Functional profile of CD4+ and CD8+ T cells in latently infected individuals and patients with active TB

Tuberculosis xxx (2013) 1e12

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IMMUNOLOGICAL ASPECTS

Functional profile of CD4þ and CD8þ T cells in latently infected individuals and patients with active TBq Nancy D. Marín a, c, Sara C. París a, Mauricio Rojas a, b, Luis F. García a, * a

Grupo de Inmunología Celular e Inmunogenética, Instituto de Investigaciones Médicas and Sede de Investigación Universitaria (SIU), Universidad de Antioquia, Medellín, Colombia Unidad de Citometría de Flujo, Sede de Investigación Universitaria, Universidad de Antioquia, Medellín, Colombia c NDM is the Recipient of a Predoctoral Scholarship from Colciencias, Bogotá, Colombia b

a r t i c l e i n f o

s u m m a r y

Article history: Received 2 October 2012 Received in revised form 17 December 2012 Accepted 22 December 2012

Tuberculosis (TB) is one of the most important infectious diseases around the world. Several studies have focused on the identification of correlates of protection against TB. Most of them have concentrated on the study of IFN-g due to its robust association with protection against TB. However, given the complexity of the immune response elicited after Mtb infection, other cytokines should also be considered. In the present study, we evaluated Th1 and Th17 responses and their association with the protection or development of active disease. Therefore, non infected individuals (nonTBi), latently infected individuals (LTBi) and patients with active TB (ATB) were studied. The evaluation of the number of cytokine producing cells by ELISPOT showed a higher number of IFN-g-producing cells in ATB patients, but no differences were found regarding the number of IL-17 producing cells among studied groups. The evaluation of IFN-g, IL-2, TNF-a and IL-17 producing CD4þ and CD8þ T cells at 1 day and 6 days of stimulation with mycobacterial antigens suggests the presence of functional signatures associated with latency or active TB. The results presented herein suggest the possible use of the evaluation of Th1-type cytokines, such as IFN-g and/or TNF-a, as a correlate of protection against TB; however, these results need to be validated for other groups. Ó 2012 Published by Elsevier Ltd.

Keywords: Polyfunctional T cells CD4þ CD8þ Tuberculosis Th1 Th17

1. Introduction Tuberculosis (TB) is still one of the most important infectious diseases with almost 2 billion infected people worldwide and was responsible for 1.4 million deaths in 2011.1 Over the last decades, considerable efforts have focused on the identification of biomarkers or correlates of protection against TB.2 The measurement of IFN-g in response to specific mycobacterial antigens such as ESAT-6 and CFP10 has proven useful in detecting latent tuberculosis infection and active TB, but not in differentiating them.3e6 Th1 cell responses play a pivotal role in the defense against Mtb infection, mainly by inducing macrophage activation and favoring the control of Mtb replication.7e9 Since the measurement of IFN-g production alone in response to specific mycobacterial antigens does

q This study was supported by Colciencias, (Bogotá, Colombia) grant 1115-40820488 and Estrategia de Sostenibilidad 2010e2011 (Comité para el Desarrollo de la Investigación), Universidad de Antioquia, Medellín, Colombia. * Corresponding author. Grupo de Inmunología Celular e Inmunogenética, Sede de Investigación Universitaria, Cra 53 No. 61-30, Lab. 410, Medellín, Colombia. Tel.: þ57 4 219 6401; fax: þ57 4 219 1060. E-mail address: [email protected] (L.F. García).

not correlate with protection,10 other immune components must also contribute to protection.11 In this regard, multiparameter flow cytometry offers important advantages by detecting specific T cells with multiple functions.12 In addition to IFN-g, TNF-a plays important roles in the protective response against Mtb by contributing to activate effector mechanisms on monocytes and maintaining the integrity of the granuloma.13,14 The importance of TNF-a has been demonstrated by the increased incidence of TB in patients receiving anti-TNF-a therapy for autoimmune diseases.15 Additionally, IL-2, which is produced almost exclusively by activated T cells, is important for the differentiation and survival of activated cells, the maintenance of an adequate effector function and T cell renewal, and the maintenance of memory cells; however, it is also associated with T cell tolerance by inducing regulatory T cells.16e18 IL-2 production has been reported to be reduced in patients with active TB (ATB) compared to healthy controls,19,20 and its levels negatively correlate with the antigen load. Furthermore, antigen specific IL-2 production has been reported to increase after TB treatment.21 The role of other cytokines, such as IL-17, produced by Th17 cells during Mtb infection is less clear. IL-17 has been proposed to play an important role during early stages of the Mtb infection by favoring granuloma formation22 and by increasing the recruitment of IFN-g-

1472-9792/$ e see front matter Ó 2012 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.tube.2012.12.002

Please cite this article in press as: Marín ND, et al., Functional profile of CD4þ and CD8þ T cells in latently infected individuals and patients with active TB, Tuberculosis (2013), http://dx.doi.org/10.1016/j.tube.2012.12.002

