Tuberculosis 94 (2014) 219e225
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IMMUNOLOGICAL ASPECTS
Mycobacterium tuberculosis-specific polyfunctional cytotoxic CD8þ T cells express CD69 Li Li a, Binyan Yang a, Xianlan Zhang b, Suihua Lao b, Changyou Wu a, * a Institute of Immunology, Zhongshan School of Medicine, Key Laboratory of Tropical Disease Control Research of Ministry of Education, Sun Yat-sen University, Guangzhou 510080, PR China b Chest Hospital of Guangzhou, Guangzhou 510095, PR China
a r t i c l e i n f o
s u m m a r y
Article history: Received 14 June 2013 Received in revised form 24 December 2013 Accepted 27 December 2013
Increasing evidences in animals and humans suggest that CD8þ T cells contribute significantly to immune defenses against Mycobacterium tuberculosis (Mtb). In the present study, we found that without any stimulation, CD8þ T cells in pleural fluid cells (PFCs) expressed significantly higher levels of CD69 than PBMCs from patients with tuberculous pleurisy (TBP). CD8þCD69þ T cells expressed significantly higher levels of CD45RO and HLA-DR and lower levels of CD45RA than CD8þCD69 T cells, demonstrating that CD8þCD69þ T cells were activated memory cells. Furthermore, we found higher expression of CCR6 and lower expression of CCR7 and CD62L on CD8þCD69þ T cells compared with CD8þCD69 T cells, suggesting that the expression of CCR6 and reduced expression of CCR7 and CD62L might facilitate the migration of circulating CD8þCD69þ T cells into tuberculous pleural space. Importantly, following stimulation with culture filtrate protein of 10 kDa (CFP10) peptides, CD8þCD69þ T cells but not CD8þCD69 T cells expressed CD107a/b, IFN-g and TNF-a, demonstrating that CD8þCD69þ T cells were MTB-specific cells. In addition, the majority of CD8þCD69þ T cells were dominated by polyfunctional T cells. In summary, we demonstrated that CD69 as a useful marker for MTB-specific CD8þ T cells in PFCs from patients with TBP enabled a direct ex vivo estimation of the quantity, as well as the quality, of MTBspecific CD8þ responses. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: CD8þ T cells Mycobacterium tuberculosis Tuberculous pleurisy CD69
1. Introduction Tuberculosis (TB) is a leading cause of infectious mortality worldwide, accounting for over 8 million new cases and 2.9 million deaths annually [1e3]. Increasing experimental evidence in the mouse tuberculosis (TB) model has suggested a protective role for CD8þ T cells in the host defense [4e6]. For example, adoptive transfer or in vivo depletion of CD8þ cells showed that this population could confer protection against subsequent challenge. b2microglobulin- deficient mice are more susceptible to Mtb and to large doses of Bacille Calmette Gue’rin infection than their wildtype littermates [5]. In murine experimental models, CD4þ T cells are essential for control of acute TB infection and CD8þ T cells are essential to maintain and control the chronic phase of TB [7]. In humans, Mtb-specific CD8þ T cells have been identified in Mtbinfected individuals [8,9].
* Corresponding author. Institute of Immunology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, PR China. Tel./fax: þ86 20 87333201. E-mail address: [email protected] ( Changyou Wu). 1472-9792/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tube.2013.12.007
It has been demonstrated that T-lymphocytes in the lungs, both in normal individuals and in those with granulomatous disease, are almost entirely CD45ROþ memory T cells and express both earlyand late-activation markers, such as CD69 and CD29 respectively [10]. CD69 is a membrane molecule transiently expressed on activated lymphocytes, and its selective expression in inflammatory infiltrates probably suggests that it plays a role in the pathogenesis of inflammatory diseases [11]. We have previously found a significant increase in CD69 expression on CD4þ T cells in PFCs and demonstrated that most of these CD4þCD69þ T cells were MTBspecific Th1 cells [12]. Moreover, we have demonstrated that CFP10-specific CD8þ T cells were present in patients with TBP [13]. In the present study, we found that without any stimulation, CD8þ T cells in PFCs expressed significantly higher levels of CD69 than PBMCs from patients with TBP. Further phenotypic analysis indicated that CD8þCD69þ T cells were activated memory T cells. In order to further characterize the biological functions of CD8þCD69þ T cells, we stimulated PFCs with CFP10 peptides and assessed the expression of cytotoxic molecules and cytokines as well. By using the protein transport inhibitor, Brefeldin A (BFA), we could block the transport of newly synthesized CD69 molecule to the cell
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surface. We could determine the function of CD8þCD69þ T cells which were induced in vivo. Our results indicated that cytotoxic and cytokine-producing CD8þ T cells were mostly enriched within CD8þCD69þ T cells, suggesting that CFP10-specific CD8þ T cells primarily expressed CD69 in vivo. CD69 could be used as a marker for the enrichment and isolation of MTB-specific CD8þ T cells for further study.
CCR7-PE (3D12), CXCR3-APC (1C6), CXCR4-PE (12G5), CD62L-PE (Dreg56), CD45RA-FITC (L48), CD45RO-FITC (UCHL1), CD127-APC (hIL-7R-M21), CD25-FITC (M-A251), HLA-DR-PE (G46-6), CD107aFITC (H4A3), CD107b-FITC (H4B4), IFN-g-APC (clone B27), TNF-aPE-Cy7 (MAb11), IL-2-APC (MQ1-17H12), and isotype-matched control antibodies. CD27-APC (O323) was obtained from Biolegend (San Diego, CA). IFN-g-FITC (45.15) was purchased from Beckman Coulter (Fullerton, CA).
