- Email: [email protected]
CD8+ αβ and Vγ2Vδ2+ T cells in HIV-negative individuals with different Mycobacterium tuberculosis infection statuses
Accepted Manuscript Characterization of CD4/CD8+ α β and Vγ2Vδ2+ T cells in HIV-negative individuals with different Mycobacterium tuberculosis infection statuses Yan Gao, Shu Zhang, Qinfang Ou, Lei Shen, Sen Wang, Jing Wu, Xinhua Weng, Zheng W. Chen, Wenhong Zhang, Lingyun Shao PII: DOI: Reference:
S0198-8859(15)00476-0 http://dx.doi.org/10.1016/j.humimm.2015.09.039 HIM 9618
To appear in:
Human Immunology
Received Date: Revised Date: Accepted Date:
23 June 2014 24 July 2015 26 September 2015
Please cite this article as: Gao, Y., Zhang, S., Ou, Q., Shen, L., Wang, S., Wu, J., Weng, X., Chen, Z.W., Zhang, W., Shao, L., Characterization of CD4/CD8+ α β and Vγ2Vδ2+ T cells in HIV-negative individuals with different Mycobacterium tuberculosis infection statuses, Human Immunology (2015), doi: http://dx.doi.org/10.1016/ j.humimm.2015.09.039
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Characterization of CD4/CD8+ αβ and Vγ2Vδ2+ T cells in HIV-negative individuals with different Mycobacterium tuberculosis infection statuses Yan Gao a, Shu Zhanga, Qinfang Oub, Lei Shena, Sen Wanga, Jing Wu a, Xinhua Wenga, Zheng W. Chenc, Wenhong Zhanga*, Lingyun Shaoa*
a
Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai
200040, China b
Department of Pulmonary Diseases, Wuxi No.5 People’s Hospital, Wuxi 214005,
China c
Department of Microbiology and Immunology, Center for Primate Biomedical
Research, University of Illinois College of Medicine, 835 S. Wolcott Avenue, MC790, Chicago, IL 60612
* Corresponding author. Mailing address: 12 Wulumuqi Zhong Road, Shanghai 200040, China. Phone: 86-21-52888123. Fax: 86-21-52888714. Email for Lingyun Shao:
[email protected];
Email
[email protected]
E-mail address: YG: [email protected] SZ: [email protected] QO: [email protected] 1
for
Wenhong
Zhang:
LS: [email protected] SW: [email protected] JW: [email protected] XW: [email protected] ZC: [email protected] WZ: [email protected] LS: [email protected]
2
Abstract Background The immune responses of T cell subsets among patients with different Mycobacterium tuberculosis (M.tb) infection statuses [i.e., active tuberculosis (ATB), latent tuberculosis infection (LTBI) and non-infection (healthy control, HC)] have not been fully elucidated in HIV-negative individuals. Specifically, data are limiting in high tuberculosis epidemic regions in China. To investigate the distributions and functions of T cell subsets (i.e., CD3+, CD4+, CD8+ αβ and Vγ2Vδ2+ T cells) in HIV-negative subjects with different M.tb infection statuses, we conducted a case-control study that enrolled 125 participants, including ATB patients (n=46), LTBI subjects (n=34), and HC (n=45). Results An IFN-γ release assay (IGRA) was employed to screen LTBI subjects. Whole blood cell surface staining and flow cytometry were used to detect phenotypic distributions of T cells phenotypic distributions in the peripheral blood mononuclear cells (PBMCs) and tuberculous pleural fluid mononuclear cells (PFMCs). PPD and the phosphorylated antigen HMBPP were employed as stimulators for the detection of M.tb antigen-specific T cell functions via intracellular cytokine staining (ICS). The absolute numbers of T cell subsets, including CD3+CD4+, CD3+CD8+ αβ and Vγ2Vδ2+ T cells, were significantly reduced in active tuberculosis compared with latent tuberculosis or the healthy controls. Importantly, PPD-specific CD3+CD4+ and CD3+CD8+ αβ T cells and HMBPP-specific Vγ2Vδ2+ T cells in ATB patients were also significantly reduced compared to the LTBI/HC subjects (P<0.05). In contrast, the proportion of CD4+ T cells in PFMCs was higher compared to PBMCs, while 3
CD8+ and Vγ2Vδ2+ T cells in PFMCs were lower compared to PBMCs (all P<0.05). PPD-specific CD4+ T cells predominated among CD3+ T cells in PFMCs. Conclusions
Cellular
immune
responses
are
impaired
in
ATB
patients.
Antigen-specific CD4+ T cell may migrate from the periphery to the lesion site, where they exert anti-tuberculosis functions. Keywords: Active tuberculosis; Latent tuberculosis infection; Immune response; T cell subsets; Interferon-gamma
4
1. Introduction Approximately one-third of the world’s population (or two billion people) is estimated to be infected with Mycobacterium tuberculosis (M.tb). About 5–10% of infected people develop TB disease during their lifetime, mostly within 5 years after a new infection[1]. The risk that a new or latent infection will progress to disease is increased by a compromised immune system. M.tb as an intracellular pathogen that has a complex relationship with the host. When the natural immune response cannot control the growth of M.tb. There is continuous exposure to M.tb antigens. Then, the organism starts to induce acquired immune responses, with a predominance of cell-mediated immune responses[2, 3]. CD4+ T cells play an important role primarily through the secretion of interferon-gamma (IFN-γ), interleukin -2 (IL-2), tumor necrosis factor-α (TNF-α) and other cytokines that are involved in immune control [4-6]. CD8+ T cells and γδ T cells play protective roles through the secretion of IFN-γ, perforin and granzyme[5-13]. Our previous studies in HIV/AIDS patients showed that cytokine secretion by M.tb antigen-specific CD8+ and Vγ2Vδ2+ T cells was greatly enhanced in subjects with latent tuberculosis infection compared with subjects with active tuberculosis. This enhanced response was likely to help the HIV-1-infected host effectively inhibit pathogen replication into a latent state[14] . However, the immune responses of T cell subsets among subjects with different M.tb infection statuses, such as active, latent and no M.tb infection, have not been fully elucidated in HIV-negative individuals. Specifically, data are limiting in the high 5
tuberculosis epidemic regions of China. Therefore, we conducted a case-controlled study to investigate the distributions and functions of T cell subsets, including CD3+, CD4+, CD8+ αβ and Vγ2Vδ2+ T cells, in HIV-negative subjects with different M.tb infection statuses. We further analyzed the immune responses at the lesion sites of M.tb infection (pleural effusion of patients with tuberculous pleurisy).