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producing cells to the lung through the up-regulation of chemokine production.23 However, IL-17 may also play a detrimental role in TB by sustaining the differentiation and recruitment of neutrophils into the lung24 through the induction of G-CSF and IL-8 production.25,26 The high levels of neutrophils in TB affected lungs,27 in addition to its deleterious proinflammatory role, may be critical for the necrosis of the granuloma,28 supporting the non-protective role of IL-17 producing cells during Mtb infection. Since the immune response against mycobacterial infections is complex and protection against TB is not mediated by a single component (i.e IFN-g), the contribution of other immune components should be further analyzed. Tcells with the capacity to carry out multiple effector functions are known as polyfunctional T cells,29 and recent studies have shown that polyfunctional T cells provide immune protection in viral infections such as HIV.30 They also correlated with protection in a murine leishmania model31 and in TB vaccines.32e35 However, their roles during Mtb infection are controversial and do not seem to have the same protective efficacy as observed in chronic viral infections.31,36 A higher frequency of cells simultaneously producing IFN-g, TNF-a and/or IL-2 have been detected in ATB compared to latently infected individuals,11,37,38 and their frequency decreased after anti-TB treatment.11 Contrariwise, Harari et al.39 and Day et al.40 found a reduced frequency of polyfunctional T cells in ATB compared to latently TB infected individuals, which is recovered with the anti-TB therapy. A recovery of dual IFN-g/ IL-2 producing cells with the anti-TB therapy was also reported.21,41 The protective immune response against mycobacterial infection according to previous studies seems to be more dependent on quality, as measured by the capacity of T cells to carry out multiple functions, than on its magnitude,42 which is related to the frequency of antigen specific T cells with a particular effector function.29 Since, so far there is no specific cytokine profile clearly associated with immune protection against ATB, the analysis of Th1 and Th17 responses may provide important information for a better understanding of the protective immune response in TB and also may help to identify correlates of protection. Thus, in non infected persons, latently infected and ATB patients, we evaluated the number of IFN-g and IL-17 producing cells by ELISPOT, the frequency of polyfunctional T cells producing IFN-g, TNF-a, IL-2 and IL-17 and the level of these cytokines secreted after 1 and 6 days of stimulation with CFP-10, PPD or MTB. Results suggest that distinct T cell functional signatures are induced in LTBi and ATB following stimulation with mycobacterial antigens. These functional signatures correlate with the state of infection and might also be indicative of the clinical activity of the disease.

than 15 years old were excluded. The study was approved by the Ethics Committee of the Instituto de Investigaciones Médicas of the Universidad de Antioquia and a written informed consent was obtained from all participants. 2.2. Sample preparation Peripheral blood was collected in heparin tubes, and peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density gradient centrifugation (Biowittaker, Walkersville, MD). Recovered PBMCs were counted using a hemocytometer and viability, as tested by trypan blue exclusion, was always 95%. 2.3. Antigens Recombinant CFP-10 was provided by the Department of Microbiology and Immunology at Colorado State University (Fort Collins, CO) through the “Tuberculosis Vaccine Testing and Research Material Contract No. HHSN26266400091C NIH, NIAID N01-AI-40091). PPD (RT50) was obtained from Statens Serum Institute (Copenhagen, Denmark). Irradiated MTB was obtained from BEI Resources (Manassas, VA). 2.4. Elispot

2. Material and methods

The frequency of IFN-g and IL-17 producing cells was evaluated by ELISPOT using human IFN-g and IL-17 ELISPOT kit (eBioscience, San Diego, CA) according to the manufacturer’s instructions, as previously described.44 Briefly, each well in the MultiScreenHTS 96-well filter plates (Millipore. Billerica, MA) was covered with either anti-IFN-g or anti-IL-17 capture antibody overnight at room temperature. Then, wells were blocked with RPMI-1640 (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen) plus Penicillin/streptomycin (Biowittaker, Walkersville, MD) at room temperature for 1e2 h. Finally, 1  105 PBMC/well were cultured in complete medium in the presence of CFP-10 (5 mg/ml) or PPD (10 mg/ml) for 48 h. Non stimulated wells were used as controls. After incubation, the plates were washed and incubated with biotin-labeled antieIFNeg or anti-IL-17 detection antibodies for 2 h at room temperature. The wells were washed again and HRP-streptavidin was added for 45 min, light protected. Finally, AEC substrate (BD Pharmingen, San Diego, CA) was added and the reaction was stopped with distilled water. The spots on the dried plates were counted in an ImmnunoSpot ReaderÒ (CTL, Shaker Heights, OH). Data from nil control were subtracted from stimulated samples. Results are reported as spot forming units (SFU)  106 cells.

2.1. Study population

2.5. Flow cytometry for multifunctional T cells

Fifty-one patients with active TB (ATB) were recruited at the Tuberculosis Control Program in Medellín (Colombia) and its metropolitan area. ATB diagnosis was based on the positive acid fast sputum smear for Mtb and studied before or within the first 2 weeks of anti-TB treatment. Thirty-one subjects with latent TB infection (LTBi) were selected according to an IFN-g positive response to CFP-10, as evaluated by ELISA in seven-day whole blood culture supernatants, as previously reported by our group.43 Eighteen individuals with negative response to CFP-10 were included as a nonTB infected (nonTBi) control group. Individuals from nonTBi and LTBi groups were healthy without clinical evidence of active tuberculosis. Eighty-four percent of ATB, 67% of LTBi and 69% of nonTBi were BCG vaccinated as evaluated by the presence of a BCG scar. Individuals infected with HIV, persons using immunosuppressive drugs, individuals with diabetes, or those younger

