T-cell responses have been demonstrated to be essential for preventing Mycobacterium tuberculosis infection. The Th1-cytokines produced by T cells, such as INF-γ, IL-2, and TNF-α, not only limit the invasion of M. tuberculosis but also eliminate the pathogen at the site of infection. Bacillus Calmette–Guérin (BCG) is known to induce Th1-type responses but the protection is inadequate. Identification of immunogenic components, in addition to those expressed in BCG, and induction of a broad spectrum of Th1-type responses provide options for generating sufficient adaptive immunity. Here, we studied human pulmonary T-cell responses induced by the M. tuberculosis-specific antigen Rv3615c, a protein with a similar size and sequence homology to ESAT-6 and CFP-10, which induced dominant CD4+ T-cell responses in human tuberculosis (TB) models. We characterized T-cell responses including cytokine profiling, kinetics of activation, expansion, differentiation, TCR usage, and signaling of activation induced by Rv3615c compared with other M. tuberculosis-specific antigens. The expanded CD4+ T cells induced by Rv3615c predominately produced Th1, but less Th2 and Th17, cytokines and displayed effector/memory phenotypes (CD45RO+CD27−CD127−CCR7−). The magnitude of expansion and cytokine production was comparable to those induced by well-characterized the 6 kDa early secreted antigenic target (ESAT-6), the 10 kDa culture filtrate protein (CFP-10) and BCG. Rv3615c contained multiple epitopes Rv3615c1–15, Rv3615c6–20, Rv3615c66–80, Rv3615c71–85 and Rv3615c76–90 that activated CD4+ T cells. The Rv3615c-specific CD4+ T cells shared biased of T-cell receptor variable region of β chain (TCR Vβ) 1, 2, 4, 5.1, 7.1, 7.2 and/or 22 chains to promote their differentiation and proliferation respectively, by triggering a signaling cascade. Our data suggest that Rv3615c is a major target of Th1-type responses and can be a highly immunodominant antigen specific for M. tuberculosis infection.
Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the leading causes of morbidity and mortality, and accounts for 10.4 million new cases and 1.4 million deaths annually . Despite the availability of anti-TB drugs and worldwide administration of Bacillus Calmette–Guérin (BCG), M. tuberculosis infection remains a major challenge to public health . The only licensed vaccine for preventing M. tuberculosis infection is BCG, which provides inadequate protection; hence, there is an urgent need for the development of new vaccines and therapeutic approaches that are effective and safe in order to end the global TB epidemic [3,4], which has become more challenging due to the constant emergence of multidrug-resistant strains.
Animal models and clinical cases have shown that M. tuberculosis-specific CD4+ T cells with effector and effector-memory phenotype provide protection against M. tuberculosis challenge and eliminate the pathogen at the site of infection [4,5]. Th1-type cytokines, in particular, IFN-γ, TNF-α, and IL-2, produced by CD4+ T cells play a key role in adaptive immunity against M. tuberculosis infection and in the control of disease progression [6,7]. In the search for new vaccine candidates, many strategies now focus on identifying M. tuberculosis-specific immunodominant epitopes, which can induce Th1-type CD4+ T-cell responses from the majority of the population, or modifying BCG to improve efficacy and provide broad protection [7,8]. Components in the region of differentiation (RD1) of M. tuberculosis have been evaluated as potential candidates. RD1 encodes two strong immunogenic proteins and virulence factors of M. tuberculosis: the 6 kDa early secreted antigenic target (ESAT-6) and the 10 kDa culture filtrate protein (CFP-10), as well as several structural components of the ESAT-6 secretion system, the (ESX)-1 type secretion system that is responsible for ESAT-6 and CFP-10 secretion. ESAT-6 and CFP-10 are required for M. tuberculosis virulence and are deleted through repeated passing of the Mycobacterium bovis BCG vaccine strain, by which the attenuation of the BCG is generated [9–13]. Both ESAT-6 and CFP-10 induce dominant Th1-type CD4+ T-cell responses against M. tuberculosis. ESAT-6 was evaluated as a leading vaccine candidate in the form of the ESAT-6–Ag85 fusion protein-specific strong and long-lived M. tuberculosis-specific T-cell responses in naïve human volunteers [14,15]; CFP-10 was the basis of T-cell-based diagnostic blood tests for M. tuberculosis infection and induced IFN-γ release of CD4+ T cells [16,17]. All focus on inducing Th1-type CD4+ T-cell adaptive immunity.
In addition to ESAT-6 and CFP-10, Rv3615c, a protein that depends on a component of RD1 for secretion, has being recently evaluated as a vaccine candidate. Rv3615c was previously identified as ESX-1 substrate protein C (EspC) and is known as a small protein with similar amino acid length and homologous sequence to ESAT-6, CFP-10, and other members of the ESAT-6 family [18,19]. The encoding region for Rv3615c is out of RD1 but its secretion is controlled by the ESAT-6 secretion system . Although not expressed in BCG, it is actively expressed and accessible in the antigen-processing process during intracellular M. tuberculosis infection in vivo [21,22]. Studies have shown that Rv3615c contained immunodominant epitopes of CD4+ T cells and induced Th1-type responses. In a mouse model, a significant number of IFN-γ-producing cells were detected in spleen cells of mice immunized with a DNA vaccine encoding Rv3615c and stimulated with the cognate antigen or H37Rv in vitro, as well as increasing the amount of Th1 cytokines, including IFN-γ, TNF-α, and IL-2, which were detected in the culture supernatants. In this model, both CD4+ and CD8+ T cells were responsible for elevated Th1 cytokine expression, and a portion of them expressed multiple cytokines . In a human study, peripheral blood mononuclear cells (PBMCs) isolated from patients with active TB and subjects with LTBI were found to have a significant number of IFN-γ-producing cells after stimulation with Rv3615c or its overlapping peptides in vitro. Rv3615 contains multiple epitopes of human T cells, the majority of which predominately induce CD4+ T-cell responses; only a few of them induced weak CD8+ T-cell responses. Among the CD4+ T-cells expressing IFN-γ, a portion of them also co-expressed IL-2 . These data suggest the potential of Rv3615c as an additional candidate for inducing adaptive immunity against TB.