2. Methods 2.3. Preparation of PFCs and PBMCs 2.1. Study participants A total of thirty-three patients with tuberculous pleurisy (12 females and 21 males, range 20e65 years of age) were recruited from the Chest Hospital of Guangzhou, China. Diagnosis of pleural effusion from TB etiology was based on one of the following criteria: (i) demonstration of MTB on pleural fluid smear (by the Ziehl-Neelsen method); (ii) pleural fluid or pleural biopsy specimens growing Mycobacterium tuberculosis on LowensteineJensen medium; or (iii) histological evidence of caseating granuloma on biopsy specimens of pleural tissue with positive staining for M. tuberculosis. Patients who had been previously diagnosed with HIV, HBV, or HCV or with a history of autoimmune diseases were excluded from the study. None of the patients was receiving MTB-related treatments at the time of the study. The study was approved by the Zhongshan School of Medicine Review Board (Guangzhou, China). 2.2. Peptides, reagents and mAbs Twenty-six 13-15mer peptides that overlapped by 11 aa and spanned the CFP10 protein were synthesized. All of the peptides were synthesized by Sangon Biotech (Shanghai)Co.,Ltd. Peptide purity was >95%, as assayed by HPLC, and their composition was verified by mass spectrometry. Lyophilized peptides were dissolved at 20 mg/ml in DMSO, aliquoted, and stored at 80 C. Purified anti-CD28 (clone CD28.2) and anti-CD49d (clone 9F10) mAbs were purchased from BD Biosciences (San Jose, CA). The following mAbs were used for phenotypic and intracellular cytokine analysis and were purchased from BD Biosciences (San Jose, CA, USA): CD3-APC-Cy7 (SK7), CD3-Pacific Blue (UCHT1), CD8-FITC (RPA-T8), CD8-APC (RPA-T8), CD8-eFluor450 (RPA-T8), CD69-PE (FN50), CD69-Pecy7 (FN50), CCR4-PE (1G1), CCR6-PE (11A9),
PFCs were isolated by lysing erythrocytes using ammonium chloride solution and resuspended to a final concentration of 2 106 cells/mL in complete RPMI 1640 medium (Invitrogen, Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum (FCS; HyClone, Logan, UT), 100 U/mL penicillin, 100 mg/mL streptomycin, 2 mM L-glutamine, and 50 mM 2-mercaptoethanol. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque gradient centrifugation of heparinized venous blood obtained from patients with tuberculous pleurisy. 2.4. Flow cytometry For surface staining, PFCs or PBMCs were washed with PBS buffer containing 0.1% BSA and 0.05% sodium azide and incubated with the respective mAbs for 30 min. The cells were thereafter washed twice and resuspended in PBS. For the detection of intracellular cytokines, cells were incubated at a concentration of 2 106 cells/mL with 1 mg/mL peptides plus 1 mg/mL anti-CD28 and 1 mg/mL anti-CD49d for 8 h in the presence of brefeldin A (BFA, 10 mg/mL; SigmaeAldrich, St Louis, MO). For the detection of CD107a/b, PFCs were stimulated with CFP10 peptides plus CD107aFITC and CD107b-FITC. One hour later, Brefeldin A and Monensin (1 mL/mL, BD Biosciences Pharmingen) were added and the plates were incubated for another 5 h. After stimulation, cells were washed with PBS containing 0.1% BSA and 0.05% sodium azide. Cells were incubated with antibodies for surface staining and then fixed with 4% paraformaldehyde, permeabilized with PBS containing 0.1% saponin and stained for intracellular cytokines. Flow cytometry was performed using BD FACSCalibur (BD Biosciences) or FACSAria Ⅱ (BD Biosciences) and the data were analyzed using FlowJo software (TreeStar, San Carlos, CA).
Figure 1. The expression of CD69 on CD8þ T cells in PBMCs and PFCs from patients with TBP without any stimulation. (A) Representative expression of CD69 on CD8þ T cells is determined by FACS in PBMCs and pleural fluid cells (PFCs) from patients with TBP. The numbers in each quadrant represent the percentages of CD69 expression in gated CD8þ T cells. (B) Summary data of CD69 expression on PBMCs (n ¼ 19) and PFCs (n ¼ 33). Each symbol represents a value from a single donor. Differences between groups are assessed by the Student’s t test. Horizontal lines represent means SEM.
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2.5. Statistical analysis The Wilcoxon matched pairs test (Two-tailed) and Student’s t test was performed to determine statistical differences between the groups using GraphPad Prism software version 5. A value of p < 0.05 was considered statistically significant. 3. Results 3.1. Significantly higher expression of CD69 on CD8þ T cells in PFCs than PBMCs from patients with TBP We first assessed the surface expression of CD69 on CD8þ T cells from PFCs and PBMCs of patients with TBP. Without any stimulation, a significantly higher level of CD69 expression was observed on CD8þ T cells from PFCs than PBMCs from TBP patients (Figure 1(A) and (B), P < 0.01). Moreover, higher levels of CD69 expression (9.09% 8.50%, mean SD) on CD8þ T cells were observed in 64% of the PFCs than on the PBMCs from individuals with TBP (3.72% 2.79%, mean SD). 3.2. Significantly higher expression of CD45RO and HLA-DR on CD8þCD69þ T cells than CD8þCD69 T cells We further assessed the phenotype of CD8þCD69þ T cells. We demonstrated that CD8þCD69þ T cells expressed significantly
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higher levels of CD45RO and lower levels of CD45RA than CD8þCD69 T cells (Figure 2(A) and (B), P < 0.05). In addition, no significant difference was observed on the expression of CD127 and CD27 (Figure 2(A) and (B)). We also assessed another two activation markers, CD25 and HLA-DR. CD8þCD69þ T cells expressed significantly higher levels of HLA-DR than CD8þCD69 T cells, a molecule which is usually induced on T cells after activation (Figure 3(A) and (B), P < 0.05). Unexpectedly, no significant difference was observed as to the expression of CD25, which is usually temporally expressed on activated T cells or constitutively expressed on regulatory T cells. 3.3. Significantly higher expression of CCR6 and lower expression of CCR7 and CD62L on CD8þCD69þ T cells than CD8þCD69 T cells In order to gain insight into the mechanisms of CD8þCD69þ T cells recruitment to the local sites of MTB infection, we analyzed the expression of a panel of chemokine receptors on CD8þCD69þ and CD8þCD69 T cells from the TBP (Figure 4(A)). Interestingly, we found that expression of CCR6 was significantly increased on CD8þCD69þ T cells compared with CD8þCD69 T cells (Figure 4(B), P < 0.05). In contrast, the expression levels of CCR4, CXCR3 and CXCR4 were not significantly different between the groups. In contrast, the expression of CCR7 and CD62L was significantly lower on CD8þCD69þ T cells than CD8þCD69 T cells (Figure 4(B), P < 0.05). Our results indicated that the higher expression of CCR6
Figure 2. The expression of naïve/memory markers on CD8þCD69þ and CD8þCD69 T cells. (A) PFCs were stained with CD8, CD69, CD45RA, CD45RO, CD127 and CD27. The expression of each marker within CD8þCD69þ and CD8þCD69 T cells is evaluated by FACS. Isotype controls are shown in gray and the expression of each marker is shown by dotted lines. The numbers represent the percentages of chemokine receptor expression in gated cells. Data are representative of six donors. (B) Mean value of expression of each marker on CD8þCD69þ and CD8þCD69 T cells is indicated. Differences between groups are assessed by the Wilcoxon matched pairs test (Two-tailed). *, P < 0.05.