In order to evaluate the capacity of CD4þ and CD8þ T cells to produce different cytokines, whole blood 24 h (1 day) and PBMC 144 h (6 days) cultures were performed as recently reported by our group.45 Briefly, for 1 day whole blood cultures, heparinized blood diluted 1:10 in 1 ml of RPMI-1640 (Invitrogen) and supplemented with penicillin/streptomycin (BioWhittakerÒ) were stimulated with 1 mg/ml of anti-CD28 plus anti-CD49d (BD Bioscience) and either CFP-10 (5 mg/ml), PPD (10 mg/ml) or MTB (5 mg/ml) for 24 h. For 6-day cultures, PBMCs were stimulated with CFP-10 (5 mg/ml), PPD (10 mg/ ml) or MTB (5 mg/ml). Non stimulated cells were used as negative control. Four hours before the end of incubation, supernatants were collected and brefeldin A (10 mg/ml) was added. Red cells from the whole blood cultures were lysed with Optilyse (Beckman Coulter) according to the manufacturer’s instructions. Subsequently, the cells were surface stained with anti-CD4-FITC (clone OKT4) (Biolegend,

Please cite this article in press as: Marín ND, et al., Functional profile of CD4þ and CD8þ T cells in latently infected individuals and patients with active TB, Tuberculosis (2013), http://dx.doi.org/10.1016/j.tube.2012.12.002

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San Diego, CA) and anti-CD8-eFluor (clone OKT8) (eBioscience). Then, the cells were fixed, permeabilized and intracellularly stained with anti-IFN-g-PECy7 (clone B27), anti-TNF-a-APC (clone MAb11), anti-IL-2- PerCPCy5 (clone MQ1-17H12) (all from Biolegend) and anti-IL-17-PE (clone eBIO64CAP17 from eBioscience). To evaluate the cell viability of long-term cultures, cells were stained with 7-Aminoactinomycin D (7-AAD). Less than 1% of CD4þ and CD8þ T cells were 7AAD positive. One hundred thousand cells were acquired in a FACS

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Canto II Flow Cytometer (Becton Dickinson Biosciences. San Diego, CA) and analyzed using Tree Star FlowJo Software v7.6.1 (Ashland, OR). For the analysis, doublets exclusion was performed followed by the gating of lymphocytes based on forward scatter (FCS) and side scatter (SSC). Then CD4þ or CD8þ T cells were plotted versus Side Scatter (SSC). Gated CD4þ and CD8þ T cells were evaluated for the frequency of IFN-g, TNF-a, IL-17 or IL-2 producing cells. The analysis with FlowJoÒ software provides the frequency of single or multiple cytokine producer cells. 2.6. Cytokine measurement by Luminex The levels of 17 cytokines and chemokines were measured in supernatants from both 1-day and 6-day cultures stimulated with mycobacterial antigens by MilliplexÒ xMAP Technology, according to the manufacturer’s instructions (Millipore. Billerica, MA). Briefly, each well in 96-well plates was prewet with assay buffer for 10 min and then removed by vacuum. Twenty-five microlitres of each standard, control or samples were added with the appropriate matrix solution and assay buffer. Premixed beads corresponding to the 17 analytes were added and incubated under agitation overnight at 4  C. After incubation, 25 ml of detection antibodies were added into each well and incubated 60 min on a plate shaker followed by incubation with streptavidin-phycoerythrin for 30 min at room temperature. Finally, 150 ml of sheath fluid was added into the wells, mixed for 5 min and the plate was read in the Bio-Plex 200 (BIO-RAD; Hercules, CA). Concentrations in pg were calculated using the standard curve provided with the kit. 2.7. ELISA The levels of interleukin-23 produced in response to mycobacterial antigens were evaluated in supernatants from 1-day cultures using the QuantikineÒ kit for human IL-23 (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. The reading was performed in an ELISA reader (ELx800NB, BioTek Instruments Inc, Winooski, VT) at 450 nm. IL-23 concentrations in the samples were calculated against the standard curve. 2.8. Statistical analysis The number of cells producing IFN-g and IL-17 by ELISPOT were compared using KruskaleWallis test followed by Dunn’s post-test. Two-way ANOVA test was used to compare the frequency of single and multiple producer cells evaluated by flow cytometry. Wilcoxon signed rank test was used to compare the functional composition of CD4þ and CD8þ T cells from 1-day and 6-day cultures. The levels of cytokines and chemokines released into supernatants were compared between LTBi and ATB by ManneWhitney test. Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, Inc, La Jolla, CA). A p < 0.05 was considered statistically significant. 3. Results 3.1. ATB patients have increased number of IFN-g producing cells in the ELISPOT

Figure 1. Number of IFN-g- and IL-17 producing cells in response to mycobacterial antigens. PBMC were stimulated with CFP-10 or PPD for 48 h and the number of IFN-g(B) and IL-17- (C) producing cells was evaluated by ELISPOT. A representative example of the IFN-g- and IL-17 producer cells elicited upon stimulation with CFP-10 and PPD in one LTBi is shown (A). Cells from nonTBi, LTBi and ATB were plated at 1.5  106 cells per well for 48 h in the presence of CFP-10 or PPD. The results are reported as spot forming units (SFU) per 1  106 cells. KruskaleWallis test with Dunn’s post-test. Lines represent median. *p < 0.05; **p < 0.01; ***p < 0.001. nonTBi n ¼ 10e11, LTBi n ¼ 12e14, ATB ¼ 14e18.