Since pulmonary T cells are the front line to encounter M. tuberculosis invasion and responsive for clearance of the pathogen, and are critical to the onset of infection and disease progression, we isolated pleural fluid mononuclear cells (PFMCs) from patients with active tuberculosis pleuritic and evaluated T-cell responses ex vivo induced by Rv3615c. We characterized those T cells by cytokine profiling, kinetics of activation, expansion, differentiation, TCR usage, and signaling of activation. We also used overlapping peptides to screen T-cell epitopes and estimated the epitope coverage among populations. We expected this characterization to provide information for the further study of Rv3615c as a vaccine candidate and shed light on understanding how human pulmonary T cells respond to M. tuberculosis infection.
Material and methods
Subjects and samples
Twenty-five patients diagnosed with tuberculosis pleurisy (TBP) and 15 healthy donors (HD) from the Chest Hospital of Guangzhou, China and Blood Center of Guangzhou, China respectively (Supplementary Table S1) were enrolled in the present study. The diagnosis of TBP was described in our previous publication , and was based on clinical symptoms, physical examination, and roentgenographic findings with positive sputum bacterial examination and positive cultures for M. tuberculosis either in cultures of pleural biopsy or pleural fluid (PF). The PF was obtained through thoracentesis operated by Dr Suihua Lao according to strict medical operation rules and methods. PF, from 130 to 1000 ml, was obtained from 25 TB patients and 160–1280 × 106 cells were recovered from the PF. The PBMCs were from HD who had a history of receiving the BCG vaccine. All PF and peripheral blood samples were obtained after less than 1 week of anti-TB treatment. All subjects were free of autoimmune diseases or co-infected HIV, HBV, or HCV infections. Informed consents were signed by all subjects, and the study was approved by Zhongshang School of Medicine Review Board and Chest Hospital of Medicine Review Board, Guangzhou, China, and The Blood Center of Medicine Review Board, Guangzhou, China.
Isolation and preparation of PFMCs and PBMCs
PFMCs and PBMCs were isolated and prepared following a previously described protocol . Briefly, PF was collected from patients with TBP and centrifuged at 2500 rpm for 20 min, the cell pellets were then collected. Cell pellets from PF, and peripheral blood from patients and HD were suspended in PBS and isolated by Ficoll-Hypaque (Tianjin HaoYang Biological Manufacture, Tianjin, China) density gradient centrifugation at 2000 rpm for 20 min. PFMCs and PBMCs were collected and washed twice with Hank’s balanced salt solution, then resuspended at a final concentration of 2 × 106 cells/ml in complete RPMI-1640 medium (Life Technologies, Grand Island, U.S.A.) supplemented with 10% heat-inactivated fetal calf serum (Sijiqing, Hangzhou, China), 100 μg/ml streptomycin, 100 U/ml penicillin, 2 mM L-glutamine, and 50 μM 2-mercaptoethanol (Life Technologies, Grand Island, U.S.A.).
Freezing and thawing of cells
When freezing cells, the cells were isolated and resuspended in cell freezing medium supplemented with 90% heat-inactivated fetal calf serum (Sijiqing, Hangzhou, China) and 10% dimethylsulfoxide (DMSO) (Zhanchen Biological Technology Co., Ltd., Guangzhou, China), and were cryopreserved in liquid nitrogen. When thawing cells, the microtubes were placed into 37°C water and shaken gently. The cell suspension was transferred into a 15 ml tube and washed twice in complete RPMI-1640 medium. Finally, the cells were resuspended at a final concentration of 2 × 106 cells/ml in complete RPMI-1640 medium.
