Journal of Immunological Methods

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1 Journal of Immunological Methods 39 (213) Contents lists available at SciVerse ScienceDirect Journal of Immunological Methods journal homepage: Research paper Pitfalls in determining the cytokine profile of human T cells Ingrid Olsen a,b,, Ludvig M. Sollid a,c a Centre for Immune Regulation and Department of Immunology, Oslo University Hospital-Rikshospitalet, 27 Oslo, Norway b Section for Immunology, Norwegian Veterinary Institute, Oslo, Norway c Centre for Immune Regulation and Department of Immunology, University of Oslo, 27 Oslo, Norway article info abstract Article history: Received 2 September 212 Received in revised form 7 December 212 Accepted 25 January 213 Available online 15 February 213 Keywords: T cells IL-1 IL-17 Cytokines PMA/Ionomycin Anti-CD3/anti-CD28 Secretion of cytokines by T cells in vitro can be influenced by the methods chosen for T cell activation. However, the awareness of this fact appears insufficient. Two of the most widely applied methods for activation of T cells are phorbol 12-myristate 13-acetate (PMA) together with Ionomycin or anti-cd3/anti-cd28 stimulation. We analyzed production of IL-4, IFN-γ, IL-17 and IL-1 by a panel of human CD4 T-cell clones isolated from intestinal biopsies using the Bio-Plex assay and also flow-cytometry for the latter three cytokines. Higher levels of IL-17 and IFN-γ were produced by stimulation with PMA/Ionomycin compared to anti-cd3/ anti-cd28. Some T-cell clones which were assigned to produce both cytokines by stimulation with PMA/Ionomycin, were only assigned to produce IFN-γ by anti-cd3/anti-cd28 stimulation. IL-1 production was higher after anti-cd3/anti-cd28 stimulation. Furthermore the dose response curve for PMA/Ionomycin differed for IL-1 compared to IL-17 and IFN-γ as it was biphasic with no IL-1 production at higher PMA/Ionomycin concentrations. These results demonstrated that the cytokine profile may be differently determined depending on the assay and conditions used and illustrate that care should be taken when designing and interpreting studies of cytokine production by T cells. 213 Elsevier B.V. All rights reserved. 1. Introduction CD4 T cells are traditionally classified as Th1, Th2, Th17 or Treg cells. In vitro differentiation of naïve CD4 T cells into various subsets has been fairly well described, and the classification of the subsets is usually based on the production of hallmark cytokines like IFN-γ, IL-4, IL-17 and IL-1 together with the expression of defined transcription factors (Zhu and Paul, 21). The paradigm considering CD4 T-cell subsets as distinct lineages has, however, been questioned with the recognition of additional subsets as Th9 and Th22 cells, and the plasticity of CD4 T helper cells has been discussed in many papers (O'Shea and Paul, 21; Lee et al., 29; Zielinski et al., 212). Cytokine production by Corresponding author at: Norwegian Veterinary Institute, Ullevaalsveien 68, 454 Oslo, Norway. Tel.: ; fax: address: ingrid.olsen@vetinst.no (I. Olsen). T cells is assessed after stimulation using a variety of protocols. If the specificity of the T cells are known, antigens in different forms ranging from synthetized peptides to whole cell extracts are used, while polyclonal stimulations are the option when the antigens are unknown. Two of the most widely applied methods for activation of T cells are phorbol 12-myristate 13-acetate (PMA) together with Ionomycin or anti-cd3/anti-cd28 stimulations. It is known that different in vitro stimulations can have an effect on the results (Baran et al., 21) and different stimulation protocols are often recommended by the manufacturer of reagents to obtain maximal cytokine production. Nevertheless, the awareness of this knowledge appears limited, especially in many studies characterizing T-cell responses in various diseases where the aim is more applied, and not to gain knowledge of basic T-cell immunology. The aim of the present study was to demonstrate how the use of PMA/Ionomycin and anti-cd3/anti-cd28 at different doses can affect the cytokine production from CD4 T-cell clones /$ see front matter 213 Elsevier B.V. All rights reserved.