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Figure 3. The expression of CD25 and HLA-DR on CD8þCD69þ and CD8þCD69 T cells. (A) Expression of CD25 and HLA-DR on CD8þCD69þ and CD8þCD69 T cells. The percentages of each marker expression within the CD8þCD69þ and CD8þCD69 T cell subsets are shown by dotted line. (B) Average value of CD25 and HLA-DR expression on CD8þCD69þ and CD8þCD69 T cells from six donors is demonstrated. *, P < 0.05.
and lower expression of CCR7 and CD62L might contribute to the selective migration of CD8þCD69þ T cells into the local sites of MTB infection. 3.4. CD107a/b, IFN-g and TNF-a were primarily produced by CD8þCD69þ T cells than CD8þCD69 T cells To further measure the biological activity of CD8þCD69þ T cells at local sites of MTB infection, we measured the expression of CD107a/b, IFN-g and TNF-a production. PFCs were stimulated with
or without CFP10 peptides in the presence of antibodies to CD107a/ b, monensin and BFA. The results showed that without any stimulation, CD8þCD69þ or CD8CD69þ T cells produced very low levels of CD107a/b, IFN-g and TNF-a (Figure 5(A)). Following stimulation with CFP10 peptides, CD8þCD69þ T cells expressed large amounts of CD107a/b, IFN-g and TNF-a (Figure 5(B)). Moreover, coexpression of CD107a/b with IFN-g or TNF-a was observed for CD8þCD69þ T cells (Figure 5(C)). In contrast, no significantly increased expression of CD107a/b, IFN-g and TNF-a was observed as to CD8þCD69 T cells.
Figure 4. The expression of chemokine receptors on CD8þCD69þ and CD8þCD69 T cells. (A) CD8þCD69þ or CD8þCD69 cells were gated and the expression of chemokine receptors is analyzed by flow cytometry. The numbers represent the percentages of chemokine receptor expression in gated cells. Open dotted histograms represent chemokine receptor staining and filled grey histograms represent isotype controls. One representative result from six donors with similar results is shown. (B) Mean value of percentages of chemokine receptors on CD8þCD69þ and CD8þCD69 cells are demonstrated. Differences between groups were assessed by the Wilcoxon matched pairs test (Two-tailed). Significant differences are indicated. *, P < 0.05.
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3.5. Polyfunctional CD8þ cells were preferentially enriched within CD8þCD69þ T cells We further aimed to examine whether direct ex vivo evaluation of surface expression of CD69 without in vitro upregulation permits direct assessment of MTB-specific CD8þ T cells. Our initial experiments indicated that stimulation of PFCs with CFP10 peptides resulted in increased surface CD69 expression (data not shown). Short-term stimulation of these cells includes incubation with
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secretion inhibitory brefeldin A (BFA), which can block the transport of newly synthesized CD69 molecules to the cell surface and thus allows for the direct assessment of the relationship between in vivo CD69 expression and cytokine production. After culturing PFCs with CFP10 peptides and BFA, we analyzed the production of IFN-g, IL-2 and TNF-a by intracellular cytokine staining. As shown in Figure 6(A), IFN-g, IL-2 and TNF-a production was primarily enriched within CD8þCD69þ T cells but not CD8þCD69 T cells. It has been suggested that T cells simultaneously producing IFN-g, IL2 and TNF-a were associated with protective immunity. We therefore compared the expression of IFN-g, IL-2 and TNF-a in CD8þCD69þ, CD8þCD69 and total CD8þ T cells after short-term in vitro stimulation with CFP10 peptides. The total CD8þ antigenspecific responses were defined as the percentages of T cells expressing any combination of IFN-g, IL-2 or TNF-a, and seven distinct functional populations could be delineated (IFN-g/IL-2/ TNF-a triple expressers; IFN-g/IL-2, IFN-g/TNF-a or TNF-a/IL-2 double expressers; or IFN-g, IL-2 or TNF-a single expressers). The distribution of the seven subsets was similar within CD8þCD69þ, CD8þCD69 and total CD8þ fractions. Polyfunctional T cells (IFN-g/ IL-2/TNF-a triple expressers) dominated the CD8þ T cell response and the percentages of IFN-g/TNF-a double expressers were less frequent (Figure 6(B)). Importantly, the percentages of polyfunctional CD8þ T cells within CD8þCD69þ fractions were significantly higher than that in total CD8þ fractions or CD8þCD69 fractions. Our results demonstrated that polyfunctional T cells were primarily enriched within CD8þCD69þ T cells. 4. Discussion
Figure 5. The expression of CD107a/b, IFN-g and TNF-a on CD8þCD69þ and CD8þCD69 T cells following stimulation with CFP10 peptides. PFCs were stimulated with or without CFP10 peptides in the presence of FITC conjugated anti-CD107a/b mAbs, monensin and BFA for 8 h. CD8þCD69þ and CD8þCD69 T cells were gated respectively. The expression of CD107a/b, IFN-g and TNF-a on CD8þCD69þ and CD8þCD69 T cells following stimulation with medium (A) or CFP10 peptides (B) were indicated. Numbers in quadrants indicate percentages of the cells in each population. Data shown are representative results of three donors with similar results. (C) Statistical analysis of IFN-g, TNF-a and CD107a/b expression by CD8þCD69 or CD8þCD69þ T cells following stimulation with medium or CFP-10 (n ¼ 3). For each plot, the median is represented by a horizontal line and the range by whiskers. Differences between groups were assessed by the Student’s t test. ***, P < 0.001.