Peptides, reagents, and mAbs
Rv3615c, ESAT-6, CFP-10, and overlapping peptides of Rv3615c were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). The sequence of the Rv3615c1–103 protein followed by: MTENLTVQPERLGVLASHHDNAAVDASSGVEAAAGLGESVAITHGPYCSQFNDTLNVYL TAHNALGSSLHTAGVDLAKSLRIAAKIYSEADEAWRKAIDGLFT. The purity of the peptides was ≥95% by HPLC, and the compositions were verified by mass spectrometry. Lyophilized peptides were dissolved in 10% DMSO to obtain a concentration of 10 mg/ml. BCG was purchased from Chengdu Institute of Biological Products (Chengdu, China). The fluorochrome-conjugated monoclonal antibodies for flow cytometry were: Phycoerythrin-Texas Red (ECD) conjugated-CD3 (UCHT1); Phycoerythrin (PE) conjugated-CD3 (UCHT1); Allophycocyanin-Cyanin 7 (APC-Cy7) conjugated-CD4 (RPAT4); Phycoerythrin-Cyanin 7 (PE-Cy7) conjugated-IFN-γ (B27); Allophycocyanin (APC) conjugated-IFN-γ (B27); Fluorescein isothiocyanate (FITC) conjugated-IFN-γ (B27); Phycoerythrin (PE) conjugated-TNF-α (MAb11); Fluorescein isothiocyanate (FITC) conjugated-TNF-α (MAb11); Peridinin chlorophyll protein Cyanin 5.5 (Percp-Cy5.5) conjugated-IL-2 (MQ1-17H12); Fluorescein isothiocyanate (FITC) conjugated-IL-2 (MQ1-17H12); Phycoerythrin-Cyanin 7 (PE-Cy7) conjugated-CD69 (FN50); Phycoerythrin (PE) conjugated-CD69 (FN50); Phycoerythrin (PE) conjugated-CD103 (Ber-ACT8); Fluorescein isothiocyanate (FITC) conjugated-CD25 (MA251); Alexa Fluor 700 (AF700) conjugated-CD45RO (UCHL1); Fluorescein isothiocyanate (FITC) conjugated-CD45RO (UCHL1); Phycoerythrin (PE) conjugated-CD62L (DREG56); Peridinin chlorophyll protein Cyanin 5.5 (Percp-Cy5.5) conjugated-CD62L (DREG56); Allophycocyanin (APC) conjugated-CD27 (MT271); Phycoerythrin-Cyanin 7 (PE-Cy7) conjugated-CCR7 (3D12); Phycoerythrin (PE) conjugated-CCR7 (3D12); Allophycocyanin (APC) conjugated-CXCR3 (1C6); Phycoerythrin-Texas Red (ECD) conjugated-T-bet (O4-46); and purified anti-CD28 (clone CD28.2) mAbs were purchased from BD Biosciences (San Jose, U.S.A.).
ELISA and ELISPOT for cytokine production
For the quantitative ELISA assay of cytokines, PFMCs were suspended in complete RPMI-1640 medium at a density of 2 × 106/ml and stimulated with or without Rv3615c, ESAT-6, CFP-10, BCG, individual Rv3615c peptide, and a Rv3615c peptide pool at a concentration of 10 μg/ml in the presence of anti-CD28 (1 μg/ml) in a round-bottom 96-well plate, 200 μl/well, at 37°C and 5% CO2 for 72 h. The culture supernatants were collected for the quantitative assessment of IFN-γ, TNF-α, IL-2, IL-4, IL-6, and IL-10 with BD OptEIA™ Human ELISA Sets (BD Biosciences, San Jose, CA, U.S.A.), and IL-17A with Human IL-17A (homodimer) ELISA Ready-SET-Go!® (eBioscience, California, U.S.A.) according to the manufacturer’s instructions. For the ELISPOT assay of cytokine-producing cells, PFMCs were suspended in complete RPMI-1640 medium at a density of 1 × 106/ml and stimulated with or without Rv3615c, ESAT-6, CFP-10, and BCG at 10 μg/ml in pre-coated BD™ ELISPOT plates, 100 μl/well, at 37°C and 5% CO2 for 24 h. The frequency of IFN-γ-producing cells was counted using an ImmunoSpot S6 Analyzer (Cellular Technology Ltd., U.S.A.) according to the manufacturer’s instructions, and the results are shown as the mean of readings from triplicate wells.
Flow cytometry analysis
The procedure for studying cell phenotype, intracellular cytokine, and transcriptional factor expression has been previously described . Briefly, for phenotyping, cells were washed with PBS containing 0.1% BSA and 0.05% sodium azide, then stained with fluorochrome-conjugated monoclonal antibodies for 30 min at 4°C in the dark. After washing with PBS twice, cells were suspended in 100 μl of PBS and applied to fluorescence-activated cell sorting (FACS) Aria II (Becton Dickinson, San Jose, U.S.A.) for data collection. For studying intracellular cytokines and transcriptional factors, cells were suspended in culture medium at a concentration of 1 × 106/ml and cultured with or without Rv3615c, ESAT-6, CFP-10, and BCG at 10 μg/ml in the presence of anti-CD28 (1 μg/ml) at 37°C and 5% CO2 for 12 h. Brefeldin A (Sigma–Aldrich, U.S.A.) was added into the culture during the final 6 h at a concentration of 10 μg/ml. The cells were washed twice with PBS containing 0.1% BSA and 0.05% sodium azide, and stained with fluorochrome-conjugated monoclonal antibodies for lineage differentiation for 30 min at 4°C in the dark. The cells were then washed with PBS, fixed with 4% paraformaldehyde, and permeabilized with 0.1% saponin overnight at 4°C. After staining for intracellular cytokines and transcriptional factors with fluorochrome-conjugated monoclonal antibodies for 30 min at 4°C in the dark, cells were applied to FACS Aria II (Becton Dickinson, San Jose, U.S.A.) for data collection. Data were analyzed using the FlowJo software (TreeStar, San Carlos, U.S.A.).