2 I. Olsen, L.M. Sollid / Journal of Immunological Methods 39 (213) Materials and methods 2.1. T-cell clones CD4+ T-cell clones were derived from intestinal biopsies of patients with Crohn's disease. The generation of T-cell clones has been described previously (Olsen et al., 29) Reagents The following reagents were used for stimulation of T cells: anti-cd3, clone UCHT1, (Biolegend); anti-cd28, Clone CD28.2 (Biolegend); Dynabeads Human T-Activator CD3/CD28 (Life technologies); PMA and Ionomycin (both from SigmaAldrich). Complete cell culture medium; RPMI 164 (Gibco) containing 1% human serum, β mercaptoethanol, penicillin and streptomycin. The following antibodies were used for analyzing T cells by flow cytometry: anti-ifnγ FITC (IgG1κ, clone B27), anti- IL-17a PE (IgG1κ, clone ebio64cap17), anti-il-1 Alexa 647(IgG1κ, clone JES3-9D7) together with relevant isotype controls, all ebioscience. The LIVE/DEAD Fixable Read Dead Cell Stain Kits (Life technologies) were used to distinguish between live and dead cells by flow cytometry Stimulations and Bio-Plex Cytokine Assay T-cell clones, at a concentration of 2 cells/well, were stimulated in vitro in complete medium with PMA/Ionomycin or anti-cd3 and anti-cd28 antibodies using different concentrations and incubation times. The anti-cd3/anti-cd28 stimulations were performed using antibody coated beads (1 bead/ cell) or platebound anti-cd3 (.2 1 μg/ml) together with soluble anti-cd28 (.1 5 μg/ml). Flat-bottomed 96 well plates were incubated with anti-cd3 in PBS at 4 C for 24 h. The plates were washed three times with PBS before addition of T cells and the anti-cd28 antibodies at various concentrations. The supernatant was removed after 6, 12, 24 and 48 h and stored at 7 C until tested. The amount of cytokines in the supernatant was measured using the Bio-Plex Cytokine Assay (Bio-Rad) according to the manufacturer's instructions. FACS CALIBUR flow cytometer (Becton Dickinson), equipped with Cell-Quest software Statistical analyzes The mean difference in cytokine production in the Bio-Plex assay was compared using the paired T-test. The number of live cells assessed by flow-cytometry was compared using the Wilcoxon matched-pairs signed rank test. 3. Results 3.1. Different cytokine profile dependent on stimulation method The different cytokine profile in response to PMA/Ionomycin and anti-cd3/anti-cd28 beads was noted in an experiment where we wanted to investigate the phenotype of CD4 T-cell clones derived from intestinal biopsies of Crohn's disease patients (Fig. 1). All the clones secreted high amounts of IFN-γ using both methods with relatively low amounts of IL-4. A majority of the T-cell clones had the ability to additionally produce IL-17 and/or IL-1. The amount of IFN-γ and IL-17 was significantly higher (pb.1 for IFN-γ and p=.45 for IL-17) after PMA/Ionomycin stimulation compared to stimulation with anti-cd3/anti-cd28 beads. Some clones would not have been recognized as IL-17 producers if only the latter method was used. Even more striking, we observed that the IL-1 production was very prominent after anti-cd3/anti-cd28 bead stimulation for many clones compared to the generally very low levels of this cytokine seen after PMA/Ionomycin stimulation (p=.2). Some clones appeared to shift phenotype from IL-17 to IL-1 production depending on the chosen protocol. IL-4 was produced at low levels, and no significant differences in the IL-4 production were detected by the two stimulation methods Stimulation and staining for intracellular cytokines T-cell clones (approximately 4, cells) were stimulated with high and low concentrations of PMA (1 and 1 ng/ml) and Ionomycin(4 μm,.4 μm), plate coated anti-cd3 (1 and 1 μg/ml) and soluble anti-cd28 (2 and.2 μm) or left unstimulated. Six clones were tested by flow-cytometry except for stimulation with low concentrations of anti-cd3/anti-cd28 (1 μg/ml/.2 μg/ml)where three clones were tested.the cells were incubated for 6 or 24 h and monensin was added for the last 5 and 18 h