Without treatment, tuberculous pleurisy (TBP) usually resolves spontaneously and thus is thought to be a useful model to study the correlates of protective immune responses at the site of infection [14e17]. We previously demonstrated that CD4þ T cells in PFCs from patients with TBP expressed significantly higher levels of CD69 than PBMCs from patients with TB or healthy donors and that these CD4þCD69þ T cells were mostly MTB-specific Th1 cells [12]. In the present study, we further found that the expression of CD69 was significantly increased on CD8þ T cells in PFCs than PBMCs from patients with TBP. We further demonstrated that CD8þCD69þ T cells expressed significantly higher levels of CD45RO and decreased levels of CD45RA compared with CD8þCD69 T cells, suggesting that these CD8þCD69þ T cells were memory T cells. Moreover, we further assessed the activation markers on these cells and found that CD8þCD69þ T cells expressed significantly higher levels of HLA-DR than CD8þCD69 T cells, indicating that these cells were mostly activated at local sites of TB infection. In order to further characterize the phenotype of CD8þCD69þ T cells, we analyzed chemokine receptor expression. We found that compared with CD8þCD69 T cells, CD8þCD69þ T cells expressed significantly higher levels of CCR6 and lower levels of CCR7 and CD62L [13,18]. We propose that the elevated expression of CCR6 may contribute to the selective migration of CD8þ T cells into local sites of TB infection while decreased expression both CCR7 and CD62L could favor the migration of CD8þ T cells from draining lymph nodes to peripheral sites of infection. CCL20, the CCR6 agonist, is constitutively expressed in skin and mucosa-associated tissues. The expression of CCR6 on CD8þCD69þ cells might immediately chemoattract to infected sites [19]. We also noticed that the expression of CCR4, CXCR3 and CXCR4 were not significantly different between CD8þCD69þ and CD8þCD69 T cells, suggesting that these chemokine receptors may not be involved in the selective migration of CD8þ T cells. In order to confirm whether these CD8þCD69þ cells were MTBspecific T cells, we performed intracellular cytokine staining. We
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Figure 6. Polyfunctional Th1 cells are primarily enriched within CD8þCD69þ T cells. (A) Cells were gated on CD8þCD69þ and CD8þCD69þ cells, respectively. Representative expression of IFN-g, TNF-a and IL-2 within gated cells is shown. The numbers in each quadrant represent the percentages of positive cells in gated T cells. (B) The average frequencies of CD8þCD69þ, CD8þCD69 and CD8þ T cells expressing each of the seven combinations of IFN-g, IL-2 and TNF-a from six donors are demonstrated.
noted that stimulation of PFCs resulted in increased surface CD69 expression. We overcame this limitation by incubation with BFA during short-term stimulation. Using this method, we were able to directly assess the relationship between in vivo CD69 expression and their biological function. CD107a and CD107b are two intracellular proteins that are normally found in lysosomes and also structural components of cytotoxic granules [20]. Following exocytosis of cytotoxic granules, CD107a/b is transiently expressed on the surfaces of cytotoxic lymphocytes. Therefore, the expression of CD107a/b was usually as markers of cytotoxic cells. Following stimulation with CFP10 peptides, we demonstrated that the expression of CD107a/b were significantly induced in CD8þCD69þ subset, whereas CD8þCD69 cells scarcely produced either CD107a/b or cytokines. We also noticed significant coexpression of CD107a/b with intracellular cytokine IFN-g and TNF-a, which might contribute to the optimal biological activity of CD8þ T cells. T cell responses are commonly determined by the expression of various cytokines, such as IFN-g, IL-2 and TNF-a for Th1 cells, after short-term stimulation with specific antigens. T cells that produce multiple factors simultaneously are termed polyfunctional T cells and have been shown to provide vaccine-induced immunity and may indeed mediate protection against TB [21e25]. In the present study, analysis of CD8þ T subpopulations indicated that the majority of total CD8þ T cells produced three cytokines (IFN-g, IL-2 and TNF-a) simutaneously. However, the percentages of polyfunctional CD8þ T cells within CD8þCD69þ fractions were significantly higher than that in total CD8þ fractions or CD8þCD69 fractions. Consistent with the results demonstrated in our previous paper [12], both CD4þCD69þ T cells and CD8þCD69þ T cells represented effector or effector memory cells and quickly
produced large amounts of cytokines or killing molecules following short-term stimulation. Combination of CD69 expression could be used to more precisely define polyclonal CD4þ or CD8þ T cells. Currently, we have not yet found any significant differences as to the expression and function of CD69 between CD4þ and CD8þ T cells. In conclusion, our findings demonstrated that CD8þCD69þ T cells were significantly increased in PFCs compared with PBMCs in patients with TBP. These cells were activated memory T cells with increased expression of CCR6 and have potential cytotoxic function and ability to produce large amounts of cytokines, which may contribute to their function against MTB infection at local sites. Financial disclosure This work was funded by the Program of China during the Twelfth Five-Year Plan Period (20BZX10003007002003), National Natural Science Foundation of China (31100632 and 31270942), the Foundation of Guangzhou Pearl River Science New Star (2011J2200078), Natural Science Foundation of Guangdong Province (S2011040004322 and S2012010009159) and Graduate Education Innovation Training Project of Guangdong Province (sybzzxm201108). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Conflict of interest disclosure The authors have declared that no conflict of interest exists.