PFMCs were washed twice with pre-warmed PBS supplemented with 0.1% BSA and resuspended in staining medium containing 5 μM carboxyfluorescein diacetate succinimidyl ester (CFSE, Invitrogen, Carlsbad, U.S.A.) at a cell density of 2 × 106/ml. After incubating at 37°C and 5% CO2 for 10 min, cells were quenched with five volumes of ice-cold complete RPMI-1640 medium for 5 min on ice and washed twice with cold RPMI-1640 medium to remove the excess dye. CFSE-labeled cells were then cultured with or without Rv3615c, ESAT-6, CFP-10, and BCG at 10 μg/ml in the presence of anti-CD28 at 1 μg/ml. At indicated time intervals, cell samples were collected and stained with fluorochrome-conjugated mAbs for phenotyping at 4°C in the dark. Cell samples were assayed by FACS Aria II (Becton Dickinson, San Jose, U.S.A.), and data were analyzed using FlowJo (TreeStar, San Carlos, U.S.A.).
TCR Vβ repertoire analysis
Assessment of T-cell receptor variable region of β chain (TCR Vβ) expression has been previously described . Briefly, PFMCs were suspended in culture medium at a density of 1 × 106/ml and cultured with or without Rv3615c (10 μg/ml, final concentration) in the presence of anti-CD28 (1 μg/ml, final concentration) at 37°C and 5% CO2 for 12 h. Brefeldin A (Sigma–Aldrich, U.S.A.) was added to the cultures at a concentration of 10 μg/ml during the final 6 h. Cells were washed with PBS and stained with fluorochrome-conjugated monoclonal antibodies for lineage differentiation and the IOTest Beta Mark TCR Vβ repertoire kit (Beckman Coulter, Inc., Brea, CA, U.S.A.) for TCR Vb expression at 4°C in the dark for 30 min. After washing with PBS, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% saponin overnight at 4°C. The following day, cells were stained with the anti-IFN-γ-APC antibody at 4°C in the dark. After washing, the prepared cell samples were assayed by flow cytometry FACS Aria II (Becton Dickinson, San Jose, U.S.A.), and the data were analyzed using FlowJo (TreeStar, San Carlos, U.S.A.).
PFMCs were suspended in complete RPMI-1640 supplemented with 1% FBS at a density of 1 × 106/ml and rested overnight. Cells were pelleted and resuspended in complete RPMI-1640 culture medium at a density of 1 × 106/ml and cultured with or without Rv3615c and individual overlapping peptides (50 μg/ml) in the presence of anti-CD28 (1 μg/ml) at 37°C and 5% CO2 for 30 min. Cells were washed and lysed in buffer containing 50 mM Tris (pH 7.5), 1% (v/v) Triton X-100, 150 mM NaCl, 10% (v/v) glycerol, 1 mM EDTA, and a protease inhibitor cocktail (R&D Systems). The cell lysates were loaded onto 10% SDS gels at 50 μg/well for electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, U.S.A.). The membranes were blotted as indicated with first Abs (1:1000) followed by HRP-conjugated second Abs (1:2000) (Cell Signaling Technology, Danvers, MA, U.S.A.), and scanned using a Tanon 5200 chemo-iluminescence image analysis system (Tanon, Shanghai, China).
All statistical tests were performed with GraphPad Prism 5 (GraphPad Software Inc., San Diego, U.S.A.). Comparisons were performed using the unpaired Student’s t-test when comparing two groups, one-way ANOVA for more than two groups, or two-way ANOVA for two variables. Data are represented as the mean or mean ± SD. ***P<0.001, **P<0.01, *P<0.05, and P>0.05, or not significant, as stated in the figure legends.
Rv3615c induced cytokine production of PFMCs from patients with TBP as potent as conventional
M. tuberculosis-specific antigens
PF, from 130 to 1000 ml, was obtained from 25 TB patients and 160–1280 × 106 cells were recovered from the PF. To test the immunogenicity of Rv3516c to pulmonary M. tuberculosis-specific T cells, we isolated PFMCs from patients with active TBP and PBMCs from HD as the control. The PFMCs and PBMCs were stimulated with or without Rv3615c, ESAT-6, CFP-10, and BCG in the presence of anti-CD28. By using quantitative ELISA, we detected significantly high levels of IFN-γ, TNF-α, and IL-2 in PFMC culture supernatants but not in PBMC culture supernatants with Rv3615c or in PFMC culture supernatants with medium control (Figure 1A). The PBMCs were from HD who had a history of receiving the BCG vaccine and the cells from HD did not respond the stimulation to the TB antigens. We also found low levels of IL-6, IL-10, and IL-17 but not IL-4 in the culture supernatants (Figure 1B). By using ELISPOT, we also counted a significant number of IFN-γ-producing cells in PFMC cultures but not in PBMC cultures with Rv3615c or in PFMC cultures with medium control (Figure 1C). Comparing responses of the PFMCs to other well-characterized M. tuberculosis-specific antigens, we found that the quantity of cytokine production and frequency of cytokine-producing cells induced by Rv3615c were similar to those by ESAT-6, CFP-10, and BCG. The responsiveness of PFMC preparations to Rv3516c was slightly higher than the responsiveness of PFMC preparations to ESAT-6 and CFP-10, but similar to the responsiveness of PFMC preparations to BCG, in both quantitative cytokine assay and frequency of cytokine-producing cells. The combination of Rv3615c with other well-characterized M. tuberculosis-specific antigens increased the rate of responsiveness. All combinations increased the quantity of cytokine production at the same protein concentration (10 μg/ml) as the individual protein alone (Figure 1D). These data together suggest that Rv3615c is a major target of Th1-type T-cell immunity in active TB.