To evaluate the specific Th1 and Th17 responses to mycobacterial antigens in nonTBi, LTBi and ATB patients, PBMC were stimulated with CFP-10 and PPD and then the number of IFN-g- and IL-17 producing cells was determined by ELISPOT. After 48 h of incubation, no spots, or a negligible number of them, were detected in unstimulated wells (Figure 1A). There was a higher number of IFNg-producing cells following CFP-10 and PPD stimulation compared

Please cite this article in press as: Marín ND, et al., Functional profile of CD4þ and CD8þ T cells in latently infected individuals and patients with active TB, Tuberculosis (2013), http://dx.doi.org/10.1016/j.tube.2012.12.002

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to the number of cells producing IL-17 (Figure 1). ATB had an increased number of IFN-g-producing cells compared to nonTBi in response to CFP-10 (p < 0.01) and PPD (p < 0.001), and also compared to LTBi after PPD stimulation (p < 0.001) (Figure 1B). No differences were observed in the number of responding cells between nonTBi and LTBi groups, irrespective of the antigen. Stimulation with PPD induced a higher number of IFN-g-producing cells in ATB compared to CFP-10 (p < 0.01). No differences were found in the number of IL-17 producing cells among the studied groups, except in ATB patients in whom PPD induced a higher number of cells compared to CFP-10 (p < 0.01) (Figure 1C). The high number of IFN-g producing cells observed in ATB using the ELISPOT assay suggests that ELISPOT may be useful in detecting ATB and discriminating ATB from latent TB infection, but cannot differentiate non infected from latently infected individuals. 3.2. PPD induces a higher Th1 response compared to CFP-10 and MTB The higher number of IFN-g producing cells observed in ATB compared to nonTBi and LTBi upon stimulation with mycobacterial antigens led us to investigate whether these cells are able to produce other cytokines and are also differentially present in ATB compared to nonTBi and LTBi. Therefore, the frequency of mono and polyfunctional CD4þ and CD8þ T cells with the capacity to produce IFN-g, TNF-a, IL-2 and IL-17 was determined by intracellular flow cytometry in 1-day and 6-day cultures after CFP-10, PPD and MTB stimulation. Figure 2 illustrates the gating strategy used to identify CD4þ and CD8þ T cells producing single or multiple cytokines. Comparative analysis of the frequency of cytokine producing cells induced after CFP-10, PPD or MTB stimulation showed that in 1-day cultures, CFP-10 (p < 0.05) and PPD (p < 0.001) stimulation induced a greater frequency of CD4þIFN-gþ T cells in ATB compared to MTB (Figure 3A). Also, stimulation with PPD induced

in ATB a greater frequency of CD4þTNF-aþ (p < 0.05) compared to the MTB stimulation in the same group (Figure 3D). At 6 days, the frequency of CD4þIFN-gþ T cells induced in LTBi and ATB after PPD stimulation was higher compared to those induced by CFP-10 (p < 0.01 for LTBi and p < 0.001 for ATB) and MTB (p < 0.05 for LTBi and p < 0.001 for ATB) (Figure 3E). PPD stimulation also induced a high frequency of CD4þTNF-a producing cells in LTBi compared to CFP-10 (p < 0.001) and MTB (p < 0.01) stimulation, whilst in ATB the frequency of CD4þTNF-aþ T cells was higher only compared to MTB stimulation (p < 0.01) (Figure 3H). No differences were found in the frequency of IL-2 and IL-17 CD4þ producing cells following CFP-10, PPD or MTB stimulation in 1-day (Figure 3B, C) or 6-day cultures (Figure 3F, G). Regarding CD8þ T cells, PPD stimulation induced a higher frequency of CD8þIL-17 þ T cells in nonTBi compared to CFP-10 (p < 0.05) or MTB (p < 0.05) stimulation. In addition, stimulation with MTB also induced a higher frequency of CD8þTNF-aþ T cells in LTBi than those induced with CFP-10 (p < 0.05) and PPD (p < 0.05) (data not shown). These results showed that CFP-10 and PPD stimulation induced a higher response compared to MTB. Likewise, LTBi and ATB showed a higher response compared to nonTBi, mainly in CD4þ T cells and, at a lesser extent, in CD8þ T cells. 3.3. CD4þ cells from ATB have a higher IFN-g response in 1-day cultures, while LTBi cells show the highest IFN-g and TNF-a response in 6-day cultures The frequency of mono and polyfunctional T cells was evaluated in 1-day and 6-day cultures. Analysis of CD4þ and CD8þ T cells following stimulation with mycobacterial antigens showed that T cells producing only one cytokine (i.e. IFN-g, IL-2, IL-17 or TNF-a) were more frequent than T cells producing different combinations of these cytokines (Figures 4 and 5). At 1 day, PPD induced a higher proportion of CD4þIFN-gþ T cells in ATB compared to nonTBi (p < 0.01), but no differences were found in

Figure 2. Representative flow cytometry analysis showing the gating strategy for identification of single and polyfunctional CD4D and CD8D T cells. Fresh whole blood and PBMC were cultured for 1 day or 6 days, respectively, in the presence of CFP-10, PPD and MTB. At the end of the cultures, cells were permeabilized and stained for IFN-g, IL-2, TNFa and IL-17 as described in materials and methods and then analyzed for intracellular cytokines. Non stimulated cultures served as control. Figure shows a representative example from one LTBi upon PPD stimulation for 6 days. The analysis was similar for CD4þ and CD8þ T cells. Briefly, after doublets exclusion, CD4þ and CD8þ T cells were gated versus FSC-A and analyzed for intracellular TNF-a, IFN-g, IL-17 and IL-2. Analysis was done with the FlowJoÒ software using the combination gate tool in order to obtain the frequency of single and multiple producer cells.