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Contents lists available at ScienceDirect
Tuberculosis journal homepage: http://intl.elsevierhealth.com/journals/tube
IMMUNOLOGICAL ASPECTS
Mycobacterium tuberculosis-specific polyfunctional cytotoxic CD8þ T cells express CD69 Li Li a, Binyan Yang a, Xianlan Zhang b, Suihua Lao b, Changyou Wu a, * a Institute of Immunology, Zhongshan School of Medicine, Key Laboratory of Tropical Disease Control Research of Ministry of Education, Sun Yat-sen University, Guangzhou 510080, PR China b Chest Hospital of Guangzhou, Guangzhou 510095, PR China
a r t i c l e i n f o
s u m m a r y
Article history: Received 14 June 2013 Received in revised form 24 December 2013 Accepted 27 December 2013
Increasing evidences in animals and humans suggest that CD8þ T cells contribute significantly to immune defenses against Mycobacterium tuberculosis (Mtb). In the present study, we found that without any stimulation, CD8þ T cells in pleural fluid cells (PFCs) expressed significantly higher levels of CD69 than PBMCs from patients with tuberculous pleurisy (TBP). CD8þCD69þ T cells expressed significantly higher levels of CD45RO and HLA-DR and lower levels of CD45RA than CD8þCD69 T cells, demonstrating that CD8þCD69þ T cells were activated memory cells. Furthermore, we found higher expression of CCR6 and lower expression of CCR7 and CD62L on CD8þCD69þ T cells compared with CD8þCD69 T cells, suggesting that the expression of CCR6 and reduced expression of CCR7 and CD62L might facilitate the migration of circulating CD8þCD69þ T cells into tuberculous pleural space. Importantly, following stimulation with culture filtrate protein of 10 kDa (CFP10) peptides, CD8þCD69þ T cells but not CD8þCD69 T cells expressed CD107a/b, IFN-g and TNF-a, demonstrating that CD8þCD69þ T cells were MTB-specific cells. In addition, the majority of CD8þCD69þ T cells were dominated by polyfunctional T cells. In summary, we demonstrated that CD69 as a useful marker for MTB-specific CD8þ T cells in PFCs from patients with TBP enabled a direct ex vivo estimation of the quantity, as well as the quality, of MTBspecific CD8þ responses. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: CD8þ T cells Mycobacterium tuberculosis Tuberculous pleurisy CD69
1. Introduction Tuberculosis (TB) is a leading cause of infectious mortality worldwide, accounting for over 8 million new cases and 2.9 million deaths annually [1e3]. Increasing experimental evidence in the mouse tuberculosis (TB) model has suggested a protective role for CD8þ T cells in the host defense [4e6]. For example, adoptive transfer or in vivo depletion of CD8þ cells showed that this population could confer protection against subsequent challenge. b2microglobulin- deficient mice are more susceptible to Mtb and to large doses of Bacille Calmette Gue’rin infection than their wildtype littermates [5]. In murine experimental models, CD4þ T cells are essential for control of acute TB infection and CD8þ T cells are essential to maintain and control the chronic phase of TB [7]. In humans, Mtb-specific CD8þ T cells have been identified in Mtbinfected individuals [8,9].
* Corresponding author. Institute of Immunology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, PR China. Tel./fax: þ86 20 87333201. E-mail address: [email protected] ( Changyou Wu). 1472-9792/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tube.2013.12.007
It has been demonstrated that T-lymphocytes in the lungs, both in normal individuals and in those with granulomatous disease, are almost entirely CD45ROþ memory T cells and express both earlyand late-activation markers, such as CD69 and CD29 respectively [10]. CD69 is a membrane molecule transiently expressed on activated lymphocytes, and its selective expression in inflammatory infiltrates probably suggests that it plays a role in the pathogenesis of inflammatory diseases [11]. We have previously found a significant increase in CD69 expression on CD4þ T cells in PFCs and demonstrated that most of these CD4þCD69þ T cells were MTBspecific Th1 cells [12]. Moreover, we have demonstrated that CFP10-specific CD8þ T cells were present in patients with TBP [13]. In the present study, we found that without any stimulation, CD8þ T cells in PFCs expressed significantly higher levels of CD69 than PBMCs from patients with TBP. Further phenotypic analysis indicated that CD8þCD69þ T cells were activated memory T cells. In order to further characterize the biological functions of CD8þCD69þ T cells, we stimulated PFCs with CFP10 peptides and assessed the expression of cytotoxic molecules and cytokines as well. By using the protein transport inhibitor, Brefeldin A (BFA), we could block the transport of newly synthesized CD69 molecule to the cell
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surface. We could determine the function of CD8þCD69þ T cells which were induced in vivo. Our results indicated that cytotoxic and cytokine-producing CD8þ T cells were mostly enriched within CD8þCD69þ T cells, suggesting that CFP10-specific CD8þ T cells primarily expressed CD69 in vivo. CD69 could be used as a marker for the enrichment and isolation of MTB-specific CD8þ T cells for further study.
CCR7-PE (3D12), CXCR3-APC (1C6), CXCR4-PE (12G5), CD62L-PE (Dreg56), CD45RA-FITC (L48), CD45RO-FITC (UCHL1), CD127-APC (hIL-7R-M21), CD25-FITC (M-A251), HLA-DR-PE (G46-6), CD107aFITC (H4A3), CD107b-FITC (H4B4), IFN-g-APC (clone B27), TNF-aPE-Cy7 (MAb11), IL-2-APC (MQ1-17H12), and isotype-matched control antibodies. CD27-APC (O323) was obtained from Biolegend (San Diego, CA). IFN-g-FITC (45.15) was purchased from Beckman Coulter (Fullerton, CA).