Rv3615 induced cytokine production of PFMCs from patients with TBP
CD4+ T cells in the PFMCs produced Th1-type cytokines in response to Rv3615c
To find out which cell subsets contributed cytokines in PFMC culture supernatants, we identified cytokine-producing cells by lineage differentiation and intracellular cytokine expression in PFMCs using flow cytometry. After stimulation with Rv3615c, the majority of cytokine-expressing cells in PFMCs were CD4+ T cells (>70%), <20% of them were CD4− T cells (mostly CD8+ T cells) (Figure 2A). These data demonstrate that CD4+ T cells play a predominant role in Rv3615c-induced Th1 cytokine production (Figure 2A and B) as well as in other well-characterized M. tuberculosis-specific antigen-induced Th1 cytokine production, including ESAT-6, CFP-10, and BCG. The frequency of cytokine-producing CD4+ T cells in response to Rv3615c was almost the same as those in response to other M. tuberculosis-specific antigens. Further analyzing the cytokine expression data, we found that >40% of the Rv3615c-induced cytokine-producing CD4+ T cells were polyfunctional and produced more than one cytokine (Figure 2C and D). The frequency of polyfunctional CD4+ T cells induced by Rv3615c was similar to those induced by other well-characterized M. tuberculosis antigens. Rv3615c-specific polyfunctional CD4+ T cells were considered functionally and qualitatively better than their cytokine-secreting counterparts.
CD4+ T cells contributed to the Th1-type cytokine production of PFMCs incubated with Rv3615c
Rv3615c activated CD4+ T cells specific to the cognate antigen and promoted their expansion
We studied the kinetics of cell activation by assessing the expression of CD69 and CD25 using flow cytometry. After being cultured with Rv3615c, CD4+ T-cell expression of CD69 was elevated from day 1. The level of CD69 expression slightly decreased on day 3 although the decline was not statistically significant (Figure 3A and B). Rv3615c was more potent in activating M. tuberculosis-specific CD4+ T cells than other M. tuberculosis antigens. The frequency of CD69 expression in Rv3615c-activated cells was significantly different from those cultured with ESAT-6, CFP-10, and BCG on day 1 and 3. The increase in CD25-expressing cells after culture with Rv3615c was not significant until day 3, compared with day 0 without Rv3615c. There was no statistical difference in CD25 expression between Rv3615c- and other M. tuberculosis-activated CD4+ T cells, suggesting that most M. tuberculosis-specific CD4+ T cells had already been activated by day 3 with M. tuberculosis antigens.
Rv3615c activated CD4+ T cells from patients with TBP and promoted their expansion
Next, we used a CFSE labeling approach to estimate cell division as a parameter for cell expansion. After culturing with Rv3615c, the CFSE intensity of a portion of CD4+ T cells decreased on day 3, and further decreased on day 5 and 7. As a consequence, the proportion of divided cells increased consistently from day 3 to 7 (Figure 3C and D). We also found that Rv3615c drove cell expansion as potently as other M. tuberculosis antigens, and there was no statistical difference among those antigen-driven expansions.
In contrast, Rv3615c had little effect on the activation and expansion of CD4− T cells (mostly CD8+ T cells). Fewer CD4− T cells exhibited elevated expression of CD69 and a decreased density of CFSE than CD4+ T cells (data not shown). A relative deficiency of the activation of CD4− T cells was also observed for other M. tuberculosis antigens tested here (including ESAT-6, CFP-10, and BCG), which was in consist with their deficiency to induce cytokine production, and suggested that those M. tuberculosis antigens might have a similar mechanism of inducing protective T-cell-based immunity.
Expanded Rv3615c-specific CD4+ T cells displayed effector or effector-memory phenotype
We identified Rv3615c-specific CD4+ T cells by their cytokine production following cognate antigen stimulation and assessed their differentiation. After culturing with Rv3615c, most cytokine-producing cells expressed CD45hi, CD62Llo, CD27lo, and CCR7lo, suggesting an effector phenotype (Figure 4A and B); a few of the cytokine-producing cells expressed CD45ROloCD62Lhi, CD45ROloCD27hi, and CD45ROloCCR7hi, suggesting an effector-memory phenotype (Figure 4C). The frequency of Rv3615c-specific CD4+ T cells expressing a combination of differentiation markers is shown in Figure 4(D). We found that Rv3615c-specific CD4+ T cells identified by IFN-γ, TNF-α, or IL-2 production had a similar expression of differentiation phenotype, which suggested that they were a homologous population with similar response dynamics and characterizations.
Rv3615c-activated CD4+ T cells expressed effector and effector-memory phenotypes
Rv3615c-specific CD4+ T cells isolated from pleural fluid displayed tissue-resident memory phenotype
The previous explanation for the expression of CD69 was that they were in an activated state, perhaps as a result of retained antigen. However, it is well known that CD69+ and/or CD103+/− expression is a generic characteristic of resting tissue-resident memory (TRM) T cells in infectious lungs . Thus, we detected the expression of TRM (CD69+ and CD103+) T-cell markers in local infected lesions. We found that TRM cells in the local infection site (PFMC) were very high, expressing high levels of CD69 and low levels of CD103. However, both TRM cell markers are rarely expressed in circulation (PBMC (TBP) and PBMC (HD)) (Figure 5A). According to the above analysis, we identified TRM cells dependent on the expression of the conventional marker CD69. In addition, we found there was a significantly large amount of Rv3615c-specific CD69+CD4+ T-cell formation in the local infection site (Figure 5A and B). Furthermore, Rv3615c-specific TRM CD4+ T cells expressed high levels of Th1-specific cytokines (IFN-γ and TNF-α), a Th1-specific chemokine receptor (CXCR3), and a Th1-specific transcription factor (T-bet) in endogenous compared with conventional CD4+ T cells (CD69−). Meanwhile, Rv3615c-specific TRM CD4+ T cells displayed an effector/effector memory phenotype (CD45RO+CCR7−CD62L−) (Figure 5C) and Rv3615c-specific CD4+ T cells (CD4+IFN-γ+) expressed higher levels of CD69 than their counterpart CD4+IFN-γ− T cells (Figure 5D). Collectively, these data suggest that Rv3615c-specific CD4+ T cells are predominately TRM T cells in the local infection site.