Please cite this article in press as: Marín ND, et al., Functional profile of CD4þ and CD8þ T cells in latently infected individuals and patients with active TB, Tuberculosis (2013), http://dx.doi.org/10.1016/j.tube.2012.12.002

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Figure 3. Frequency of single cytokine producer CD4 T cells stimulated with CFP-10, PPD and MTB in 1-day and 6-day cultures. One-day whole blood cultures (AeD) and 6-day PBMC cultures (EeH) from nonTBi (white bars), LTBi (grey bars) and ATB (black bars) were stimulated with CFP-10, PPD or MTB. CD4þ T cells were stained as described in materials and methods, and then analyzed for intracellular IFN-g (A, E), TNF-a (B, F), IL-17 (C, G) and IL-2 (D, H) expression. Single cytokine producer cells were selected as shown in Figure 2. Two-way ANOVA with Bonferroni post-test. *p < 0.05; **p < 0.01; ***p < 0.001. Bars represent Mean  95% IC. nonTBi n ¼ 4, LTBi n ¼ 6, ATB ¼ 11.

the frequency of IL-2, IL-17 or TNF-a single producer T cells, nor in the frequency of polyfunctional T cells among the studied groups (Figure 4B). Also, there were no differences in the frequency of single or polyfunctional T cells induced by CFP-10 (Figure 4A) or MTB (Figure 4C) stimulation among the groups. At 6 days, CFP-10 stimulation induced a high frequency of CD4þTNF-aþ T cells in ATB compared to nonTBi (p < 0.05) (Figure 4D). In contrast, the frequency of CD4þIFN-gþ (p < 0.05) and CD4þTNF-aþ (p < 0.001) T cells induced in LTBi following PPD stimulation was higher compared to nonTBi (Figure 4E), but

no differences were found compared to ATB. MTB stimulation also induced a lower frequency of CD4þIFN-gþ (p < 0.001), CD4þTNF-aþ (p < 0.001) and CD4þIFN-gþTNF-aþ (p < 0.001) T cells in nonTBi and ATB, compared to LTBi (Figure 4F). The proportions of CD4þ T cell subsets producing 1, 2, 3 or 4 cytokines in nonTBi, LTBi and ATB after in vitro stimulation with CFP10 are shown in Figure 4G. Cells producing one cytokine were the most common, whereas the proportion of cells producing 3 or 4 cytokines were the least common T cell subsets. No differences were found in the frequency of polyfunctional T cells between

Please cite this article in press as: Marín ND, et al., Functional profile of CD4þ and CD8þ T cells in latently infected individuals and patients with active TB, Tuberculosis (2013), http://dx.doi.org/10.1016/j.tube.2012.12.002

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Figure 4. Frequency of single or polyfunctional CD4D T cells stimulated with CFP-10, PPD and MTB in 1 day and 6 days cultures. One-day whole blood cultures (AeC) and 6 days PBMC cultures (DeF) from nonTBi (white bars), LTBi (grey bars) and ATB (black bars) were stimulated with CFP-10, PPD or MTB. Pie charts show the relative proportions of CD4þ T cells producing 4, 3, 2 or 1 cytokine in nonTBi, LTBi and ATB after in vitro stimulation with CFP-10 (G). CD4þ T cells were stained as described in materials and methods, and then analyzed for intracellular IFN-g, TNF-a, IL-17 and IL-2. Single producer cells were selected as shown in Figure 2 and then analyzed using the combination gate tool from FlowJoÒ software. Two-way ANOVA with Bonferroni post-test. *p < 0.05; **p < 0.01; ***p < 0.001. Bars represent Mean  95% CI. nonTBi n ¼ 4e5, LTBi n ¼ 6, ATB ¼ 11.

LTBi and ATB, except after MTB stimulation, where LTBi had a higher frequency of CD4þIFN-gþTNF-aþ T cells (p < 0.001). 3.4. CD8þ cells from LTBi show a higher IFN-g response in 1-day cultures, while ATB cells show the highest IFN-g response following CFP-10 and PPD stimulation in 6-day cultures Regarding CD8þ T cells, the frequency of CD8þIFN-gþ T cells was higher than the frequency of CD8þ T cells producing other

cytokines or a combination of them (Figure 5). At 1 day, CFP-10 stimulation induced a lower frequency of CD8þIFN-gþ T cells in ATB compared to LTBi (p < 0.05) (Figure 5A), and PPD also had the same effect in nonTBi (p < 0.01) and ATB compared to LTBi (p < 0.01) (Figure 5B). No differences were found in the frequency of CD8þ T cells producing more than one cytokine among the studied groups after CFP-10 or PPD stimulation, nor in the frequency of mono and polyfunctional T cells induced after MTB stimulation (Figures 5Ae5C). At 6 days, the frequency of

Please cite this article in press as: Marín ND, et al., Functional profile of CD4þ and CD8þ T cells in latently infected individuals and patients with active TB, Tuberculosis (2013), http://dx.doi.org/10.1016/j.tube.2012.12.002

N.D. Marín et al. / Tuberculosis xxx (2013) 1e12

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Figure 5. Frequency of single or polyfunctional CD8D T cells stimulated with CFP-10, PPD and MTB in 1-day and 6-day cultures. One-day whole blood cultures (AeC) and 6day PBMC cultures (DeF) from nonTBi (white bars), LTBi (grey bars) and ATB (black bars) were stimulated with CFP-10, PPD or MTB. Pie charts show the relative proportions of CD4þ T cells producing 4, 3, 2 or 1 cytokine in nonTBi, LTBi and ATB after in vitro stimulation with CFP-10 (G). CD4þ T cells were stained as described in materials and methods, and then analyzed for intracellular IFN-g, TNF-a, IL-17 and IL-2 expression. Single producer cells were selected as shown in Figure 1 and then analyzed using the combination gate tool from FlowJoÒ software. Two-way ANOVA with Bonferroni post-test. *p < 0.05; **p < 0.01. Bars represent Mean  95% CI. nonTBi n ¼ 4e5, LTBi n ¼ 6, ATB ¼ 11.