respectively. The cells were then stained with LIVE/DEAD Fixable Read Dead Cell Stain according to the manufactures instructions followed by staining for intracellular cytokines. Briefly the cells were incubated with the LIVE/DEAD reagent (1/1 dilution in PBS) on ice for 2 min. The cells were washed twice in PBS followed by fixation in 1% paraformaldehyde for 1 h and permeabilization in PBS with 2% FCS and.2% saponin for 3 min. The cells were stained with antibodies against IFN-γ, IL-1 and IL-17a and analyzed on a Fig. 1. Production of IFN-γ, IL-4, IL-17 and IL-1 by CD4+ T-cell clones (n=17) after stimulation with PMA/Ionomycin (5 ng/2 μm) and anti-cd3/anti-cd28 beads (1 bead/cell). The cells were stimulated in duplicates for 24 h. The cytokine production in the supernatant was measured in the Bio-Plex assay. The individual clones are depicted together with the mean±sem. There was significantly less IL-1 and more IFN-γ and IL-17 after PMA/Ionomycin stimulation compared to anti-cd3/anti-cd28 stimulation using the paired t-test.

3 18 I. Olsen, L.M. Sollid / Journal of Immunological Methods 39 (213) Effect of incubation time In our standard protocol we assay cytokine production after 24 h for anti-cd3/anti-cd28 stimulation while we generally used 6 h for PMA/Ionomycin stimulation. We thus wanted to exclude that the disparate observations were a result of incubation time. Seven T-cell clones with different cytokine profiles and high production of IL-4, IL-1 or IL-17 were included. The clones were stimulated with anti-cd3/anti-cd28 and PMA/Ionomycin, and the production of cytokines was measured after 6, 12, 24 and 48 h. The kinetics varied between the different clones, however secretion of IL-17, IFN-γ and IL-1 generally peaked at h while IL-4 that showed a continuous increase in response also peaked at 48 h. The two clones with the highest production of IL-4, IL-17 and IL-1 and the two clones with the lowest IFN-γ production are shown in Fig. 2 since the amount of IFN-γ was above the standard curve for most clones. Stimulation with PMA/Ionomycin appeared to induce a somewhat faster response than anti-cd3/anti-cd28 for IL-17 and IFN-γ. The differences in IL-1 and IL-17 production by the two stimulation methods could not be explained by incubation time Effect of concentration We next wanted to see if the observed differences could be explained by the strength of the stimulation signal, and we thus titrated the amount of PMA/Ionomycin used for stimulation. Based on the time-curve described above, 24 h of stimulation was chosen in this experiment. Five T-cell clones with different cytokine profiles and high production of IL-4, IL-1 or IL-17 were included. The production of IL-4, IL-17 and IFN-γ increased with higher concentrations of PMA/Ionomycin. However, surprisingly, we noted that IL-1 production was induced in some clones when stimulated with the lower concentration of PMA/Ionomycin, but this response waned as the concentrations increased giving biphasic dose response curves (Fig. 3). These clones also secreted large amounts IL-1 after anti-cd3/anti- CD28 bead stimulation. To see if a similar dose response curve could be achieved in these two clones by different doses of anti-cd3/anti-cd28, we decided to cross-titrate the amount of the two antibodies using various concentrations of platebound anti-cd3 together with soluble anti-cd28. The amount of anti-cd28 had minimal effect on the cytokine secretion by the twoclones,whichbothproducedlarge amount of cytokines by anti-cd3 stimulation alone (data not shown). Titration of anti-cd3 stimulation showed monophasic dose response curves forall thefourcytokines(fig. 4). Finally, we also tested cytokine production in PBMC from two healthy donors after stimulation with different doses of both PMA/Ionomycin and anti-cd3/ anti-cd28 for 24 h. Much lower levels of cytokines were detected and classical monophasic dose response curves were observed for all four cytokines (data not shown) Intracellular staining and flow cytometry The