6
2. Materials and Methods 2.1. Study population One hundred and twenty-five individuals were recruited in this study, including active tuberculosis (ATB) patients (n=46), latent tuberculosis infection (LTBI) subjects (n=34), and healthy controls (HC) (n=45). The ATB patients were recruited from January 1, 2011, to October 31, 2011, from Wuxi No.5 People’s Hospital. LTBI subjects were recruited from the close contacts of ATB patients, and the healthy controls were recruited from volunteers at Fudan University. This study was approved with written consent by the Ethics Committee of Huashan Hospital, Fudan University, with approval number of 2011-247. Written informed consent was obtained from all of the participants. 2.2. Criteria for ATB, LTBI and HC inclusion We employed an IFN-γ release assay (IGRA) for tuberculosis to distinguish BCG vaccination from M.tb infection. The individuals were divided into three groups based on the IGRA assay. ATB patents included patients with active pulmonary tuberculosis (n=27) and tuberculous pleurisy (n=19). All patients with pulmonary tuberculosis were sputum acid-fast bacillus (AFB) smear- or culture-positive, and treatment naïve or anti-tuberculosis treated with a duration of less than 1 week. Confirmed tuberculous pleurisy was diagnosed with M.tb culture-positive in the pleural fluid and/or pleural biopsy. Thirty-four individuals were diagnosed with LTBI based on a positive IGRA and no evidence of active tuberculosis (e.g., clinical manifestations of pulmonary and extrathoracic tuberculosis and abnormal chest radiographs). Forty-five 7
individuals were healthy controls who had negative IGRA results and no evidence of active tuberculosis. All enrolled participants were HIV-negative, had not been diagnosed with cancer, diabetes, autoimmune diseases or other chronic infections (i.e., chronic HBV/HCV infection), and had not received immune modulator treatments. 2.3. Immunofluorescence staining and flow cytometric analysis Blood samples collected freshly from all groups of participants were handled, and analyzed by phenotyping and intracellular cytokine staining (ICS) at the biocontainment laboratory[12]. Peripheral blood mononuclear cells (PBMCs) were isolated from heparin-anticoagulated blood by density gradient sedimentation using Lympholyte-H (Cedarlane Laboratories Ltd, Ontario, Canada). For cell-surface staining, 100 µL of anticoagulated blood was treated with red blood cell (RBC) lysis buffer and washed twice with 5% fetal bovine serum (FBS)-phosphate-buffered saline (PBS) prior to staining[14]. PBMCs were stained with up to four Abs (conjugated to FITC, PE, allophycocyanin, pacific blue, and PE-Cy7) for at least 10 min at room temperature. After staining, the cells were fixed with 2% formaldehyde-PBS prior to analysis on a BD FACS Aria flow cytometer (BD Bioscience, San Diego, CA, USA). Lymphocytes were gated based on forward-scatter and side-scatter properties; at least 20,000 gated events were analyzed using the FCS EXPRESS 3 Software (De Novo Software, Glendale, CA, USA). Absolute cell numbers were calculated based on flow cytometry data and complete blood counts. The following mouse anti-human mAbs were used: Vγ2 (7A5) and Vδ2 (15D) (Thermo Scientific, Rockford, MD, USA); CD3 (SP34, SP34-2), CD8 (RPA-T8), CD28 (CD28.2), CD49d (9F10) and IFN-γ (4S.B3) 8
(BD Pharmingen, San Diego, CA, USA); and CD4 (OKT4) (BD Bioscience, San Diego, CA, USA). The secondary Ab (PE-conjugated goat anti-mouse IgG; Beckman Coulter, Marseille, France) was used for indirect staining. 2.4. Intracellular cytokine staining ICS was performed using the standard protocol as recently described[13-15]. For the ICS assay, 106 PBMCs plus the costimulatory mAbs CD28 (1 µg/mL) and CD49d (1 µg/mL) were incubated with purified protein derivative (PPD) (25 µg/mL), phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) (40 ng/mL) or media alone in a 200 µL final volume for 1 h at 37°C in a 5% CO2 atmosphere, followed by an additional 5 h of incubation in the presence of brefeldin A (GolgiPlug;
BD
Bioscience).
After
staining
the
cell
surface
markers
CD3/CD4/CD8/Vγ2 for at least 15 min at room temperature, the cells were permeabilized for 45 min (Cytofix/cytoperm; BD Biosciences) at 4°C and stained another 45 min for IFN-γ at room temperature prior to resuspending in 2% formaldehyde-PBS. HMBPP belongs to phosphoantigen
and only TCRs bearing
both the Vγ2 and Vδ2 elements can recognize HMBPP. The phosphoantigen compound HMBPP used in the study was 98% pure and specifically stimulated the activation/expansion of Vγ2Vδ2 T cells but not other cell subpopulations[12]. 2.5. IFN-γ release assay (IGRA) The IGRA used in this study was the QuantiFERON-TB Gold In-Tube (QFT-GIT) test. The QFT-GIT was performed according to the manufacturer’s instructions (QuantiFERON-TB Gold In-Tube, Cellestis Ltd., Carnegie, Australia). Briefly, a 3 9
mL venous blood sample was collected from each participant on the day of pleural effusion collection and aliquoted into three tubes (TB-specific antigen, mitogen and nil tubes, respectively). The samples were incubated at 37°C in a humidified 5% CO2 incubator for 24 hours. On the second day, the tubes were centrifuged at 3000 rcf for 10 minutes, and the plasma was collected and stored at 4°C until the IFN-γ assay was performed using an enzyme-linked immunosorbent assay (ELISA). The optical density (OD) of each test was read using a 450 nm filter with a 620 nm reference filter using the ELISA plate reader. The results were interpreted as positive, negative or indeterminate using the QFT-GIT analysis software developed by the company (QFT-GIT, Cellestis Ltd., Carnegie, Australia). If IFN-γ secretion in response to TB antigen was ≥0.35 IU/mL after subtracting the nil control, the sample was considered positive for QFT-GIT. If the value was <0.35 IU/mL, it was considered negative. If the negativity was associated with a poor PHA response (i.e., IFN-γ secretion in response to mitogen was <0.5 IU/mL), it was considered an indeterminate or invalid result for QFT-GIT. Subjects with IFN-γ secretion >8.0 IU/mL in the nil control samples were also considered indeterminate for QFT-GIT. 2.6. Statistical analysis Statistical analysis was performed with GraphPad Prism software (version 5.01; GraphPad Software, Inc.). The data were compared using the non-parametric Mann-Whitney test or Student’s t-test. Significance was inferred for P<0.05.
10
3. Results 3.1. Clinical characteristics of enrolled participants Based on the final diagnosis, the 125 enrolled participants were divided into three groups: the ATB group (n=46) with diagnosis of confirmed active pulmonary tuberculosis (n=27) and tuberculous pleurisy (n=19), the LTBI group (n=34) and the HC group (n=45). In the ATB group, patients with confirmed tuberculous pleurisy (n=19) were culture-positive for M.tb in the pleural fluid (n=5) and/or histologically confirmed to have tuberculosis by pleural biopsy under a thoracoscope (n=14). The median age of the enrolled patients was 47.6 years and 13 out of 19 were male (68.5%). Thirty (65.2%) participants received TB treatment; the treatment duration at enrollment ranged from 1-7 days. Three out of 45 (6.5%) patients had a history of exposure to active tuberculosis. The median age of the LTBI group was 44.8 years and 16 out of 34 were male. Among them, 16 (47.1%) had a history of exposure to active tuberculosis. In the HC group, the median age was 34.8 years and 18 out of 45 were male. Thirteen had a history of exposure to active tuberculosis (Table 1). More than 80% of the participants received the BCG vaccination, and all were screened by IGRA and chest X-ray or CT scan. The characteristics of the 3 groups were described in Table 1. 1 2 3
3.2. The numbers of T cell subsets of participants with different M.tb infection statuses To investigate the distributions of T cell subsets among the ATB, LTBI and HC groups, 11
1
whole blood cell surface staining and flow cytometry were used to detect the
2
percentages of peripheral blood CD3+, CD3+CD4+, CD3+CD8+ αβ and Vγ2Vδ2+ T
3
cells. Absolute cell numbers were calculated based on flow cytometry data and
4
complete blood counts. We found that the median numbers of CD3+ T cells in the
5
ATB, LTBI and HC groups were 890/µL, 1379/µL and 1326/µL, respectively, with the
6
ATB group significantly lower than both the LTBI and HC groups (P=0.0030
7
compared with LTBI and P=0.0009 compared with HC); while there was no
8
significant difference between the LTBI and HC groups (P = 0.167) (Figure 1).