2. Methods 2.3. Preparation of PFCs and PBMCs 2.1. Study participants A total of thirty-three patients with tuberculous pleurisy (12 females and 21 males, range 20e65 years of age) were recruited from the Chest Hospital of Guangzhou, China. Diagnosis of pleural effusion from TB etiology was based on one of the following criteria: (i) demonstration of MTB on pleural fluid smear (by the Ziehl-Neelsen method); (ii) pleural fluid or pleural biopsy specimens growing Mycobacterium tuberculosis on LowensteineJensen medium; or (iii) histological evidence of caseating granuloma on biopsy specimens of pleural tissue with positive staining for M. tuberculosis. Patients who had been previously diagnosed with HIV, HBV, or HCV or with a history of autoimmune diseases were excluded from the study. None of the patients was receiving MTB-related treatments at the time of the study. The study was approved by the Zhongshan School of Medicine Review Board (Guangzhou, China). 2.2. Peptides, reagents and mAbs Twenty-six 13-15mer peptides that overlapped by 11 aa and spanned the CFP10 protein were synthesized. All of the peptides were synthesized by Sangon Biotech (Shanghai)Co.,Ltd. Peptide purity was >95%, as assayed by HPLC, and their composition was verified by mass spectrometry. Lyophilized peptides were dissolved at 20 mg/ml in DMSO, aliquoted, and stored at 80 C. Purified anti-CD28 (clone CD28.2) and anti-CD49d (clone 9F10) mAbs were purchased from BD Biosciences (San Jose, CA). The following mAbs were used for phenotypic and intracellular cytokine analysis and were purchased from BD Biosciences (San Jose, CA, USA): CD3-APC-Cy7 (SK7), CD3-Pacific Blue (UCHT1), CD8-FITC (RPA-T8), CD8-APC (RPA-T8), CD8-eFluor450 (RPA-T8), CD69-PE (FN50), CD69-Pecy7 (FN50), CCR4-PE (1G1), CCR6-PE (11A9),
PFCs were isolated by lysing erythrocytes using ammonium chloride solution and resuspended to a final concentration of 2 106 cells/mL in complete RPMI 1640 medium (Invitrogen, Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum (FCS; HyClone, Logan, UT), 100 U/mL penicillin, 100 mg/mL streptomycin, 2 mM L-glutamine, and 50 mM 2-mercaptoethanol. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque gradient centrifugation of heparinized venous blood obtained from patients with tuberculous pleurisy. 2.4. Flow cytometry For surface staining, PFCs or PBMCs were washed with PBS buffer containing 0.1% BSA and 0.05% sodium azide and incubated with the respective mAbs for 30 min. The cells were thereafter washed twice and resuspended in PBS. For the detection of intracellular cytokines, cells were incubated at a concentration of 2 106 cells/mL with 1 mg/mL peptides plus 1 mg/mL anti-CD28 and 1 mg/mL anti-CD49d for 8 h in the presence of brefeldin A (BFA, 10 mg/mL; SigmaeAldrich, St Louis, MO). For the detection of CD107a/b, PFCs were stimulated with CFP10 peptides plus CD107aFITC and CD107b-FITC. One hour later, Brefeldin A and Monensin (1 mL/mL, BD Biosciences Pharmingen) were added and the plates were incubated for another 5 h. After stimulation, cells were washed with PBS containing 0.1% BSA and 0.05% sodium azide. Cells were incubated with antibodies for surface staining and then fixed with 4% paraformaldehyde, permeabilized with PBS containing 0.1% saponin and stained for intracellular cytokines. Flow cytometry was performed using BD FACSCalibur (BD Biosciences) or FACSAria Ⅱ (BD Biosciences) and the data were analyzed using FlowJo software (TreeStar, San Carlos, CA).
Figure 1. The expression of CD69 on CD8þ T cells in PBMCs and PFCs from patients with TBP without any stimulation. (A) Representative expression of CD69 on CD8þ T cells is determined by FACS in PBMCs and pleural fluid cells (PFCs) from patients with TBP. The numbers in each quadrant represent the percentages of CD69 expression in gated CD8þ T cells. (B) Summary data of CD69 expression on PBMCs (n ¼ 19) and PFCs (n ¼ 33). Each symbol represents a value from a single donor. Differences between groups are assessed by the Student’s t test. Horizontal lines represent means SEM.
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2.5. Statistical analysis The Wilcoxon matched pairs test (Two-tailed) and Student’s t test was performed to determine statistical differences between the groups using GraphPad Prism software version 5. A value of p < 0.05 was considered statistically significant. 3. Results 3.1. Significantly higher expression of CD69 on CD8þ T cells in PFCs than PBMCs from patients with TBP We first assessed the surface expression of CD69 on CD8þ T cells from PFCs and PBMCs of patients with TBP. Without any stimulation, a significantly higher level of CD69 expression was observed on CD8þ T cells from PFCs than PBMCs from TBP patients (Figure 1(A) and (B), P < 0.01). Moreover, higher levels of CD69 expression (9.09% 8.50%, mean SD) on CD8þ T cells were observed in 64% of the PFCs than on the PBMCs from individuals with TBP (3.72% 2.79%, mean SD). 3.2. Significantly higher expression of CD45RO and HLA-DR on CD8þCD69þ T cells than CD8þCD69 T cells We further assessed the phenotype of CD8þCD69þ T cells. We demonstrated that CD8þCD69þ T cells expressed significantly
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higher levels of CD45RO and lower levels of CD45RA than CD8þCD69 T cells (Figure 2(A) and (B), P < 0.05). In addition, no significant difference was observed on the expression of CD127 and CD27 (Figure 2(A) and (B)). We also assessed another two activation markers, CD25 and HLA-DR. CD8þCD69þ T cells expressed significantly higher levels of HLA-DR than CD8þCD69 T cells, a molecule which is usually induced on T cells after activation (Figure 3(A) and (B), P < 0.05). Unexpectedly, no significant difference was observed as to the expression of CD25, which is usually temporally expressed on activated T cells or constitutively expressed on regulatory T cells. 3.3. Significantly higher expression of CCR6 and lower expression of CCR7 and CD62L on CD8þCD69þ T cells than CD8þCD69 T cells In order to gain insight into the mechanisms of CD8þCD69þ T cells recruitment to the local sites of MTB infection, we analyzed the expression of a panel of chemokine receptors on CD8þCD69þ and CD8þCD69 T cells from the TBP (Figure 4(A)). Interestingly, we found that expression of CCR6 was significantly increased on CD8þCD69þ T cells compared with CD8þCD69 T cells (Figure 4(B), P < 0.05). In contrast, the expression levels of CCR4, CXCR3 and CXCR4 were not significantly different between the groups. In contrast, the expression of CCR7 and CD62L was significantly lower on CD8þCD69þ T cells than CD8þCD69 T cells (Figure 4(B), P < 0.05). Our results indicated that the higher expression of CCR6
Figure 2. The expression of naïve/memory markers on CD8þCD69þ and CD8þCD69 T cells. (A) PFCs were stained with CD8, CD69, CD45RA, CD45RO, CD127 and CD27. The expression of each marker within CD8þCD69þ and CD8þCD69 T cells is evaluated by FACS. Isotype controls are shown in gray and the expression of each marker is shown by dotted lines. The numbers represent the percentages of chemokine receptor expression in gated cells. Data are representative of six donors. (B) Mean value of expression of each marker on CD8þCD69þ and CD8þCD69 T cells is indicated. Differences between groups are assessed by the Wilcoxon matched pairs test (Two-tailed). *, P < 0.05.