effect of various stimulation protocols was also tested by flow cytometry. The number of cytokine producing cells after stimulation with high and low concentrations of anti-cd3/anti-cd28 and PMA/Ionomycin after 6 and 24 h were measured. The highest numbers of cytokine producing cells were generally observed after 6 h. This was particularly pronounced for IL-17 and IFN-γ production after stimulation with PMA/Ionomycin while less so for IL-1 production after stimulation with anti-cd3/anti-cd28 (data not shown). The apparent difference in kinetics between intracellular staining and the Bio-Plex assay can easily be explained by the continuous accumulation of cytokines measured in the latter. For IL-17 and IFN-γ, a higher response was observed after stimulation with the high concentrations of both anti-cd3/ anti-cd28 and PMA/Ionomycin compared to the low concentrations (Fig. 5A). Furthermore PMA/Ionomycin induced higher numbers of IL-17 and IFN-γ producing cells compared to anti-cd3/anti-cd28 which was in agreement with the results IFN-γ IL anti-cd3/cd28, TCC955.A.N.6 anti-cd3/cd28, TCC955.A.E.1 PMA/Ionomycin, TCC955.A.N.6 PMA/Ionomycin, TCC955.A.E.1 anti-cd3/cd28, TCC955.A.N.6 anti-cd3/cd28, TCC918.B.N.3 PMA/Ionomycin, TCC955.A.N.6 PMA/Ionomycin, TCC918.B.N.3 IL-17 IL anti-cd3/cd28, TCC955.A.N.6 anti-cd3/cd28, TCC918.B.N.3 PMA/Ionomycin, TCC955.A.N.6 PMA/Ionomycin, TCC918.B.N.3 anti-cd3/cd28, TCC918.B.N.1 anti-cd3/cd28, TCC918.B.N.3 PMA/Ionomycin, TCC918.B.N.1 PMA/Ionomycin, TCC918.B.N time (hrs) time (hrs) Fig. 2. Production of IFN-γ, IL-4, IL-17 and IL-1 by CD4+ T-cell clones after stimulation with PMA/Ionomycin (5 ng/2 μm) and anti-cd3/anti-cd28 beads (1 bead/cell). The cells were stimulated in triplicates for, 6, 12, 24 and 48 h. The triplicates were pooled and the cytokine production in the supernatant was measured in the Bio-Plex assay.

4 I. Olsen, L.M. Sollid / Journal of Immunological Methods 39 (213) IFN-γ IL IL-17 IL TCC918.B.M.1 TCC99.A TCC946.A.8.2b.17 TCC954.A TCC955.A.M.3 3ng/.125μM 12.5ng/.5μM 5ng/2μM PMA/Ionomycin 2ng/8μM 3ng/.125μM 12.5ng/.5μM 5ng/2μM PMA/Ionomycin 2ng/8μM Fig. 3. Cytokine production by five CD4+ T-cell clones after stimulation with various concentrations of PMA/Ionomycin. The cells were stimulated in duplicates for 24 h. The cytokine production in the supernatant was measured in the Bio-Plex assay. The mean+sd is depicted. from the Bio-Plex assay. In comparison, only anti-cd3/ anti-cd28 and low doses of PMA/Ionomycin induced detectable amounts of IL-1 (Fig. 5B). A shift from IL-1 to IL-17 production when stimulated with high concentrations of anti-cd3/anti-cd28 and PMA/Ionomycin respectively (Fig. 6) was demonstrated which was in agreement with the results from the assay. Furthermore, the intracellular staining also demonstrated that IL-17 and IL-1 generally were coproduced with IFN-γ (Fig. 5), while IL-1 and IL-17 were not produced by the same cells (Fig. 6). A LIVE/DEAD stain was also included to assess the number of live cells after the different stimulation protocols (Fig. 7). The only significant differences in the number of live cells between the two time-points were in the unstimulated samples using the Wilcoxon matched-pairs signed rank test. At 6 h, there were significantly lower numbers of live cells after stimulation with the high concentrations of PMA/Ionomcyin compared to unstimulated samples and cells stimulated with low concentrations of PMA/Ionomycin. No significant differences between the various stimulation protocols where seen at 24 h. cytokine pg/ml Anti-CD3 (µg/ml) TCC918.B.M.1 IL-17 TCC918.B.M.1 IL-1 TCC918.B.M.1 IFN- TCC918.B.M.1 IL-4 TCC955A.M.3a IL-17 TCC955A.M.3a IL-1 TCC955A.M.3a IFN- TCC955A.M.3a IL-4 Fig. 4. Cytokine production in two T-cell clones after stimulation with various concentration of platebound anti-cd3 and soluble anti-cd28 (.2 μg/ml). The cells were stimulated in duplicates for 24 h. The cytokine production in the supernatant was measured in the Bio-Plex assay. The mean+sd is depicted.