Rv3615c-specific CD4+ T cells have a profile of TRM phenotype
The Rv3615c protein contained multiple epitopes that were broadly recognized by CD4+ T cells from patients with TBP
To determine the location and immunodominance of T-cell epitopes with Rv3615c, we prepared overlapping peptides of Rv3615c for screening epitopes recognized by M. tuberculosis-specific CD4+ T cells. A total of 19 overlapping peptides span the entire Rv3615c, each with 15 mer and overlapping 11 amino acids. We tested 25 of the PFMC preparations from patients with TBP for their responsiveness to each peptide (Figure 6A). There were 11 PFMC preparations that responded to four or more peptides, six PFMC preparations responded to three peptides, five PFMC preparations responded to two peptides, and three PFMC preparations responded to one peptide. These data suggest that Rv3615c induces CD4+ T-cell responses from PFMC preparations by recognizing one or more epitopes. Two of the PFMC preparations responded slightly to any of the peptides (8%, 2/25). The most recognized epitopes were located in regions of Rv3615c1–20 and Rv3615c66–90 (Figure 6B), which were represented by Rv3615c1–15 (40%, 10/25), Rv3615c6–20 (36%, 9/25), Rv3615c66–80 (88%, 22/25), Rv3615c71–85 (52%, 13/25), and Rv3615c76–90 (72%, 18/25). Few CD8+ T cells responded to those peptides (Figure 6C), consistent with the results shown in Figure 2. Our data suggest that Rv3615c could induce M. tuberculosis-specific CD4+ T-cell responses in the majority of populations.
Rv3615c contained multiple epitopes of CD4+ T cells
TCR Vβ usage of Rv3615c-specific CD4+ T cells varied among patients with TBP
The variation of epitope recognition and magnitude of responses suggested that different TCR Vβ was used by polyclonal CD4+ T cells in response to Rv3615c. To screen TCR Vβ expression, we identified Rv3615c-specific CD4+ T cells by lineage differentiation and IFN-γ expression in response to the cognate antigen, and assessed the TCR Vβ expression with fluorochrome-conjugated anti-TCR Vβ monoclonal antibodies (Figure 7A). We found that there was a great variation of TCR Vβ usage among CD4+ T cells from patients with TBP, including the type of TCR Vβs, the number of TCR Vβs, and the frequency of each TCR Vβ usage in each patient (Figure 7B). Among the TCR Vβs being screened, seven TCR Vβs (TCR Vβ1, TCR Vβ2, TCR Vβ4, TCR Vβ5.1, TCR Vβ7.1, TCR Vβ7.2, and TCR Vβ22) were frequently expressed on Rv3615c-specific IFN-γ+CD4+ T cells (Figure 7C). These variations might be due to the difference in HLA haplotype of patients and their CD4+ T-cell clonality.
CD4+ T cells responding to Rv3615c had a broad TCR Vβ usage among patients with TBP
Rv3615c promoted CD4+ T-cell expansion and cytokine production by triggering the signaling cascade of activation
T-cell activation and subsequent expansion, differentiation, and function are triggered by TCR engagement with a cognate antigen, which activates molecules associated with the antigen receptor and initiates a signaling cascade. Zeta-chain-associated protein kinase 70 (ZAP-70) is a member of the protein-tyrosine kinase family and is expressed near the surface membrane of T cells. It binds to the zeta chain of CD3, which is the most important member of the CD3 family, as it is a key component of the T-cell receptor complex and plays a critical role in T-cell signaling (Figure 8C) . Rv3615c activated CD4+ T cells by increasing phosphorylated ZAP-70 expression in a time-dependent manner (Figure 8A). The signaling pathway initiated by ZAP-70 and the transduction was well characterized and illustrated (Figure 8B and C). There is a list of molecules that play a role in transduction. We tested Rv3615c and its overlapping peptides that were mostly recognized by PFMCs for their activation of signaling molecules in this pathway (Figure 8B and C). We found elevated expression and phosphorylation of most signaling molecules, from upstream to downstream, in Rv3615c-specific CD4+ T cells after culturing with Rv3615c and with individual peptides, except Fos, which showed significant phosphorylation only with Rv3615c71–85 peptide, Rv3615c76–90 peptide, and Rv3615c. These data demonstrate that Rv3615c could trigger a signaling cascade to activate CD4+ T cells and to promote their expansion, differentiation, and function. The difference in expression of individual signaling molecules suggests that epitopes engaging with different TCRs might trigger a signaling cascade with different intensity or different pathways of transduction.