CD8þIFN-gþ T cells induced by CFP-10 (p < 0.01) and PPD (p < 0.05) was higher in ATB compared to nonTBi (Figure 5D, 5E). Unexpectedly, stimulation with PPD induced a higher frequency of CD8þIL-17 þ T cells in nonTBi compared to ATB (p < 0.05) (Figure 5E). Finally, stimulation with MTB induced a lower frequency of CD8þIFN-gþ T cells in ATB compared to LTBi (p < 0.05), and a higher frequency of CD8þTNF-aþ T cells in nonTBi compared to ATB (p < 0.01) (Figure 5F). The change of CD8þ profile at 1 day and 6 days following CFP-10 and PPD

stimulation is noteworthy; at 1 day, LTBi had a higher response, while at 6 days, ATB had the higher response. CD8þ T cells producing one cytokine were the most common and the proportion of CD8þ T cells producing 3 or 4 cytokines were the least common subset (Figure 5). These results suggest that the evaluation of Th1-type cytokines produced by CD4þ and CD8þ T cells after in vitro stimulation for 1 and 6 days with mycobacterial antigens may help to identify T cell functional signatures associated with latency or active disease. Specifically, the high IFN-g

Please cite this article in press as: Marín ND, et al., Functional profile of CD4þ and CD8þ T cells in latently infected individuals and patients with active TB, Tuberculosis (2013), http://dx.doi.org/10.1016/j.tube.2012.12.002

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and TNF-a response of CD4þ T cells observed in LTBi following MTB stimulation confirms the importance of these Th1molecules in the protective immunity to Mtb. 3.5. Long-term stimulation increases the frequency of single and double-producer cells The use of short- and long-term cultures allows the evaluation of different T cell subsets; while short-term cultures are proposed to measure the contribution of effector or effector memory cells, in

long-term cultures responding cells are mainly central memory cells.46 The evaluation of changes in the functional composition of CD4þ and CD8þ T cells in 1-day and 6-day cultures did not show differences in the frequency of mono or polyfunctional CD4þ or CD8þ T cells elicited by stimulation with CFP-10. However, PPD induced a significant increase in the frequency of CD4þ T cells single (p < 0.01) (Figure 6A) and double (p < 0.05) cytokine producer T cells in 6-day cultures compared to 1-day cultures (Figure 6B) in LTBi, and also in the frequency of single producer cells (p < 0.001) in ATB (Figure 6A). MTB stimulation increased the

Figure 6. Functional composition of CD4D and CD8D cells associated with the time culture in nonTBi, LTBi and ATB. Changes in the functional composition of CD4þ T cells were evaluated by comparing the T cell frequency of CD4þ T cells producing one (A, D), two (B, E) or three (C, F) cytokines after 1 day (D1) and 6 days (D6) of in vitro stimulation with PPD (AeC) and MTB (DeF). Wilcoxon signed rank test *p < 0.05; **p < 0.01; ***p < 0.001. nonTBi n ¼ 4e5, LTBi n ¼ 6, ATB ¼ 11.

Please cite this article in press as: Marín ND, et al., Functional profile of CD4þ and CD8þ T cells in latently infected individuals and patients with active TB, Tuberculosis (2013), http://dx.doi.org/10.1016/j.tube.2012.12.002

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frequency of single (p < 0.05) (Figure 6D) and triple (p < 0.05) (Figure 6F) producer T cells in LTBi at 6 days compared to the response in 1-day cultures, whereas the change was only significant in the frequency of double-producer cells in ATB (Figure 6E). Regarding CD8þ T cells, PPD stimulation (p < 0.05) increased the frequency of double-producer CD8þ T cells in LTBi in long-term cultures only after PPD stimulation. In the same group, the frequency of single producer CD8þ T cells elicited after CFP-10 stimulation was reduced in 6-day cultures compared to 1-day cultures (data not shown). These results show that increasing the culture time also increased the frequency of single and doublecytokine producer T cells in LTBi and ATB after PPD and MTB stimulation, suggesting that long-term cultures may be better to evaluate the functional capacities of T cells. 3.6. ATB patients have decreased levels of IFN-g in supernatants from long-term cultures stimulated with PPD To investigate the contribution of other cytokines and chemokines to the immune response in tuberculosis, 17 molecules were measured by Luminex in supernatants from 1-day and 6-day cultures from LTBi and ATB after stimulation with CFP-10 and PPD (Figure 7). At 1 day, there were not differences in the levels of cytokines and chemokines released into the supernatants from whole blood cultures after CFP-10 or PPD stimulation (data not shown). At 6 days, stimulation with PPD induced higher levels of IFN-g (p < 0.01) in LTBi compared to ATB, and IL-17 tended to be higher in LTBi compared to ATB, but it did not reach statistical significance (p < 0.0533). No differences were found in the other molecules evaluated after PPD stimulation. 4. Discussion This study provides an analysis of the frequency of cytokine producing cells in nonTBi, LTBi and ATB. Initially, we evaluated the