5 11 I. Olsen, L.M. Sollid / Journal of Immunological Methods 39 (213) Fig. 5. Intracellular staining for production of cytokines. Theclones were stimulated with high (1 ng/4 μm) and low (1 ng/.4 μm) concentrations of PMA/Ionomycin and high (1 μg/ml, 2 μg/ml) and low (1 μg/ml,.2 μg/ml) concentrations of platebound anti-cd3 and soluble anti-cd28. The samples were stimulated for 6 h and protein transport was blocked by adding monensin for the last 5 h. The plots are gated on live cells using forward scatter and the LIVE/DEAD Fixable Read Dead Cell Stain (FL3). The percentages of cells in each quadrant are shown. A) Production of IFN-γ and IL-17 from clone TCC918.B.M.1. B) Production of IFN-γ and IL-1 from clone TCC955A.M.3a. 4. Discussion We have demonstrated that different methods of T-cell stimulation had profound effect on cytokine production by in vitro expanded human CD4 T-cell clones. PMA/Ionomycin induced higher IFN-γ and particularly IL-17 production, while anti-cd3/anti-cd28 induced large amounts of IL-1. Furthermore, the IL-1 response to PMA/Ionomycin had a surprising biphasic, bell-shaped curve with low IL-1 secretion in response to higher concentrations. Stimulation with anti-cd3/anti-cd28 activates T cells via the T-cell receptor (TCR) complex. Studies have shown that this can lead to enhanced IL-1 production byth1 cells particularly at high antigen doses and repeated triggering (Saraiva et al., 29). This is in line with our results where higher doses of anti-cd3/anti-cd28 led to higher IL-1 production. In comparison, PMA activates Protein Kinase C while Ionomycin is a calcium ionophore, and stimulation with these compounds bypasses the TCR complex and will lead to activation of several intracellular signaling pathways (Truneh et al., 1985). Based on our results it appears that the strength of the stimulation signal when using PMA/Ionomycin can determine the cytokine profile. A pro-inflammatory profile was seen after high doses, while lower concentration resulted in more IL-1 production and less IFN-γ and IL-17. A dual production of pro- and anti-inflammatory cytokines from individual Th1 clones has been described in the literature and is believed to be crucial in the auto-regulation of immune responses to avoid immunopathology during infection (Cope et al., 211; Jankovic et al., 27; Gerosa et al., 1999) Whether the observed cytokine profile in the present study was a result of the in vitro expansion or reflected a phenotype present in vivo is not known. However, the clones demonstrated distinct phenotypes despite being expanded using the same protocol. IL-17 and IL-1 were also detected only in a proportion of the clones suggesting that the stimulation by PMA/Ionomycin or anti-cd3/anti-cd28 alone was not sufficient to induce these cytokine. It is therefore plausible that the observed cytokine pattern was influenced by the patient and inflammatory environment where the clones were derived from. PMA/Ionomycincanbetoxictocellathighconcentrations and long incubation times and stimulations for 24 h are usually not recommended. One could thus speculate that the observed lack of IL-1 production could be caused by PMA/Ionomycin toxicity. In the present study, there were no significant differences in the number of live cells at 6 and 24 h after PMA/ Ionomycin stimulation. However, the use of a high dose of PMA/ Ionomycin did result in lower percentages of live cells after 6 h compared to the other stimulation protocols, while this was not evidentat24h.thepercentagesofil-17andifn-γ producing cells when gated on the live population were higher for the high dose of PMA/Ionomycin suggestion that the stronger signal resulted in activation of the proinflammatory pathway at the expense of anti-inflammatory pathways. Whether this was caused by selective death of potential IL-1 producing cells or a shift in cytokine production in the live cells is unclear.