9
We investigated the absolute numbers of T cell subsets, including CD3+CD4+,
10
CD3+CD8+ αβ and Vγ2Vδ2+ T cells, in the 3 groups. The median numbers of CD4+
11
T cells were 441/µL, 564/µL and 639/µL, respectively. Similar to the CD3+ T cells,
12
the ATB group had significantly lower numbers (P=0.0278 compared with LTBI and
13
P=0.0004 compared with HC) (Figure 1). The median numbers of CD8+ T cells were
14
325/µL, 420/µL and 381/µL, respectively. The number in the LTBI group was
15
significantly higher than the numbers in the ATB and HC groups (P=0.0460 and
16
P=0.0368, respectively) (Figure 1). Finally, the numbers of Vγ2Vδ2+ T cells in the 3
17
groups were 22/µL, 34/µL and 59/µL, respectively, with ATB exhibiting significantly
18
lower numbers compared to HC (P=0.0460) (Figure 1).
19
Thus, not only T cell numbers but also T cell subsets (including the αβ and γδ cell
20
numbers) were reduced during active tuberculosis compared with latent tuberculosis
21
or healthy controls. Therefore, it was worth exploring the changes in T cell functions
22
in subjects with different statuses of tuberculosis infection. 12
1 2
3.3. Antigen-specific T cell subsets of participants with different M.tb infection statuses
3
We compared the antigen-specific T cell subsets in the 3 groups using an ICS assay.
4
For αβ T cells (including CD4+ and CD8+ T cells), we employed PPD as the
5
tuberculosis antigen for in vitro stimulation. For γδ T cells, the phosphoantigen
6
HMBPP was used as the in vitro stimulator. As expected, the median number of
7
PPD-specific IFN-γ-secreting CD4+ T cells in ATB was significantly reduced (8/µL)
8
compared with the LTBI (median: 14/µL) and HC (median: 10/µL) groups (P=0.0001
9
and P=0.0027, respectively) (Figure 2). Similarly, the median numbers of
10
PPD-specific IFN-γ-secreting CD8+ T cells in the ATB, LTBI and HC groups were
11
14/µL, 27/µL and 18/µL, respectively, with the numbers in the ATB group
12
significantly lower than the LTBI group (P=0.0305) (Figure 2). Surprisingly, the
13
median numbers of HMBPP-specific IFN-γ-secreting Vγ2+ T cells were relatively
14
high in all 3 groups (19/µL, 48/µL and 51/µL, respectively), although the number in
15
the ATB group was significantly lower compared to the other 2 groups (P=0.0002
16
compared with LTBI and P<0.0001 compared with HC) (Figure 2).
17
These results were consistent with our previous study in TB-HIV coinfection subjects
18
where we demonstrated impaired T cell immune responses in HIV/AIDS patients
19
coinfected with active tuberculosis compared with subjects with latent tuberculosis or
20
without tuberculosis infection[14]. In this study, we confirmed that both a decline in
21
numbers and antigen-specific T cell immune responses correlated with active M.tb
22
infection. 13
1 2
3.4. The distributions and functions of T cell subsets at the tuberculous pleurisy lesion site.
3
To investigate the T cell immune responses at the tuberculosis lesion site, we
4
collected both peripheral blood and pleural effusion from 19 cases with tuberculous
5
pleurisy. Cell surface staining showed that the proportion of CD4+ T cells in the
6
pleural fluid mononuclear cells (PFMCs) was higher compared to the PBMCs
7
(P=0.0046), while CD8+ and Vγ2Vδ2+ T cells in the PFMCs were both lower
8
compared to the PBMCs (P=0.0029 and P=0.0042, respectively) (Figure 3A). The
9
ratio of CD4+/ CD8+ T cells in PFMCs was higher compared to PBMCs (median:
10
2.93 vs. 1.34, P<0.0001) (Figure 3B). Thus, CD4+ T cells may migrate from the
11
peripheral blood to the lesion site.
12
To investigate antigen-specific T cell immune responses at the lesion site, we detected
13
PPD-specific IFN-γ-secreting CD4+ and CD8+ T cells and HMBPP-specific
14
IFN-γ-secreting Vγ2Vδ2+ T cells in the PFMCs of patients with tuberculous pleurisy.
15
The ICS results indicated that antigen-specific CD4+ T cells predominated among
16
CD3+ T cells in the PFMCs, while antigen-specific CD8+ and Vγ2Vδ2+ T cells only
17
accounted for a small proportion (Figure 3C, 3D). Thus, we propose that CD4+ T
18
cells may migrate from the peripheral blood to the lesion site to exert anti-tuberculosis
19
immunity.
20 21
4. Discussion
22
Multiple cell subsets need to coordinate their interactions; for example, macrophages 14
1
and lymphocytes play a key role in controlling the growth of M.tb [11, 16-19].
2
However, the types of T cell subsets that mediate the onset of active TB or
3
maintenance of latent infection in humans remain unclear. Studies on T cell subsets in
4
patients with active tuberculosis found that peripheral CD3+, CD3+CD4+ and
5
CD3+CD8+ T cells were reduced compared to healthy controls[20, 21], which was
6
consistent with our results. However, data on the distributions of T cell subsets in
7
latent M.tb infection were limited. In our study, we simultaneously investigated T cell
8
subsets (including αβ and γδ T cells) in subjects with active TB, latent TB infection
9
and healthy controls. We found that CD3+ T cells in subjects with active tuberculosis
10
were significantly lower compared to both LTBI and healthy controls, indicating that
11
CD3+ T cell proliferation in active tuberculosis was inhibited. Moreover, CD4+,
12
CD8+ αβ and Vγ2Vδ2+ T cells showed similar changes in the 3 groups with different
13
M.tb infection statuses. Thus, we conclude that the numbers of T cell subsets are
14
reduced in active tuberculosis compared with latent tuberculosis infection and healthy
15
controls.
16
Our previous study on TB-HIV coinfection demonstrated that secretion of cytokines
17
by antigen-specific CD8+ and Vγ2Vδ2+ T cells was greatly enhanced, which likely
18
helped the host effectively inhibit pathogen replication and maintain the disease in a
19
latent state[14]. In vivo studies in a non-human primate model demonstrated that
20
CD4+ T cells were required to sustain multiple CD8+ T cell and CD3+ lymphocyte
21
effector functions and to prevent rapid TB progression during M.tb infection. CD8 T
22
cells also play a major role in anti-tuberculosis immunity[22, 23]. However, the 15
1
differences in antigen-specific T cells in subjects with different M.tb infection statuses
2
in humans are not clear. Most studies have focused on differences between active
3
tuberculosis and healthy individuals [23-25]. Studies on the distribution of T cell
4
subsets in populations with different M.tb infection statuses were limited[26-28]. We
5
employed PPD and HMBPP as stimulators to simultaneously detect antigen-specific
6
immune response in subjects with different M.tb infection statuses. Interestingly,
7
PPD-specific CD4+ and CD8+ T cells and HMBPP-specific Vγ2Vδ2+ T cells were
8
decreased in active tuberculosis compared with latent tuberculosis infection and
9
healthy controls, which was similar to the changes in the numbers of T cell subsets.
10
Surprisingly, the median number of HMBPP-specific Vγ2Vδ2+ T cells was even
11
higher compared to the PPD-specific CD4+ and CD8+ T cells. Non-human primate
12
model studies suggest that Vγ2Vδ2+ T effector cells may potentially enhance
13
antigen-specific antibody responses and CD4+ or CD8+ αβ T-cell responses in M.tb
14
infections[29]. Thus, we propose that the impaired function of CD4+ CD8+ and
15
Vγ2Vδ2+ T cells may be key factors for the progression from LTBI to ATB.