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magnitude of Mtb specific T cell responses by enumerating single Mtb specific cells producing IFN-g and IL-17 by ELISPOT, and then we expanded our assays to other cytokines by evaluating the frequency of polyfunctional T cells at 1 day and 6 days post-stimulation with mycobacterial antigens. Results show that evaluation of the frequency of IFN-g producing cells by ELISPOT after PPD stimulation might be an effective tool to detect active disease since it discriminated infected individuals from active ATB. In contrast, CFP-10 stimulation discriminated non infected individuals from ATB, but it did not discriminate non infected individuals from LTBi individuals, nor LTBi from ATB. The lack of differences in the IFN-g response to CFP-10 between LTBi and ATB is in agreement with Harari et al.39 who reported similar CFP-10 specific T cell responses in LTBi and ATB. In contrast, significant differences in the number of IFN-g producing cells elicited in ATB and contacts following CFP-10 stimulation have also been reported.47 The high frequency of IFN-g producing cells observed in ATB following CFP-10 and PPD stimulation supports the hypothesis that T cell responses to Mtb specific antigens, as measured by ELISPOT, may be indicative of disease activity.47,48 In line with this assumption, a reduction in the frequency of CFP-10 specific-IFN-g-producing cells has been observed during anti-TB treatment follow-up.21 Besides the differences in the immune profile exhibited by the studied groups, the magnitude of the IFN-g and IL-17 response to CFP-10 or PPD stimulation was also different. As expected, the magnitude of the response elicited by PPD was higher compared to that which was induced by CFP-10. These results confirm that the complex composition of PPD, which consists of hundreds of proteins,49 favors a higher response, whilst CFP-10, a protein made up of 100-amino acids, has fewer epitopes available to be recognized by antigen specific T cells, which explains the lower frequency of circulating CFP-10 specific T cells, even in ATB.50 The high PPD responses may also be related to cross reactivity with environmental mycobacteria. Although ELISPOT results showed that the magnitude of the response in ATB was higher, it has been proposed that quality is more

Figure 7. Cytokine and chemokine levels in supernatants from PBMC cultured for 6 days with CFP-10 or PPD. PBMC samples from ATB (black bars) and LTBi (grey bars) were stimulated with CFP-10 (A) or PPD (B) for 6 days. Supernatants were collected and frozen at 70  C until its use. Cytokines and chemokines were evaluated by Luminex. Antigen specific cytokine levels were determined by subtracting levels from unstimulated cultures. ManneWhitney test *p < 0.05; **p < 0.01. LTBi n ¼ 7e8, ATB ¼ 9e10. For box and whiskers plots, the vertical line represents the median, the boxes represent the interquartile range, and the whiskers represent the min and max value.

Please cite this article in press as: Marín ND, et al., Functional profile of CD4þ and CD8þ T cells in latently infected individuals and patients with active TB, Tuberculosis (2013), http://dx.doi.org/10.1016/j.tube.2012.12.002

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important than magnitude regarding the immune response against infections.29 Thus, the evaluation of only one or two immunological components actively participating in the defense against Mtb infection may underestimate the complexity of the host immune response to Mtb. Therefore, we quantified cells producing Th1-type cytokines such as IFN-g, TNF-a and IL-2, along with IL-17, a Th17type cytokine, by polychromatic flow cytometry after 1 day or 6 days post-stimulation with CFP-10, PPD or MTB. Of note, the use of whole blood for the 1-day (short-term) cultures and PBMC for 6-day (long-term) cultures were set up following the recommendation by Hanekom et al.,46 that whole blood cultures allow a more reliable measure of what is occurring in the host, since those cultures detect mainly effector memory T cell responses of recently activated lymphocytes, whereas long-term cultures (6 days) would allow the expansion and activation of central memory T cells. In preliminary experiments we also found long-term stimulation of whole blood results in a reduced frequency of cytokine producing cells and increased cell death compared to PBMC (data not shown). The proportion of Mtb specific CD4þ and CD8þ T cells producing only one cytokine was higher compared to those producing multiple cytokines, irrespective of the studied group and the antigen used in both 1-day and 6-day cultures. The main differences were found in the frequency of IFN-g or TNF-a producing cells among the groups, while IL-17 showed differences in some cases, and no differences were observed in the frequency of IL-2 producer cells. Our results show a biased response of CD4þ T cells toward cells expressing IFN-g in PPD stimulated short-term cultures from ATB patients. In contrast, CFP-10 and PPD induced a higher IFN-g response by CD8þ T cells in LTBi. Hanekom et al.51 suggested that, in short-term cultures, the responding cells are mainly recently activated T cells, which may correspond to cells actively participating in the response to Mtb in ATB patients. However, an opposite pattern was found in LTBi in whom a higher frequency of CD8þIFN-gþ T cells was found, compared to ATB or ATB and nonTBi after CFP-10 and PPD stimulation, respectively. Similar results were also reported by our group and others,52,53 supporting the important role of CD8þ T cells as IFN-g producers, beside their cytotoxic capabilities, in the immune defense against Mtb infection. Nevertheless, the reduced frequency of circulating CD8þ T cells producing IFN-g in ATB may be related to an increased recruitment of these cells into infected tissues.54 Reports from HIV and chronic viral infections have associated polyfunctional T cells producing IFN-g, TNF-a and IL-2 with an efficient control of infection,30,36 and have also been used to define correlates of protection mediated by vaccination against Leishmania31 and Mtb.32,33 However, unlike previous results,38,39 we did not find differences in the frequency of CD4þ T cells producing three or more cytokines after short-term stimulation with mycobacterial antigens. In this regard, the low frequency of IL-2 and IL17 producing cells dramatically reduces the overall response of cells producing 3 or 4 cytokines. IL-2 promotes T cell expansion and therefore serves to amplify the effector T cell response, although it also favors the expansion of Tregs.16 Meanwhile, the role of IL-17 during the different stages of Mtb infection is less understood.55 It has been proposed that the pathogenic role of Th17 cells is mainly associated with the recruitment of neutrophils into the lungs,56 which in the specific case of Mtb infection, has been associated with the development of pathology.28 The low frequency of these cells after in vitro stimulation with mycobacterial antigens may reflect the reduced frequency of circulating cells committed to IL-2 and IL-17 production. Nevertheless, Han et al.57 have recently shown that the polyfunctional response of T cells results from a sequential release of cytokines, with very short periods of time in which cells secrete multiple cytokines simultaneously. These

