cells was analyzed T cells had clear differences in terms of IL-2–dependent CD86 by flow cytometry. Only IL-15 was able to upregulate CD86 on upregulation (Fig. 2). IL-2 exposure to naive CD8+, but not naive T cells similarly to IL-2, whereas IL-7 did not show such CD86 CD4+, T cells led to a marked increase in CD86 expression. upregulation (Fig. 6C). Taken together, the results indicated that However, in the case of effector/memory CD4+ and CD8+ T cells, The Journal of Immunology 7

FIGURE 6. CD86+ T cells have higher levels of p-STAT5, and IL- 15 induces CD86 expression on T cells. A and B, CD86+ T cells have higher levels of p-STAT5. PBMCs were cultured in vitro with 100 IU/ml IL-2 for 10 d; thereafter, levels of p- STAT5 and p-STAT3 were analyzed by ﬂow cytometry after intracellular staining. C, PBMCs were cultured in vitro with 100 U/ml of IL-7, IL-2, or IL-15 for 10 d and analyzed by ﬂow cytometry for CD86 expression on CD3+CD4+ and CD3+CD8+ T cells (n = 4).

exposure to IL-2 caused a marked increase in CD86 expression may occur in vivo, the IL-2–dependent increase in CD86 ex- on both of these T cell subsets. This observation showed addi- pression does not seem to be explained by such a mechanism. tional differences within CD4+ and CD8+ T cell subsets in terms of IL-2–dependent CD86 upregulation. Results from our ex- Mechanisms of IL-2–dependent CD86 upregulation in T cells periments indicated that these differences might be due to the Experiments with purified T cells showed that CD86 expression on differences in the expression levels of IL-2Ra,IL-2Rb,and CD4+ and CD8+ T cells is not dependent on the presence of by- IL-2Rg. stander cells; rather, it resulted from increased expression of CD86 Because exposure to IL-2 resulted in a similar or higher per- mRNA in IL-2–treated CD86+ T cells (Fig. 3C). We also evalu- centage of CD86+CD8+ T cells compared with CD8+ T cells, ated whether the occurrence of the high percentage of CD86+ which were cultured alone without CD4+ cells (Fig. 3B), the effect T cells after 7–10 d of IL-2 exposure resulted from the expansion of IL-2 on CD86 in isolated CD4+ or CD8+ T cells appeared to be of the initially existing CD86+ T cells. Our experiments with 2 2 independent of the presence of both T cell subsets. This finding is purified CD25 CD86 T cells showed that IL-2 exposure can in contradiction to earlier work with HIV patients, in which it was upregulate CD86 expression, even on T cells, despite the absence suggested that the upregulation of CD86 expression on CD8+ of CD25+ or CD86+ T cells (Fig. 3D). T cell might be dependent on the presence or absence of CD4+ To further investigate the mechanism of IL-2–mediated CD86 T cells (8). Earlier studies suggested that T cells can acquire CD86 upregulation in T cells, we applied the pharmacological inhibitors and other surface proteins simply by membrane exchange with CsA and Rapa, which are known to interfere with IL-2 signaling in APCs (32, 33) or by exosome uptake (34). To rule out such by- T cells. CsA is known to prevent the dephosphorylation of NFAT stander effects, we performed supplementary in vitro experiments by binding to cyclophilin. Our experiments showed that CsA to assess the expression of CD86 on T cells using highly purified prevented IL-2–induced CD86 expression on T cells, suggesting CD4+ and CD8+ T cells (Fig. 3B). A notable increase in CD86+ that NFAT is involved in CD86 induction. Unlike CsA, Rapa is T cells was observed within 10 d (Fig. 3), suggesting that, al- known to inhibit IL-2 signaling and to block T cell activation by though some acquisition of CD86 molecules from adjacent cells inhibiting the mTOR pathway. A significant reduction in IL-2–

FIGURE 7. IL-2–upregulated CD86 on T cells does not provide costimulatory signals. CFSE-stained naive T cells from different donors were cultured or not with anti-CD3–conjugated beads, anti-CD3/CD28–conjugated beads, or anti-CD3–conjugated beads in combination with PFA-ﬁxed CD86+ T cells or PFA-ﬁxed CD862 T cells. For negative controls, CFSE-stained naive T cells were cultured or not with only anti-CD3–conjugated beads. Representative results from one of eight different experiments are shown. 8 IL-2 UPREGULATES CD86 ON HUMAN CD4+ AND CD8+ T CELLS