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Figure 3. The expression of CD25 and HLA-DR on CD8þCD69þ and CD8þCD69 T cells. (A) Expression of CD25 and HLA-DR on CD8þCD69þ and CD8þCD69 T cells. The percentages of each marker expression within the CD8þCD69þ and CD8þCD69 T cell subsets are shown by dotted line. (B) Average value of CD25 and HLA-DR expression on CD8þCD69þ and CD8þCD69 T cells from six donors is demonstrated. *, P < 0.05.
and lower expression of CCR7 and CD62L might contribute to the selective migration of CD8þCD69þ T cells into the local sites of MTB infection. 3.4. CD107a/b, IFN-g and TNF-a were primarily produced by CD8þCD69þ T cells than CD8þCD69 T cells To further measure the biological activity of CD8þCD69þ T cells at local sites of MTB infection, we measured the expression of CD107a/b, IFN-g and TNF-a production. PFCs were stimulated with
or without CFP10 peptides in the presence of antibodies to CD107a/ b, monensin and BFA. The results showed that without any stimulation, CD8þCD69þ or CD8CD69þ T cells produced very low levels of CD107a/b, IFN-g and TNF-a (Figure 5(A)). Following stimulation with CFP10 peptides, CD8þCD69þ T cells expressed large amounts of CD107a/b, IFN-g and TNF-a (Figure 5(B)). Moreover, coexpression of CD107a/b with IFN-g or TNF-a was observed for CD8þCD69þ T cells (Figure 5(C)). In contrast, no significantly increased expression of CD107a/b, IFN-g and TNF-a was observed as to CD8þCD69 T cells.
Figure 4. The expression of chemokine receptors on CD8þCD69þ and CD8þCD69 T cells. (A) CD8þCD69þ or CD8þCD69 cells were gated and the expression of chemokine receptors is analyzed by flow cytometry. The numbers represent the percentages of chemokine receptor expression in gated cells. Open dotted histograms represent chemokine receptor staining and filled grey histograms represent isotype controls. One representative result from six donors with similar results is shown. (B) Mean value of percentages of chemokine receptors on CD8þCD69þ and CD8þCD69 cells are demonstrated. Differences between groups were assessed by the Wilcoxon matched pairs test (Two-tailed). Significant differences are indicated. *, P < 0.05.
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3.5. Polyfunctional CD8þ cells were preferentially enriched within CD8þCD69þ T cells We further aimed to examine whether direct ex vivo evaluation of surface expression of CD69 without in vitro upregulation permits direct assessment of MTB-specific CD8þ T cells. Our initial experiments indicated that stimulation of PFCs with CFP10 peptides resulted in increased surface CD69 expression (data not shown). Short-term stimulation of these cells includes incubation with
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secretion inhibitory brefeldin A (BFA), which can block the transport of newly synthesized CD69 molecules to the cell surface and thus allows for the direct assessment of the relationship between in vivo CD69 expression and cytokine production. After culturing PFCs with CFP10 peptides and BFA, we analyzed the production of IFN-g, IL-2 and TNF-a by intracellular cytokine staining. As shown in Figure 6(A), IFN-g, IL-2 and TNF-a production was primarily enriched within CD8þCD69þ T cells but not CD8þCD69 T cells. It has been suggested that T cells simultaneously producing IFN-g, IL2 and TNF-a were associated with protective immunity. We therefore compared the expression of IFN-g, IL-2 and TNF-a in CD8þCD69þ, CD8þCD69 and total CD8þ T cells after short-term in vitro stimulation with CFP10 peptides. The total CD8þ antigenspecific responses were defined as the percentages of T cells expressing any combination of IFN-g, IL-2 or TNF-a, and seven distinct functional populations could be delineated (IFN-g/IL-2/ TNF-a triple expressers; IFN-g/IL-2, IFN-g/TNF-a or TNF-a/IL-2 double expressers; or IFN-g, IL-2 or TNF-a single expressers). The distribution of the seven subsets was similar within CD8þCD69þ, CD8þCD69 and total CD8þ fractions. Polyfunctional T cells (IFN-g/ IL-2/TNF-a triple expressers) dominated the CD8þ T cell response and the percentages of IFN-g/TNF-a double expressers were less frequent (Figure 6(B)). Importantly, the percentages of polyfunctional CD8þ T cells within CD8þCD69þ fractions were significantly higher than that in total CD8þ fractions or CD8þCD69 fractions. Our results demonstrated that polyfunctional T cells were primarily enriched within CD8þCD69þ T cells. 4. Discussion
Figure 5. The expression of CD107a/b, IFN-g and TNF-a on CD8þCD69þ and CD8þCD69 T cells following stimulation with CFP10 peptides. PFCs were stimulated with or without CFP10 peptides in the presence of FITC conjugated anti-CD107a/b mAbs, monensin and BFA for 8 h. CD8þCD69þ and CD8þCD69 T cells were gated respectively. The expression of CD107a/b, IFN-g and TNF-a on CD8þCD69þ and CD8þCD69 T cells following stimulation with medium (A) or CFP10 peptides (B) were indicated. Numbers in quadrants indicate percentages of the cells in each population. Data shown are representative results of three donors with similar results. (C) Statistical analysis of IFN-g, TNF-a and CD107a/b expression by CD8þCD69 or CD8þCD69þ T cells following stimulation with medium or CFP-10 (n ¼ 3). For each plot, the median is represented by a horizontal line and the range by whiskers. Differences between groups were assessed by the Student’s t test. ***, P < 0.001.