16
Current studies on M.tb-specific immune responses in PFMCs are rare[30-32]. To
17
further clarify the function of T cell subsets in M.tb infected lesions, we compared T
18
cell numbers and functions between the peripheral blood and pleural effusion from the
19
same patients with tuberculous pleurisy. Our study indicated that antigen-specific
20
CD4+ T cell might migrate from the periphery to the lesion site to exert
21
anti-tuberculosis functions. However, a recent study found that phosphoantigen/IL-2
22
administration
specifically
induced
major 16
expansion
and
pulmonary
1
trafficking/accumulation of phosphoantigen-specific Vγ2Vδ2+ T cells[33]. In our
2
study, the proportion of HMBPP-specific Vγ2Vδ2+ T cells was low compared with αβ
3
T cells in PFMCs and even lower compared to PBMCs. This discordancy might be
4
due to the difference between the lesion sites of the lung and pleural effusion. Thus, a
5
well-designed human study needs to be conducted to further demonstrate T cell
6
immune responses in different lesion sites of tuberculosis infection.
7
Conclusions
8
In summary, various T cells subsets are involved in the immune response to M.tb
9
infection. Both the numbers and functions of CD3+, CD4+, CD8+ αβ and Vγ2Vδ2+ T
10
cell are reduced in ATB compared with LTBI and HC. Moreover, the number and
11
function of CD4+ T cells predominate in PFMCs and are higher than PBMCs.
12
Therefore, antigen-specific CD4+ T cells may migrate from the periphery to the lesion
13
site to exert an anti-tuberculosis function. However, further studies are required to
14
clarify the mechanisms underlying the changes in CD8+ and Vγ2Vδ2+ T cells in
15
tuberculosis.
16
17
Competing interests
18
The authors declare that they have no competing interests.
19
20
Authors' contributions
21
YG performed T cell subset detection, clinical data analysis and drafted the 17
1
manuscript. SZ and QO participated in patient collection and data analysis. LS
2
performed the immunoassays. SW and JW performed the immunoassays and
3
statistical analysis. XW and WZ designed the study and helped draft the manuscript.
4
YZ helped draft the manuscript. XW participated in the design of the study. WZ and
5
LS designed the study, helped with patient collection and drafted the manuscript. All
6
authors read and approved the final manuscript.
7 8
Acknowledgement
9
Financial support. The present study was supported in part by the Key Technologies
10
Research
and
Development
Program
11
(2013ZX10003007-001-002).
12
Potential conflicts of interest. All authors: no conflicts.
13
18
for
Infectious
Diseases
of
China
1
Figure legends
2
Figure 1. T cell subset distributions in participants with different M.tb infection
3
statuses. The short transverse lines represent median numbers. ATB, active
4
tuberculosis; LTBI, latent tuberculosis infection; HC, healthy control.
5
Figure 2: Antigen-specific IFN-γ-secreting T cells in participants with different
6
M.tb infection statuses. The horizontal lines represent the median cell numbers. ATB,
7
active tuberculosis; LTBI, latent tuberculosis infection; HC, healthy control.
8
Figure 3. Distribution of numbers and antigen-specific IFN-γ-secreting T cells in
9
the peripheral blood and pleural effusion from patients with tuberculous
10
pleurisy. A. The percentages of CD3+CD4+, CD3+CD8+ and Vγ2Vδ2+ T cells in
11
CD3+ T cells in PBMCs and PFMCs. B. The ratio of CD3+CD4+/CD3+CD8+ T cells
12
in PBMCs and PFMCs. The horizontal lines represent the medians. C. The
13
representative histograms of PPD-specific IFN-γ-secreting T cells in PFMCs from a
14
patient with tuberculous pleurisy. A): PFMC-gated IFN-γ+ T cells with red dots
15
representing CD3+IFN-γ+ T cells; dots in the green rectangle represent CD3+ T cells.
16
B): The distribution of CD3-gated CD4+ and CD8+ T cells; the red dots represent
17
IFN-γ-secreting T cells. C): PFMC-gated IFN-γ+ T cells with red dots representing
18
CD3+IFN-γ+ T cells and dots in the red rectangle representing CD3+IFN-γ+ T cells.
19
D): The distribution of CD3+IFN-γ-gated CD4+ and CD8+ T cells ; the red dots
20
represent IFN-γ-secreting T cells. D. The percentages of IFN-γ-secreting T cell
21
subsets in PFMCs. PBMCs: peripheral blood mononuclear cells; PFMCs: pleural fluid
22
mononuclear cells. 19
Table 1. The demographic and clinical characteristics of study participants ATB
Pulmonary
Tuberculous
LTBI
HC
P value*
Total tuberculosis
pleurisy
27
19
46
34
45
--
16/11
13/6
29/17
16/18
18/27
0.081
43.1 (19-76)
47.6 (23-85)
44.8 (19-85)
41.1 (18-62)
34.8 (18-70)
0.108
BCG vaccination history, n (%)
23 (85.2)
15 (81.3)
38 (82.6)
28 (82.4)
42 (93.3)
0.338
Previous TB treatment, n (%)
21 (77.8)
9 (47.4)
30 (65.2)
--
--
2 (7.4)
1 (5.3)
3 (6.5)
26 (76.5)
13 (28.9)
27 (100.0)
1 (5.3)
28 (60.9)
--
--
n Male/Female
Age, median (range)
History of exposure to active TB, n (%)
Sputum AFB smear or culture positive, n (%)
20
0.553
Pleural effusion AFB smear/culture positive or --
19 (100)
19 (100)
--
--
confirmed TB by pleural biopsy, n (%)
Note：BCG: bacillus Calmette-Guerin; AFB: acid fast bacillus; ATB: Active tuberculosis; LTBI: Latent tuberculosis infection; HC: Healthy control. * Compared among groups of ATB, LTBI and HC
21
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[16] van Crevel R, Ottenhoff TH, van der Meer JW. Innate immunity to Mycobacterium tuberculosis. Advances in experimental medicine and biology 2003;531:241-7. [17] Schwander S, Dheda K. Human lung immunity against Mycobacterium tuberculosis: insights into pathogenesis and protection. American journal of respiratory and critical care medicine 2011;183:696-707. [18] Serbina NV, Jia T, Hohl TM, Pamer EG. Monocyte-mediated defense against microbial pathogens. Annual review of immunology 2008;26:421-52. [19] Chen K, Kolls JK. T cell-mediated host immune defenses in the lung. Annual review of immunology 2013;31:605-33. [20] Wu YE, Zhang SW, Peng WG, Li KS, Li K, Jiang JK, et al. Changes in lymphocyte subsets in the peripheral blood of patients with active pulmonary tuberculosis. The Journal of international medical research 2009;37:1742-9. [21] Henao-Tamayo MI, Ordway DJ, Irwin SM, Shang S, Shanley C, Orme IM. Phenotypic definition of effector and memory T-lymphocyte subsets in mice chronically infected with Mycobacterium tuberculosis. Clinical and vaccine immunology : CVI 2010;17:618-25. [22] Chen CY, Huang D, Wang RC, Shen L, Zeng G, Yao S, et al. A critical role for CD8 T cells in a nonhuman primate model of tuberculosis. PLoS pathogens 2009;5:e1000392. [23] Yao S, Huang D, Chen CY, Halliday L, Wang RC, Chen ZW. CD4+ T cells contain early extrapulmonary tuberculosis (TB) dissemination and rapid TB progression and sustain multieffector functions of CD8+ T and CD3- lymphocytes: mechanisms of CD4+ T cell immunity. Journal of immunology 2014;192:2120-32. [24] Sauzullo I, Scrivo R, Mengoni F, Ermocida A, Coppola M, Valesini G, et al. Multi-functional flow cytometry analysis of CD4(+) T cells as an immune biomarker for latent tuberculosis status in patients treated with tumour necrosis factor (TNF) antagonists. Clinical and experimental immunology 2014;176:410-7. [25] Prezzemolo T, Guggino G, La Manna MP, Di Liberto D, Dieli F, Caccamo N. Functional Signatures of Human CD4 and CD8 T Cell Responses to Mycobacterium tuberculosis. Frontiers in immunology 2014;5:180. [26] Qiu Z, Zhang M, Zhu Y, Zheng F, Lu P, Liu H, et al. Multifunctional CD4 T cell responses in patients with active tuberculosis. Scientific reports 2012;2:216. [27] Marin ND, Paris SC, Rojas M, Garcia LF. Functional profile of CD4± and CD8± T cells in latently infected individuals and patients with active TB. Tuberculosis 2013;93:155-66. [28] Yang J, He J, Huang H, Ji Z, Wei L, Ye P, et al. Molecular characterization of T cell receptor beta variable in the peripheral blood T cell repertoire in subjects with active tuberculosis or latent tuberculosis infection. BMC infectious diseases 2013;13:423. [29] Chen ZW. Multifunctional immune responses of HMBPP-specific Vgamma2Vdelta2 T cells in M. tuberculosis and other infections. Cellular & molecular immunology 2013;10:58-64. [30] Mitra DK, Sharma SK, Dinda AK, Bindra MS, Madan B, Ghosh B. Polarized 23