6 I. Olsen, L.M. Sollid / Journal of Immunological Methods 39 (213) Fig. 6. Intracellular staining demonstrating the shift from IL-1 to IL-17 production dependent on stimulation method. T-cell clones were stimulated with platebound anti-cd3 and soluble anti-cd28 (1 μg/ml, 2 μg/ml) and with PMA/Ionomycin (1 ng/4 μm). The upper panel depicts clone TCC955A.M.3a and the lower panel depicts clone TCC918.B.M.1. The samples were stimulated for 6 h and protein transport was blocked by adding monensin for the last 5 h. The plots are gated on live cells using the LIVE/DEAD Fixable Read Dead Cell Stain (FL3). The percentages of cells in each quadrant are shown. Regardless of the mechanisms, the consequence is that the method chosen to characterize cytokine production can have profound effect on the results. The fact that different in vitro stimulations can have an effect on the results is not entirely novel (Baran et al., 21), and different stimulation protocols for the various cytokines are Fig. 7. Percentages of live cells after various stimulation protocols. T cell clones were with; high concentrations of PMA/Ionomycin (1 ng/4 μm; n=6), low concentrations of PMA/Ionomycin (1 ng/.4 μm; n=6) and high (1 μg/ml, 2 μg/ml; n=6) and low (1 μg/ml,.2 μg/ml; n=3) concentrations of platebound anti-cd3 and soluble anti-cd28. The T-cell clones were stimulated for 6 and 24 h and protein transport was blocked by adding monensin for the last 5 and 18 h respectively. The cells were stained with the LIVE/DEAD Fixable Read Dead Cell Stain before permeabilization and staining for intracellular cytokines. Calculations of live cells were based on gating on forward and side scatter plot together with a low signal in FL3 (low uptake of LIVE/DEAD stain). The means± SEM and p-values (Wilcoxon matched-pairs signed rank test) are shown.

7 112 I. Olsen, L.M. Sollid / Journal of Immunological Methods 39 (213) often recommended by the manufacturer of reagents to obtain maximal cytokine production. Expansion of T cells under polarized conditions is sometimes recommended for the detection of IL-1 (Caraher et al., 2), however, there is little focus on the method of choice for the final stimulation step. PMA/Ionomycin was used to activate T cells in the hallmark paper characterizing human Th17 cells (Annunziato et al., 27). Furthermore, Dong et al. used PMA/Ionomycin when suggesting that ex vivo isolated human IL-1 producing T cells were unable to maintain IL-1 expression upon secondary restimulation (Dong et al., 27). The conclusions on these papers may have been modified if anti-cd3/anti-cd28 had been used. In the present study, all the T cells would have been described as pro-inflammatory Th1/Th17 clones if only stimulated with PMA/Ionomycin. PMA/Ionomycin is regarded as rather unphysiological, and it could be argued that anti-cd3/ anti-cd28 stimulation should be the preferred option. However, PMA/Ionomycin is better suited if the aim is to characterize the T cells' ability to secret IL-17. Ex vivo stimulation can never completely mimic the environment present at the site of the effector T cells, and our data demonstrated that care should be taken in the design and interpretation of studies characterizing cytokine production by T cells in vitro. 5. Conclusions All the four cytokines measured could be induced by both PMA/Ionomycin and anti-cd3/anti-cd28 stimulations. However, PMA/Ionomycin induced higher levels of IFN-γ and particularly IL-17 compared to anti-cd3/anti-cd28 stimulation, while IL-1 production was prominent after anti-cd3/anti-cd28 stimulation. Furthermore, the IL-1 response to PMA/Ionomycin had a biphasic, bell-shaped curve with no IL-1 production at higher PMA/Ionomycin concentrations. 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