Table II. Increased expression of CD86 on T cells of patients during lated: 1) our results from the RT-PCR experiments and flow IL-2 therapy cytometric analysis showed that CD86 mRNA and protein expression on T cells was markedly lower compared with mature DCs + + CD86 CD3 T Cells (%) or monocytes (Figs. 1A,3C). Therefore, it is also likely that CD86 Patient Information Day 0 Day 7 expressed on T cells was unable to provide the necessary costimulatory signal simply because of their lower expression levels Patient 1 (mRCC) 1.8 41.2 Patient 2 (melanoma) 4.5 32.7 on T cells. 2) In contrast, it is possible that the interactions of Patient 3 (melanoma) 2.1 34.1 CD86 expressed on T cells with CTLA-4 is stronger compared Patient 4 (melanoma) 4.3 48.7 with binding with CD28. However, binding to CTLA-4 is not mRCC, Metastatic renal cell carcinoma. likely the main cause, because we did not note an inhibition of basal proliferation of responder cells (Fig. 7). Preliminary experiments showed that CD86 on IL-2–treated T cells did not bind induced CD86 expression on T cells by Rapa also indicated the to CD28, but it had significant binding affinity for CTLA-4 (data involvement of mTOR in CD86 upregulation in T cells. Moreover, not shown). Our experiments showed that CD28-Ig and CTLA-4– + in our studies, we noted that STAT5 levels in IL-2–treated CD86 Ig bind equally to monocytic cells. Moreover, a polyclonal goat 2 T cells were significantly higher compared with their CD86 coun- anti-CD86 Ab blocked the binding of CTLA-4–Ig to the CD86+ terparts, indicating a role for STAT5 proteins in IL-2–mediated T cells, indicating that only CD86 on these T cells contributed to CD86 upregulation in T cells. However, the exact interactions of CTLA-4 binding (data not shown). CTLA-4 plays a critical role in the STAT5 pathway with the NFAT and/or mTOR cascades in this the regulation of activated T cells by providing inhibitory effects regulation are unclear. IFN-a was shown to affect IL-2–induced (43), and it possesses greater binding affinity toward CD86 than changes in human T cells (36, 37). Our current observations in- CD28. The level of CTLA-4 expression in naive T cells is known dicated that IFN-a negatively modulates the IL-2–induced to be significantly lower, and it increases significantly after suc- upregulation of CD86 expression on T cells, which correlates with cessful activation (44, 45). Especially at sites of ongoing inflam- these earlier reports (36, 37). However, because of the pleiotropic mation densely populated by activated T cells, CD86 expressed on nature of IFN-a and differential effects on different cells types, T cells might play a regulatory role simply by interacting with further studies are necessary to understand the detailed cross-talk CTLA-4 expressed on activated T cells (45). Therefore, it is likely of the signaling cascades by IL-2 and IFN-a in T cells and the that IL-2–mediated overexpression of CD86 on T cells might play potential relevance. a role in controlling the IL-2–mediated autoimmunity and in- Clinical implications of the IL-2–dependent CD86 creased inflammatory response, and it may help to prevent tissue upregulation on T cells damage. Future in-depth studies should reveal more on this issue. It is important to note that, in addition to our in vitro data, we Increased expression of CD86 on T cells might have far-reaching noted a marked upregulation of CD86 on T cells in all IL-2–treated implications for the T cell-mediated immune response. It is well patients tested (Table II). Similar to our in vitro observations, we known that interactions of peptide-MHCs with TCRs are modu- were unable to detect significant changes in the expression of lated by CD28-CD80/CD86 binding, which can augment the CD80 on T cells in these IL-2–treated patients. Because our study upregulation of Bcl-xL (intrinsic cell survival factor) and IL-2 in showed that IL-2 upregulates CD86 on T cells in vitro, as well as T cells (38–41). Increased and/or uncontrolled expression of CD86 in vivo, this event is likely to have major physiological signifi- and CD80 in specific pathological conditions may cause autoim- cance and might have an important overall impact on the clinical mune responses (4–7). Similarly, Stephan et al. (42) demonstrated outcome. In this regard, it is also remarkable that IL-2 was the first that the expression of costimulatory molecules, such as CD80 and cytokine to be approved by the U.S. Food and Drug Administra- 4-1BBL, can cause auto- and trans-costimulation in T cells, which tion (in 1992) for the treatment of advanced kidney cancer (46). significantly improved immune responses against tumors in ex- Since then, this cytokine has been widely used for the treatment perimental conditions. IL-2 is known to be important for T cell of different malignant and infectious diseases (17, 23–26, 47). activation and proliferation in vivo and in vitro; therefore, it was However, positive clinical responses after IL-2 therapy remained initially termed “T cell growth factor” (18, 19). Because of the limited. Recently, it was discovered that IL-2 also plays a crucial present observations, it is tempting to suggest that IL-2 may not role in immunoregulation (48). Thus, a so-called “yin-and-yang only act as a T cell growth factor, but may also indirectly enhance effect” of this cytokine (48) has added new dimensions and raised the immune response by inducing the expression of CD86 on concerns about the therapeutic use of IL-2. However, the existing T cells, which, in turn, could support T cell activation. evidence of its effectiveness (22, 46, 49–51) cannot be completely Our in vitro costimulation assays indicated that, in the absence of ignored. It is also noteworthy that moderate, reversible autoim- any supplementary cytokines, CD86 expressed on T cells did not munity during cytokine therapy has been reported and was asso- show apparent costimulatory effects. Two reasons can be postu- ciated with better cure and survival in cancer patients (51). Thus, it is tempting to speculate that IL-2–induced CD86 expression and Table III. Increase in CD86 expression on CD4+ and CD8+ T cells its interactions with CTLA-4 expressed on activated T cells might during IL-2 therapy be a mechanism involved in controlling the excessive inflammatory responses. At the same time, this potential blunting effect + + + + CD86 CD4 T Cells (%) CD86 CD8 T Cells (%) might be a reason for the limited curative response associated Patients Day 0 Day 7 Day 0 Day 7 with IL-2 therapy. Further studies are necessary to evaluate such effects. In recent years, in addition to IL-2, other members of Patient 1 3.4 21.8 1.2 42.0 common g-chain cytokines, especially IL-7 and IL-15, have been Patient 2 2.0 12.4 0.6 38.7 Patient 3 1.2 32.0 0.9 40.8 tested or used to boost immune responses for the treatment of Patient 4 4.6 48.2 1.1 49.4 various diseases (17, 52, 53). Thus, the IL-2–like effect of IL-15 PBMCs isolated from blood of patients (n = 4) undergoing IL-2 therapy were on CD86 expression on T cells (Fig. 6C) might be relevant to analyzed by flow cytometry for expression of CD86 before and after 1 wk of therapy. understanding the clinical outcome of their therapeutic usage. The Journal of Immunology 9

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