helper T cells in tubercular pleural effusion: phenotypic identity and selective recruitment. European journal of immunology 2005;35:2367-75. [31] Caramori G, Lasagna L, Casalini AG, Adcock IM, Casolari P, Contoli M, et al. Immune response to Mycobacterium tuberculosis infection in the parietal pleura of patients with tuberculous pleurisy. PloS one 2011;6:e22637. [32] Li Q, Li L, Liu Y, Fu X, Wang H, Lao S, et al. Biological functions of Mycobacterium tuberculosis-specific CD4+T cells were impaired by tuberculosis pleural fluid. Immunology letters 2011;138:113-21. [33] Chen CY, Yao S, Huang D, Wei H, Sicard H, Zeng G, et al. Phosphoantigen/IL2 expansion and differentiation of Vgamma2Vdelta2 T cells increase resistance to tuberculosis in nonhuman primates. PLoS pathogens 2013;9:e1003501.
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S0198-8859(15)00476-0 http://dx.doi.org/10.1016/j.humimm.2015.09.039 HIM 9618
To appear in:
Human Immunology
Received Date: Revised Date: Accepted Date:
23 June 2014 24 July 2015 26 September 2015
Please cite this article as: Gao, Y., Zhang, S., Ou, Q., Shen, L., Wang, S., Wu, J., Weng, X., Chen, Z.W., Zhang, W., Shao, L., Characterization of CD4/CD8+ α β and Vγ2Vδ2+ T cells in HIV-negative individuals with different Mycobacterium tuberculosis infection statuses, Human Immunology (2015), doi: http://dx.doi.org/10.1016/ j.humimm.2015.09.039
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Characterization of CD4/CD8+ αβ and Vγ2Vδ2+ T cells in HIV-negative individuals with different Mycobacterium tuberculosis infection statuses Yan Gao a, Shu Zhanga, Qinfang Oub, Lei Shena, Sen Wanga, Jing Wu a, Xinhua Wenga, Zheng W. Chenc, Wenhong Zhanga*, Lingyun Shaoa*
a
Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai
200040, China b
Department of Pulmonary Diseases, Wuxi No.5 People’s Hospital, Wuxi 214005,
China c
Department of Microbiology and Immunology, Center for Primate Biomedical
Research, University of Illinois College of Medicine, 835 S. Wolcott Avenue, MC790, Chicago, IL 60612
* Corresponding author. Mailing address: 12 Wulumuqi Zhong Road, Shanghai 200040, China. Phone: 86-21-52888123. Fax: 86-21-52888714. Email for Lingyun Shao:
[email protected];
[email protected]
E-mail address: YG: [email protected] SZ: [email protected] QO: [email protected] 1
for
Wenhong
Zhang:
LS: [email protected] SW: [email protected] JW: [email protected] XW: [email protected] ZC: [email protected] WZ: [email protected] LS: [email protected]
2
Abstract Background The immune responses of T cell subsets among patients with different Mycobacterium tuberculosis (M.tb) infection statuses [i.e., active tuberculosis (ATB), latent tuberculosis infection (LTBI) and non-infection (healthy control, HC)] have not been fully elucidated in HIV-negative individuals. Specifically, data are limiting in high tuberculosis epidemic regions in China. To investigate the distributions and functions of T cell subsets (i.e., CD3+, CD4+, CD8+ αβ and Vγ2Vδ2+ T cells) in HIV-negative subjects with different M.tb infection statuses, we conducted a case-control study that enrolled 125 participants, including ATB patients (n=46), LTBI subjects (n=34), and HC (n=45). Results An IFN-γ release assay (IGRA) was employed to screen LTBI subjects. Whole blood cell surface staining and flow cytometry were used to detect phenotypic distributions of T cells phenotypic distributions in the peripheral blood mononuclear cells (PBMCs) and tuberculous pleural fluid mononuclear cells (PFMCs). PPD and the phosphorylated antigen HMBPP were employed as stimulators for the detection of M.tb antigen-specific T cell functions via intracellular cytokine staining (ICS). The absolute numbers of T cell subsets, including CD3+CD4+, CD3+CD8+ αβ and Vγ2Vδ2+ T cells, were significantly reduced in active tuberculosis compared with latent tuberculosis or the healthy controls. Importantly, PPD-specific CD3+CD4+ and CD3+CD8+ αβ T cells and HMBPP-specific Vγ2Vδ2+ T cells in ATB patients were also significantly reduced compared to the LTBI/HC subjects (P<0.05). In contrast, the proportion of CD4+ T cells in PFMCs was higher compared to PBMCs, while 3
CD8+ and Vγ2Vδ2+ T cells in PFMCs were lower compared to PBMCs (all P<0.05). PPD-specific CD4+ T cells predominated among CD3+ T cells in PFMCs. Conclusions
Cellular
immune
responses
are
impaired
in
ATB
patients.
Antigen-specific CD4+ T cell may migrate from the periphery to the lesion site, where they exert anti-tuberculosis functions. Keywords: Active tuberculosis; Latent tuberculosis infection; Immune response; T cell subsets; Interferon-gamma
4
1. Introduction Approximately one-third of the world’s population (or two billion people) is estimated to be infected with Mycobacterium tuberculosis (M.tb). About 5–10% of infected people develop TB disease during their lifetime, mostly within 5 years after a new infection[1]. The risk that a new or latent infection will progress to disease is increased by a compromised immune system. M.tb as an intracellular pathogen that has a complex relationship with the host. When the natural immune response cannot control the growth of M.tb. There is continuous exposure to M.tb antigens. Then, the organism starts to induce acquired immune responses, with a predominance of cell-mediated immune responses[2, 3]. CD4+ T cells play an important role primarily through the secretion of interferon-gamma (IFN-γ), interleukin -2 (IL-2), tumor necrosis factor-α (TNF-α) and other cytokines that are involved in immune control [4-6]. CD8+ T cells and γδ T cells play protective roles through the secretion of IFN-γ, perforin and granzyme[5-13]. Our previous studies in HIV/AIDS patients showed that cytokine secretion by M.tb antigen-specific CD8+ and Vγ2Vδ2+ T cells was greatly enhanced in subjects with latent tuberculosis infection compared with subjects with active tuberculosis. This enhanced response was likely to help the HIV-1-infected host effectively inhibit pathogen replication into a latent state[14] . However, the immune responses of T cell subsets among subjects with different M.tb infection statuses, such as active, latent and no M.tb infection, have not been fully elucidated in HIV-negative individuals. Specifically, data are limiting in the high 5
tuberculosis epidemic regions of China. Therefore, we conducted a case-controlled study to investigate the distributions and functions of T cell subsets, including CD3+, CD4+, CD8+ αβ and Vγ2Vδ2+ T cells, in HIV-negative subjects with different M.tb infection statuses. We further analyzed the immune responses at the lesion sites of M.tb infection (pleural effusion of patients with tuberculous pleurisy).