results, along with differences in methodological aspects such as culture time, the use of peptide pools or fusion proteins compared to recombinant proteins used in our study, the criteria to select latently infected individuals or the clinical conditions of the patients, might explain the discordant results, and should be taken into account. Short- and long-term cultures have been proposed to evaluate the contribution of different components of the adaptive immune response, namely effector or effector memory T cells in short-term cultures, and central memory T cells in long-term cultures.46,58 Most of the studies evaluating the functional capacity of T cells have been conducted in short-term cultures, but due to the higher sensitivity of long-term cultures in detecting latent TB infection compared to short-term cultures,59,60 the evaluation of the functional capacity of CD4þ and CD8þ T cells was also measured in 6day cultures. Similar to Southerland et al.,38 our results using short-term cultures, long-term stimulation with CFP-10 induced a higher frequency of TNF-a producing CD4þ T cells from ATB. Meanwhile, PPD and MTB stimulation induced a higher frequency of IFN-gþ, TNF-aþ or double-producers, CD4þIFN-gþTNF-aþ, T cells in LTBi compared to nonTBi and ATB. TNF-a is considered a central mediator of a broad range of biological activities.61 During ATB, this cytokine plays an important role in the immune defense against Mtb.62 However, high levels of TNF-a are also associated with increased TB pathology63 and it is also responsible for some of the clinical symptoms observed during the active disease. Of note, PPD and MTB, which are complex antigenic preparations, promoted a mixed production of Th1-type cytokines including IFN-g and TNFa. In this regard, and given the importance of IFN-g and TNF-a in controlling Mtb replication, it is possible to suggest that the production of both IFN-g and TNF-a plays a protective role and may be required for an effective control of mycobacterial growth, as observed in latently infected individuals. In this regard, Denis et al.64 demonstrated that these two Th1-type cytokines synergize to activate mice macrophages, which in turn activate reactive nitrogen products to kill Mtb. However, Day et al.40 reported a higher frequency of CD4þIFN-gþTNF-aþ T cells in AFB smear þ TB patients compared to LTBi. Unlike short-term cultures, long-term stimulation of CD8þ T cells with CFP-10 and PPD induced a high proportion of cells producing IFN-g alone in ATB. The production of IFN-g alone has been associated to a terminal effector phenotype of T cells as a result of a chronic antigen exposure,29 as it may occur during ATB. Regarding the magnitude of the cytokine response elicited by CFP-10, PPD or MTB, in short-term cultures there was not a clear tendency in the response elicited by any particular antigen in CD4þ or CD8þ T cells. At 6 days, it was clear that PPD induced a higher cytokine response in CD4þ T cells compared to CFP-10 and MTB, whereas in the case of CD8þ T cells, PPD and MTB seem to induce the cytokine production in a high proportion compared to CFP-10. As mentioned above, these results may be associated with differences in the biology and composition of these molecules. PPD is a complex antigenic mixture of mycobacterial proteins49 which may be recognized by a wide range of cells and this may also be occurring with MTB. However, in the specific case of MTB, killing of the bacteria by irradiation may leave mycobacterial structural components which modulate the immune response intact, and therefore, elicit a different functional profile on T cells. The evaluation of cytokines and chemokines by Luminex secreted in 6-day culture supernatants demonstrated only significantly high levels of IFN-g by LTBi compared to ATB. Similar to our results, reduced levels of secreted IFN-g have been reported in ATB patients after stimulation with mycobacterial antigens.65,66 Altogether, these results suggest that ATB patients exhibit decreased Th1 responses and that distinct T cell functional

Please cite this article in press as: Marín ND, et al., Functional profile of CD4þ and CD8þ T cells in latently infected individuals and patients with active TB, Tuberculosis (2013), http://dx.doi.org/10.1016/j.tube.2012.12.002

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signatures are present in LTBi and ATB, and they may be detected using different times of in vitro stimulation with mycobacterial antigens. These functional signatures correlated with the state of infection and might also be indicative of the clinical activity of the disease. Thus, the results presented herein should be validated by other authors before proposing any functional signature as a possible biomarker candidate for TB. Acknowledgments The authors thank the ATB patients, non infected and latently infected individuals that agreed to participate in this study. The authors would also like to thank the Tuberculosis Control Programs of the Servicio Seccional de Salud de Antioquia and the Secretaria de Salud de Medellín that contributed in the recruitment of the patients and allowed us to have access to the patients’ clinical records. This study was supported by Colciencias, (Bogotá, Colombia) grant 1115-408-20488 and Estrategia de Sostenibilidad (Comité para el desarrollo de la Investigación) Universidad de Antioquia, Medellín, Colombia. Ethical approval: Funding:

Not required.

None.

Competing interests:

None declared.

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Please cite this article in press as: Marín ND, et al., Functional profile of CD4þ and CD8þ T cells in latently infected individuals and patients with active TB, Tuberculosis (2013), http://dx.doi.org/10.1016/j.tube.2012.12.002