Rv3615c initiated activation signaling cascades of CD4+ T cells
We identified Rv3615c as a potent immunogen to pulmonary CD4+ T cells isolated from patients with active TB. Rv3615c-activated T cells expanded, differentiated into a functional subset, and produced Th1 cytokines. The magnitude of proliferation, frequency of cytokine-producing cells, and quantity of cytokine production was similar to those induced by well-characterized immunodominant M. tuberculosis-specific antigens, such as ESAT-10, CFP-10, and BCG. Th1 cytokines, such as IFN-γ, IL-2, and TNF-α, have been reported to be critical for adaptive immunity against M. tuberculosis infection and prevention of disease migration to latency. In addition, Th1 cytokines activated Mϕ and protected the host from M. tuberculosis through apoptosis, autophagy, inflammasome formation, and nitric oxide production [31,32], and subjects deficient in the receptors for those cytokines were susceptible to M. tuberculosis infection [33,34]. Candidates for TB vaccines, such as ESAT-6 and CFP-10, were potent at inducing Th1-type responses. Here, we found that Rv3615c induced CD4+ T-cell production of IFN-γ, IL-2, and TNF-α, with a profile similar to those induced by CFP-10 and BCG, but different from ESAT-10 in IL-6, IL-10, and IL-17 production. IFN-γ and IL-2 production of CD4+ T cells in response to Rv3615c has been reported in human PBMC and mouse pulmonary T-cell responses . We found that pulmonary CD4+ T cells produced significant amounts of IFN-γ, IL-2, and TNF-α, and to a lesser extent, IL-6, IL-10, and IL-17, but not IL-4. IL-17 could trigger the recruitment of neutrophils and IFN-γ producing CD4+ T cells to synergize the effector function of the Th1-type response against M. tuberculosis [35,36]. More than 40% of cytokine-producing cells induced by Rv3615c were polyfunctional, expressing more than one cytokine. These polyfunctional T cells have been reported to be sufficient to protect mice from M. tuberculosis infection [37,38]. Since pulmonary T cells are in the front line to encounter M. tuberculosis invasion and to eliminate pathogens at the site of infection, the predominant Th1 cytokine production suggests the potency of Rv3615c-specific CD4+ T cells in the establishment of adaptive immunity against M. tuberculosis infection; the limited IL-6, IL-10, and IL-17 production suggests minimal tissue damage mediated by inflammatory response.
In response to pathogens, the timing of adaptive immunity is crucial for the onset of infection, the progress of disease, and the severity of illness. Early response efficiently restricts pathogen replication, reduces the infection-induced illness, limits the immunopathology, and shortens disease duration . Rv3615c-induced CD4+ T cells rapidly increased the expression of CD69 on day 1 post-encounter of the cognate antigen, with the frequency of CD69 expression much higher than that induced by other M. tuberculosis-specific antigens [39,40]. Although the frequency of CD69 expression declined slightly on day 3, the difference to other M. tuberculosis antigen-specific CD4+ T cells was still significant. These data suggest that Rv3615c-specific CD4+ T cells have the capability to rapidly respond to M. tuberculosis challenge and establish an efficient adaptive immunity at an early stage of pathogen invasion.
After encountering a cognate antigen, activated T cells underwent expansion and differentiation to establish a functional subset. Among these differentiated cells, effector and effector-memory phenotypes have been found to play a crucial role in adaptive immunity. CD4+ T cells with effector/effector-memory phenotype efficiently prevented mice from M. tuberculosis challenge . The effector-memory phenotype was a subpopulation of CD8+ T cells with particularly good functional activity. For example, CD8+ T cells expressing an effector-memory phenotype were shown to be critical for protection from mucosal SIV challenge , and could clear virus-infected cells at an early stage of infection . Here, we found that most pulmonary Rv3615c-specific CD4+ T cells expressed effector or effector-memory phenotype, suggesting that TCR engagement elicited a program driving cells toward expansion and differentiation into a functional subset that was potent in the control of M. tuberculosis infection.
Both CD4+ and CD8+ T cells are required for immunity against M. tuberculosis infection and the control of disease progression . Our study showed that pulmonary T cells responding to Rv3615c were dominated by CD4+ T cells; only a few CD4− cells expressed CD69, proliferated and produced cytokines at a level barely above background (using MFI of flow cytometry data). The dominance of CD4+ T cells was also seen in pulmonary T-cell responses to ESAT-6, CFP-10, and BCG, as well as in PBMC of patients with an active TB response to Rv3615c. Fewer CD8+ (CD4−) T cells in response to Rv3515c might indicate there were few dominant CD8+ T-cell epitopes, a small cell proportion in PFMC preparation, or exhaustion of function. There were only approximately 30% of CD4− cells in the T-cell population of PFMC, fewer than the average of 50–60% (or 30% CD8) of T cells in PBMC of Asians . CD8+ T cells isolated from the site of infection with active inflammation showed weak response to stimulation of cognate antigen in vivo. Terminal differentiation and lack of cytolytic capacity have been previously demonstrated to be a characteristic of lung-derived CD8+ T cells in response to viral infections . The differentiated CD8+ T cells had limited cytolytic activity and were unable to control influenza and Sendai virus infection . The terminal differentiation and loss of effector function have been associated with prolonged TCR ligation and were inflammatory cytokine-driven [47–49]. Persistent engagement of TCR triggered down-regulation of CD127 on CD8+ T cells , and adversely affected the control of viral replication , as well as increased inhibitory molecule expression and diminished polyfunctional activity against M. tuberculosis [52,53]. In contrast, CD8+ T cells isolated from PBMC showed greater response to Rv3615c than those isolated from PFMC, although the magnitude of response and frequency of cytokine-producing cells were still lower than CD4+ T cells. It would be interested and informational to further characterize pulmonary Rv3615c-specific CD8+ T cells in response to M. tuberculosis and their role in immunity against the infection.