6
2. Materials and Methods 2.1. Study population One hundred and twenty-five individuals were recruited in this study, including active tuberculosis (ATB) patients (n=46), latent tuberculosis infection (LTBI) subjects (n=34), and healthy controls (HC) (n=45). The ATB patients were recruited from January 1, 2011, to October 31, 2011, from Wuxi No.5 People’s Hospital. LTBI subjects were recruited from the close contacts of ATB patients, and the healthy controls were recruited from volunteers at Fudan University. This study was approved with written consent by the Ethics Committee of Huashan Hospital, Fudan University, with approval number of 2011-247. Written informed consent was obtained from all of the participants. 2.2. Criteria for ATB, LTBI and HC inclusion We employed an IFN-γ release assay (IGRA) for tuberculosis to distinguish BCG vaccination from M.tb infection. The individuals were divided into three groups based on the IGRA assay. ATB patents included patients with active pulmonary tuberculosis (n=27) and tuberculous pleurisy (n=19). All patients with pulmonary tuberculosis were sputum acid-fast bacillus (AFB) smear- or culture-positive, and treatment naïve or anti-tuberculosis treated with a duration of less than 1 week. Confirmed tuberculous pleurisy was diagnosed with M.tb culture-positive in the pleural fluid and/or pleural biopsy. Thirty-four individuals were diagnosed with LTBI based on a positive IGRA and no evidence of active tuberculosis (e.g., clinical manifestations of pulmonary and extrathoracic tuberculosis and abnormal chest radiographs). Forty-five 7
individuals were healthy controls who had negative IGRA results and no evidence of active tuberculosis. All enrolled participants were HIV-negative, had not been diagnosed with cancer, diabetes, autoimmune diseases or other chronic infections (i.e., chronic HBV/HCV infection), and had not received immune modulator treatments. 2.3. Immunofluorescence staining and flow cytometric analysis Blood samples collected freshly from all groups of participants were handled, and analyzed by phenotyping and intracellular cytokine staining (ICS) at the biocontainment laboratory[12]. Peripheral blood mononuclear cells (PBMCs) were isolated from heparin-anticoagulated blood by density gradient sedimentation using Lympholyte-H (Cedarlane Laboratories Ltd, Ontario, Canada). For cell-surface staining, 100 µL of anticoagulated blood was treated with red blood cell (RBC) lysis buffer and washed twice with 5% fetal bovine serum (FBS)-phosphate-buffered saline (PBS) prior to staining[14]. PBMCs were stained with up to four Abs (conjugated to FITC, PE, allophycocyanin, pacific blue, and PE-Cy7) for at least 10 min at room temperature. After staining, the cells were fixed with 2% formaldehyde-PBS prior to analysis on a BD FACS Aria flow cytometer (BD Bioscience, San Diego, CA, USA). Lymphocytes were gated based on forward-scatter and side-scatter properties; at least 20,000 gated events were analyzed using the FCS EXPRESS 3 Software (De Novo Software, Glendale, CA, USA). Absolute cell numbers were calculated based on flow cytometry data and complete blood counts. The following mouse anti-human mAbs were used: Vγ2 (7A5) and Vδ2 (15D) (Thermo Scientific, Rockford, MD, USA); CD3 (SP34, SP34-2), CD8 (RPA-T8), CD28 (CD28.2), CD49d (9F10) and IFN-γ (4S.B3) 8
(BD Pharmingen, San Diego, CA, USA); and CD4 (OKT4) (BD Bioscience, San Diego, CA, USA). The secondary Ab (PE-conjugated goat anti-mouse IgG; Beckman Coulter, Marseille, France) was used for indirect staining. 2.4. Intracellular cytokine staining ICS was performed using the standard protocol as recently described[13-15]. For the ICS assay, 106 PBMCs plus the costimulatory mAbs CD28 (1 µg/mL) and CD49d (1 µg/mL) were incubated with purified protein derivative (PPD) (25 µg/mL), phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) (40 ng/mL) or media alone in a 200 µL final volume for 1 h at 37°C in a 5% CO2 atmosphere, followed by an additional 5 h of incubation in the presence of brefeldin A (GolgiPlug;
BD
Bioscience).
After
staining
the
cell
surface
markers
CD3/CD4/CD8/Vγ2 for at least 15 min at room temperature, the cells were permeabilized for 45 min (Cytofix/cytoperm; BD Biosciences) at 4°C and stained another 45 min for IFN-γ at room temperature prior to resuspending in 2% formaldehyde-PBS. HMBPP belongs to phosphoantigen
and only TCRs bearing
both the Vγ2 and Vδ2 elements can recognize HMBPP. The phosphoantigen compound HMBPP used in the study was 98% pure and specifically stimulated the activation/expansion of Vγ2Vδ2 T cells but not other cell subpopulations[12]. 2.5. IFN-γ release assay (IGRA) The IGRA used in this study was the QuantiFERON-TB Gold In-Tube (QFT-GIT) test. The QFT-GIT was performed according to the manufacturer’s instructions (QuantiFERON-TB Gold In-Tube, Cellestis Ltd., Carnegie, Australia). Briefly, a 3 9
mL venous blood sample was collected from each participant on the day of pleural effusion collection and aliquoted into three tubes (TB-specific antigen, mitogen and nil tubes, respectively). The samples were incubated at 37°C in a humidified 5% CO2 incubator for 24 hours. On the second day, the tubes were centrifuged at 3000 rcf for 10 minutes, and the plasma was collected and stored at 4°C until the IFN-γ assay was performed using an enzyme-linked immunosorbent assay (ELISA). The optical density (OD) of each test was read using a 450 nm filter with a 620 nm reference filter using the ELISA plate reader. The results were interpreted as positive, negative or indeterminate using the QFT-GIT analysis software developed by the company (QFT-GIT, Cellestis Ltd., Carnegie, Australia). If IFN-γ secretion in response to TB antigen was ≥0.35 IU/mL after subtracting the nil control, the sample was considered positive for QFT-GIT. If the value was <0.35 IU/mL, it was considered negative. If the negativity was associated with a poor PHA response (i.e., IFN-γ secretion in response to mitogen was <0.5 IU/mL), it was considered an indeterminate or invalid result for QFT-GIT. Subjects with IFN-γ secretion >8.0 IU/mL in the nil control samples were also considered indeterminate for QFT-GIT. 2.6. Statistical analysis Statistical analysis was performed with GraphPad Prism software (version 5.01; GraphPad Software, Inc.). The data were compared using the non-parametric Mann-Whitney test or Student’s t-test. Significance was inferred for P<0.05.