Without treatment, tuberculous pleurisy (TBP) usually resolves spontaneously and thus is thought to be a useful model to study the correlates of protective immune responses at the site of infection [14e17]. We previously demonstrated that CD4þ T cells in PFCs from patients with TBP expressed significantly higher levels of CD69 than PBMCs from patients with TB or healthy donors and that these CD4þCD69þ T cells were mostly MTB-specific Th1 cells [12]. In the present study, we further found that the expression of CD69 was significantly increased on CD8þ T cells in PFCs than PBMCs from patients with TBP. We further demonstrated that CD8þCD69þ T cells expressed significantly higher levels of CD45RO and decreased levels of CD45RA compared with CD8þCD69 T cells, suggesting that these CD8þCD69þ T cells were memory T cells. Moreover, we further assessed the activation markers on these cells and found that CD8þCD69þ T cells expressed significantly higher levels of HLA-DR than CD8þCD69 T cells, indicating that these cells were mostly activated at local sites of TB infection. In order to further characterize the phenotype of CD8þCD69þ T cells, we analyzed chemokine receptor expression. We found that compared with CD8þCD69 T cells, CD8þCD69þ T cells expressed significantly higher levels of CCR6 and lower levels of CCR7 and CD62L [13,18]. We propose that the elevated expression of CCR6 may contribute to the selective migration of CD8þ T cells into local sites of TB infection while decreased expression both CCR7 and CD62L could favor the migration of CD8þ T cells from draining lymph nodes to peripheral sites of infection. CCL20, the CCR6 agonist, is constitutively expressed in skin and mucosa-associated tissues. The expression of CCR6 on CD8þCD69þ cells might immediately chemoattract to infected sites [19]. We also noticed that the expression of CCR4, CXCR3 and CXCR4 were not significantly different between CD8þCD69þ and CD8þCD69 T cells, suggesting that these chemokine receptors may not be involved in the selective migration of CD8þ T cells. In order to confirm whether these CD8þCD69þ cells were MTBspecific T cells, we performed intracellular cytokine staining. We
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Figure 6. Polyfunctional Th1 cells are primarily enriched within CD8þCD69þ T cells. (A) Cells were gated on CD8þCD69þ and CD8þCD69þ cells, respectively. Representative expression of IFN-g, TNF-a and IL-2 within gated cells is shown. The numbers in each quadrant represent the percentages of positive cells in gated T cells. (B) The average frequencies of CD8þCD69þ, CD8þCD69 and CD8þ T cells expressing each of the seven combinations of IFN-g, IL-2 and TNF-a from six donors are demonstrated.
noted that stimulation of PFCs resulted in increased surface CD69 expression. We overcame this limitation by incubation with BFA during short-term stimulation. Using this method, we were able to directly assess the relationship between in vivo CD69 expression and their biological function. CD107a and CD107b are two intracellular proteins that are normally found in lysosomes and also structural components of cytotoxic granules [20]. Following exocytosis of cytotoxic granules, CD107a/b is transiently expressed on the surfaces of cytotoxic lymphocytes. Therefore, the expression of CD107a/b was usually as markers of cytotoxic cells. Following stimulation with CFP10 peptides, we demonstrated that the expression of CD107a/b were significantly induced in CD8þCD69þ subset, whereas CD8þCD69 cells scarcely produced either CD107a/b or cytokines. We also noticed significant coexpression of CD107a/b with intracellular cytokine IFN-g and TNF-a, which might contribute to the optimal biological activity of CD8þ T cells. T cell responses are commonly determined by the expression of various cytokines, such as IFN-g, IL-2 and TNF-a for Th1 cells, after short-term stimulation with specific antigens. T cells that produce multiple factors simultaneously are termed polyfunctional T cells and have been shown to provide vaccine-induced immunity and may indeed mediate protection against TB [21e25]. In the present study, analysis of CD8þ T subpopulations indicated that the majority of total CD8þ T cells produced three cytokines (IFN-g, IL-2 and TNF-a) simutaneously. However, the percentages of polyfunctional CD8þ T cells within CD8þCD69þ fractions were significantly higher than that in total CD8þ fractions or CD8þCD69 fractions. Consistent with the results demonstrated in our previous paper [12], both CD4þCD69þ T cells and CD8þCD69þ T cells represented effector or effector memory cells and quickly
produced large amounts of cytokines or killing molecules following short-term stimulation. Combination of CD69 expression could be used to more precisely define polyclonal CD4þ or CD8þ T cells. Currently, we have not yet found any significant differences as to the expression and function of CD69 between CD4þ and CD8þ T cells. In conclusion, our findings demonstrated that CD8þCD69þ T cells were significantly increased in PFCs compared with PBMCs in patients with TBP. These cells were activated memory T cells with increased expression of CCR6 and have potential cytotoxic function and ability to produce large amounts of cytokines, which may contribute to their function against MTB infection at local sites. Financial disclosure This work was funded by the Program of China during the Twelfth Five-Year Plan Period (20BZX10003007002003), National Natural Science Foundation of China (31100632 and 31270942), the Foundation of Guangzhou Pearl River Science New Star (2011J2200078), Natural Science Foundation of Guangdong Province (S2011040004322 and S2012010009159) and Graduate Education Innovation Training Project of Guangdong Province (sybzzxm201108). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Conflict of interest disclosure The authors have declared that no conflict of interest exists.
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