The hallmark of adaptive immunity is epitope-specific memory response. The priming of epitope-specific T cells is restricted by individual HLA expression. Immunization-provided protection could cover the majority of the human population only if the immunogen is broadly recognized by an individual’s immune system. Rv3615c contained multiple epitopes that were selectively recognized by CD4+ T cells from 82% of patients, by responding to at least one epitope-carrying peptide. This coverage is as broad as BCG and extended to those not covered by BCG although these two mostly overlapped. Rv3615c was encoded in BCG , but the expression was defective in the live BCG strain conventionally used for vaccination [9–13]. The combination of Rv3615c and BCG increased the coverage, from 82% responding to Rv3615c or BCG alone to 100% responding to the combination. This combination also increased the frequency of cytokine-producing T cells and the quantity of cytokines. In a mouse model, priming with BCG and boosting with a Rv3615c-containing viral vector vaccine increased polyfunctional Th1-cytokine-producing CD4+ T cells, lowered bacterial load, and reduced lung pathology in response to BCG challenge . Data suggest that incorporating additional epitopes into BCG not only increased the responsiveness by a broad range of the population but also potentially increased the efficacy of immunization.
In our study of pulmonary cells, epitopes of CD4+ T cells were mostly estimated near both terminal regions of Rv3615c (within Rv3615c1–20 and Rv3615c66–90 regions), which is slightly different from the study of CD4+ T cells in circulation. Using PBMCs from patients with active TB, the epitopes were estimated among the entire region of Rv3615c, many of them within Rv3615c6–20 and Rv3615c46–90 regions . This variation suggests that Rv3615c-specific CD4+ T cells in pulmonary tissue and in peripheral circulation might have different TCR usage, which could influence the pattern of response and effector function. This variation might also be due to sampling from different patient populations and ethnic groups with different HLA halotype profiles. Nevertheless, both studies showed the same population coverage of Rv3615c. Analysis of the TCR Vβ repertoires has been widely used to characterize alterations of T-cell repertoires, which ranged from extensive diversity to expansion of single T-cell clones . Although there were some distinct TCR Vβ repertoires in different patients with TBP, we found that Rv3615c-specific CD4+ T cells from PFMCs had biased usage of TCR Vβ 1, 2, 4, 5.1, 7.1, 7.2, and/or 22 chains. One hypothesis stated that Rv3615c might be processed into peptides that were differently presented by different HLA molecules. Some variations of Rv3615c-specific TCR Vβ repertoires in PFMCs were observed among individuals, which might be due to differences in the HLA background. Additionally, human T-cell responses, even those to immunodominant epitopes, might be quite diverse with respect to TCR usage, which was a hypothesis consistent with our results.
We have demonstrated the capability of human pulmonary Rv3615-specific CD4+ T cells for expansion and Th1 cytokine production when encountering a cognate antigen, and provided information on Rv3615c-specific CD4+ T-cells responding to M. tuberculosis in lung tissue in addition to previous studies on human circulating CD4+ T cells in a mouse model [24,55]. Data suggest the potential of Rv3615C as a vaccine candidate to induce adaptive immunity against M. tuberculosis infection, and guaranteed a further study of Rv3615c-induced CD4+ T cells in immune protection and immunopathology of M. tuberculosis. Although its efficacy was not as high as BCG in a mouse challenge model in the form of a triple-antigen fusion DNA vaccine , the protection provided by Rv3615c needs to be further characterized and evaluated in other forms, in more animal models, particularly in non-human primates, as well as in human efficacy studies. Our data suggest that further characterization of the Rv3615c-induced response and evaluation of the protective efficiency would provide information for new TB vaccine development.
Bacillus Calmette–Guérin (BCG) provides inadequate protection, and there is an urgent need for the development of new vaccines that are effective and safe to achieve the goal of ending the global tuberculosis (TB) epidemic.
Pulmonary T-cell responses have been demonstrated to be essential for preventing Mycobacterium tuberculosis infection, not only limiting the invasion of M. tuberculosis but also eliminating pathogens at the site of infection. The results indicate that Rv3615c is a major target of Th1-type responses and can be a highly immunodominant antigen specific for M. tuberculosis infection.
Our data suggest that Rv3615c can be a new TB vaccine candidate, and further characterization of Rv3615c-induced response and evaluation of the protective efficiency will provide information for new TB vaccine development.
This work was supported by the China National Science and Technology Plan Projects (973) [grant number 2013CB531506]; and Guangdong Key Laboratory of Organ Donation & Transplantation Immunology [grant number 2013A061401007].
C.W. and J.Li designed the research; J.Li analyzed data; J.Li, J.S., and X.L. performed the research; C.W., J.L., and J.Li wrote the paper; S.L. provided clinical samples and consultation. J.Li developed the software necessary to perform and record experiments.
The authors declare that there are no competing interests associated with the manuscript.
carboxyfluorescein diacetate succinimidyl ester
the 10 kDa culture filtrate protein
the 6 kDa early secreted antigenic target
ESX-1 substrate protein C
fluorescence-activated cell sorting
peripheral blood mononuclear cell
pleural fluid mononuclear cell
region of differentiation 1
- TCR Vβ
T-cell receptor variable region of β chain
zeta-chain-associated protein kinase 70