10
3. Results 3.1. Clinical characteristics of enrolled participants Based on the final diagnosis, the 125 enrolled participants were divided into three groups: the ATB group (n=46) with diagnosis of confirmed active pulmonary tuberculosis (n=27) and tuberculous pleurisy (n=19), the LTBI group (n=34) and the HC group (n=45). In the ATB group, patients with confirmed tuberculous pleurisy (n=19) were culture-positive for M.tb in the pleural fluid (n=5) and/or histologically confirmed to have tuberculosis by pleural biopsy under a thoracoscope (n=14). The median age of the enrolled patients was 47.6 years and 13 out of 19 were male (68.5%). Thirty (65.2%) participants received TB treatment; the treatment duration at enrollment ranged from 1-7 days. Three out of 45 (6.5%) patients had a history of exposure to active tuberculosis. The median age of the LTBI group was 44.8 years and 16 out of 34 were male. Among them, 16 (47.1%) had a history of exposure to active tuberculosis. In the HC group, the median age was 34.8 years and 18 out of 45 were male. Thirteen had a history of exposure to active tuberculosis (Table 1). More than 80% of the participants received the BCG vaccination, and all were screened by IGRA and chest X-ray or CT scan. The characteristics of the 3 groups were described in Table 1. 1 2 3
3.2. The numbers of T cell subsets of participants with different M.tb infection statuses To investigate the distributions of T cell subsets among the ATB, LTBI and HC groups, 11
1
whole blood cell surface staining and flow cytometry were used to detect the
2
percentages of peripheral blood CD3+, CD3+CD4+, CD3+CD8+ αβ and Vγ2Vδ2+ T
3
cells. Absolute cell numbers were calculated based on flow cytometry data and
4
complete blood counts. We found that the median numbers of CD3+ T cells in the
5
ATB, LTBI and HC groups were 890/µL, 1379/µL and 1326/µL, respectively, with the
6
ATB group significantly lower than both the LTBI and HC groups (P=0.0030
7
compared with LTBI and P=0.0009 compared with HC); while there was no
8
significant difference between the LTBI and HC groups (P = 0.167) (Figure 1).
9
We investigated the absolute numbers of T cell subsets, including CD3+CD4+,
10
CD3+CD8+ αβ and Vγ2Vδ2+ T cells, in the 3 groups. The median numbers of CD4+
11
T cells were 441/µL, 564/µL and 639/µL, respectively. Similar to the CD3+ T cells,
12
the ATB group had significantly lower numbers (P=0.0278 compared with LTBI and
13
P=0.0004 compared with HC) (Figure 1). The median numbers of CD8+ T cells were
14
325/µL, 420/µL and 381/µL, respectively. The number in the LTBI group was
15
significantly higher than the numbers in the ATB and HC groups (P=0.0460 and
16
P=0.0368, respectively) (Figure 1). Finally, the numbers of Vγ2Vδ2+ T cells in the 3
17
groups were 22/µL, 34/µL and 59/µL, respectively, with ATB exhibiting significantly
18
lower numbers compared to HC (P=0.0460) (Figure 1).
19
Thus, not only T cell numbers but also T cell subsets (including the αβ and γδ cell
20
numbers) were reduced during active tuberculosis compared with latent tuberculosis
21
or healthy controls. Therefore, it was worth exploring the changes in T cell functions
22
in subjects with different statuses of tuberculosis infection. 12
1 2
3.3. Antigen-specific T cell subsets of participants with different M.tb infection statuses
3
We compared the antigen-specific T cell subsets in the 3 groups using an ICS assay.
4
For αβ T cells (including CD4+ and CD8+ T cells), we employed PPD as the
5
tuberculosis antigen for in vitro stimulation. For γδ T cells, the phosphoantigen
6
HMBPP was used as the in vitro stimulator. As expected, the median number of
7
PPD-specific IFN-γ-secreting CD4+ T cells in ATB was significantly reduced (8/µL)
8
compared with the LTBI (median: 14/µL) and HC (median: 10/µL) groups (P=0.0001
9
and P=0.0027, respectively) (Figure 2). Similarly, the median numbers of
10
PPD-specific IFN-γ-secreting CD8+ T cells in the ATB, LTBI and HC groups were
11
14/µL, 27/µL and 18/µL, respectively, with the numbers in the ATB group
12
significantly lower than the LTBI group (P=0.0305) (Figure 2). Surprisingly, the
13
median numbers of HMBPP-specific IFN-γ-secreting Vγ2+ T cells were relatively
14
high in all 3 groups (19/µL, 48/µL and 51/µL, respectively), although the number in
15
the ATB group was significantly lower compared to the other 2 groups (P=0.0002
16
compared with LTBI and P<0.0001 compared with HC) (Figure 2).
17
These results were consistent with our previous study in TB-HIV coinfection subjects
18
where we demonstrated impaired T cell immune responses in HIV/AIDS patients
19
coinfected with active tuberculosis compared with subjects with latent tuberculosis or
20
without tuberculosis infection[14]. In this study, we confirmed that both a decline in
21
numbers and antigen-specific T cell immune responses correlated with active M.tb
22
infection. 13
1 2
3.4. The distributions and functions of T cell subsets at the tuberculous pleurisy lesion site.
3
To investigate the T cell immune responses at the tuberculosis lesion site, we
4
collected both peripheral blood and pleural effusion from 19 cases with tuberculous
5
pleurisy. Cell surface staining showed that the proportion of CD4+ T cells in the
6
pleural fluid mononuclear cells (PFMCs) was higher compared to the PBMCs
7
(P=0.0046), while CD8+ and Vγ2Vδ2+ T cells in the PFMCs were both lower
8
compared to the PBMCs (P=0.0029 and P=0.0042, respectively) (Figure 3A). The
9
ratio of CD4+/ CD8+ T cells in PFMCs was higher compared to PBMCs (median:
10
2.93 vs. 1.34, P<0.0001) (Figure 3B). Thus, CD4+ T cells may migrate from the
11
peripheral blood to the lesion site.
12
To investigate antigen-specific T cell immune responses at the lesion site, we detected
13
PPD-specific IFN-γ-secreting CD4+ and CD8+ T cells and HMBPP-specific
14
IFN-γ-secreting Vγ2Vδ2+ T cells in the PFMCs of patients with tuberculous pleurisy.
15
The ICS results indicated that antigen-specific CD4+ T cells predominated among
16
CD3+ T cells in the PFMCs, while antigen-specific CD8+ and Vγ2Vδ2+ T cells only
17
accounted for a small proportion (Figure 3C, 3D). Thus, we propose that CD4+ T
18
cells may migrate from the peripheral blood to the lesion site to exert anti-tuberculosis
19
immunity.
20 21
4. Discussion
22
Multiple cell subsets need to coordinate their interactions; for example, macrophages 14
1
and lymphocytes play a key role in controlling the growth of M.tb [11, 16-19].
2
However, the types of T cell subsets that mediate the onset of active TB or
3
maintenance of latent infection in humans remain unclear. Studies on T cell subsets in
4
patients with active tuberculosis found that peripheral CD3+, CD3+CD4+ and
5
CD3+CD8+ T cells were reduced compared to healthy controls[20, 21], which was
6
consistent with our results. However, data on the distributions of T cell subsets in
7
latent M.tb infection were limited. In our study, we simultaneously investigated T cell
8
subsets (including αβ and γδ T cells) in subjects with active TB, latent TB infection
9
and healthy controls. We found that CD3+ T cells in subjects with active tuberculosis
10
were significantly lower compared to both LTBI and healthy controls, indicating that
11
CD3+ T cell proliferation in active tuberculosis was inhibited. Moreover, CD4+,
12
CD8+ αβ and Vγ2Vδ2+ T cells showed similar changes in the 3 groups with different
13
M.tb infection statuses. Thus, we conclude that the numbers of T cell subsets are
14
reduced in active tuberculosis compared with latent tuberculosis infection and healthy
15
controls.
16
Our previous study on TB-HIV coinfection demonstrated that secretion of cytokines
17
by antigen-specific CD8+ and Vγ2Vδ2+ T cells was greatly enhanced, which likely
18
helped the host effectively inhibit pathogen replication and maintain the disease in a
19
latent state[14]. In vivo studies in a non-human primate model demonstrated that
20
CD4+ T cells were required to sustain multiple CD8+ T cell and CD3+ lymphocyte
21
effector functions and to prevent rapid TB progression during M.tb infection. CD8 T
22
cells also play a major role in anti-tuberculosis immunity[22, 23]. However, the 15
1
differences in antigen-specific T cells in subjects with different M.tb infection statuses
2
in humans are not clear. Most studies have focused on differences between active
3
tuberculosis and healthy individuals [23-25]. Studies on the distribution of T cell
4
subsets in populations with different M.tb infection statuses were limited[26-28]. We
5
employed PPD and HMBPP as stimulators to simultaneously detect antigen-specific
6
immune response in subjects with different M.tb infection statuses. Interestingly,
7
PPD-specific CD4+ and CD8+ T cells and HMBPP-specific Vγ2Vδ2+ T cells were
8
decreased in active tuberculosis compared with latent tuberculosis infection and
9
healthy controls, which was similar to the changes in the numbers of T cell subsets.
10
Surprisingly, the median number of HMBPP-specific Vγ2Vδ2+ T cells was even
11
higher compared to the PPD-specific CD4+ and CD8+ T cells. Non-human primate
12
model studies suggest that Vγ2Vδ2+ T effector cells may potentially enhance
13
antigen-specific antibody responses and CD4+ or CD8+ αβ T-cell responses in M.tb
14
infections[29]. Thus, we propose that the impaired function of CD4+ CD8+ and
15
Vγ2Vδ2+ T cells may be key factors for the progression from LTBI to ATB.
16
Current studies on M.tb-specific immune responses in PFMCs are rare[30-32]. To
17
further clarify the function of T cell subsets in M.tb infected lesions, we compared T
18
cell numbers and functions between the peripheral blood and pleural effusion from the
19
same patients with tuberculous pleurisy. Our study indicated that antigen-specific
20
CD4+ T cell might migrate from the periphery to the lesion site to exert
21
anti-tuberculosis functions. However, a recent study found that phosphoantigen/IL-2
22
administration
specifically
induced
major 16
expansion
and
pulmonary
1
trafficking/accumulation of phosphoantigen-specific Vγ2Vδ2+ T cells[33]. In our
2
study, the proportion of HMBPP-specific Vγ2Vδ2+ T cells was low compared with αβ
3
T cells in PFMCs and even lower compared to PBMCs. This discordancy might be
4
due to the difference between the lesion sites of the lung and pleural effusion. Thus, a
5
well-designed human study needs to be conducted to further demonstrate T cell
6
immune responses in different lesion sites of tuberculosis infection.
7
Conclusions
8
In summary, various T cells subsets are involved in the immune response to M.tb
9
infection. Both the numbers and functions of CD3+, CD4+, CD8+ αβ and Vγ2Vδ2+ T
10
cell are reduced in ATB compared with LTBI and HC. Moreover, the number and
11
function of CD4+ T cells predominate in PFMCs and are higher than PBMCs.
12
Therefore, antigen-specific CD4+ T cells may migrate from the periphery to the lesion
13
site to exert an anti-tuberculosis function. However, further studies are required to
14
clarify the mechanisms underlying the changes in CD8+ and Vγ2Vδ2+ T cells in
15
tuberculosis.
16
17
Competing interests
18
The authors declare that they have no competing interests.
19
20
Authors' contributions
21
YG performed T cell subset detection, clinical data analysis and drafted the 17
1
manuscript. SZ and QO participated in patient collection and data analysis. LS
2
performed the immunoassays. SW and JW performed the immunoassays and
3
statistical analysis. XW and WZ designed the study and helped draft the manuscript.
4
YZ helped draft the manuscript. XW participated in the design of the study. WZ and
5
LS designed the study, helped with patient collection and drafted the manuscript. All
6
authors read and approved the final manuscript.
7 8
Acknowledgement
9
Financial support. The present study was supported in part by the Key Technologies
10
Research
and
Development
Program
11
(2013ZX10003007-001-002).
12
Potential conflicts of interest. All authors: no conflicts.
13
18
for
Infectious
Diseases
of
China
1
Figure legends
2
Figure 1. T cell subset distributions in participants with different M.tb infection
3
statuses. The short transverse lines represent median numbers. ATB, active
4
tuberculosis; LTBI, latent tuberculosis infection; HC, healthy control.
5
Figure 2: Antigen-specific IFN-γ-secreting T cells in participants with different
6
M.tb infection statuses. The horizontal lines represent the median cell numbers. ATB,
7
active tuberculosis; LTBI, latent tuberculosis infection; HC, healthy control.
8
Figure 3. Distribution of numbers and antigen-specific IFN-γ-secreting T cells in
9
the peripheral blood and pleural effusion from patients with tuberculous
10
pleurisy. A. The percentages of CD3+CD4+, CD3+CD8+ and Vγ2Vδ2+ T cells in
11
CD3+ T cells in PBMCs and PFMCs. B. The ratio of CD3+CD4+/CD3+CD8+ T cells
12
in PBMCs and PFMCs. The horizontal lines represent the medians. C. The
13
representative histograms of PPD-specific IFN-γ-secreting T cells in PFMCs from a
14
patient with tuberculous pleurisy. A): PFMC-gated IFN-γ+ T cells with red dots
15
representing CD3+IFN-γ+ T cells; dots in the green rectangle represent CD3+ T cells.
16
B): The distribution of CD3-gated CD4+ and CD8+ T cells; the red dots represent
17
IFN-γ-secreting T cells. C): PFMC-gated IFN-γ+ T cells with red dots representing
18
CD3+IFN-γ+ T cells and dots in the red rectangle representing CD3+IFN-γ+ T cells.
19
D): The distribution of CD3+IFN-γ-gated CD4+ and CD8+ T cells ; the red dots
20
represent IFN-γ-secreting T cells. D. The percentages of IFN-γ-secreting T cell
21
subsets in PFMCs. PBMCs: peripheral blood mononuclear cells; PFMCs: pleural fluid
22
mononuclear cells. 19
Table 1. The demographic and clinical characteristics of study participants ATB
Pulmonary
Tuberculous
LTBI
HC
P value*
Total tuberculosis
pleurisy
27
19
46
34
45
--
16/11
13/6
29/17
16/18
18/27
0.081
43.1 (19-76)
47.6 (23-85)
44.8 (19-85)
41.1 (18-62)
34.8 (18-70)
0.108
BCG vaccination history, n (%)
23 (85.2)
15 (81.3)
38 (82.6)
28 (82.4)
42 (93.3)
0.338
Previous TB treatment, n (%)
21 (77.8)
9 (47.4)
30 (65.2)
--
--
2 (7.4)
1 (5.3)
3 (6.5)
26 (76.5)
13 (28.9)
27 (100.0)
1 (5.3)
28 (60.9)
--
--
n Male/Female
Age, median (range)
History of exposure to active TB, n (%)
Sputum AFB smear or culture positive, n (%)
20
0.553
Pleural effusion AFB smear/culture positive or --
19 (100)
19 (100)
--
--
confirmed TB by pleural biopsy, n (%)
Note：BCG: bacillus Calmette-Guerin; AFB: acid fast bacillus; ATB: Active tuberculosis; LTBI: Latent tuberculosis infection; HC: Healthy control. * Compared among groups of ATB, LTBI and HC
21
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