Effect of HMBPP and peptide E7 on IL-6 production by γδ T cells in pleural fluid of tuberculosis patients and umbilical cord blood

doi:10.3877/cma.j.issn.1674-0785.2020.08.002

Abstract

Abstract:

Objective

To investigate the effect of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) and peptide E7 on the production of interleukin-6 (IL-6) by γδ T cells in the pleural fluid of tuberculosis patients and umbilical cord blood.

Methods

Eighteen samples of pleural fluid were collected from patients who were clinically diagnosed with tuberculous pleurisy at Guangzhou Chest Hospital and 17 samples of umbilical cord blood were collected at the Third Affiliated Hospital of Sun Yat-sen University. γδ T cells and CD277⁺ CD14⁺ monocyte-macrophages were sorted from the pleural fluid and umbilical cord blood samples by fluorescence-activated cell sorting. They were co-cultured and stimulated with HMBPP or E7. The percentages of γδ T cells secreting IL-6 and CD277⁺ CD14⁺ monocyte-macrophages expressing programmed cell death ligand-1 (PD-L1) were detected by flow cytometry. Expression of IL-6 and PD-L1 mRNA in cells was detected by RT-PCR. Secretion of IL-6 was measured by ELISA. PD-L1 specific small interfering RNA (siRNA) was used to knock down the expression of PD-L1 to detect whether PD-L1 is involved in the production of IL-6. After stimulation with HMBPP or E7, the difference in the above mean values was compared between the control group and the treatment group by independent sample t-test.

Results

In the pleural fluid of the tuberculosis patients and umbilical cord blood, after 24 hours of stimulation with HMBPP or E7, the percentages of IL-6⁺ γδ T cells were significantly higher than that of the control group. After 12 hours of stimulation with HMBPP or E7, the relative expression of IL-6 in γδ T cells was significantly increased at the mRNA and protein levels. After HMBPP or E7 stimulation, the relative expression of PD-L1 in CD277⁺ CD14⁺ monocyte-macrophages were significantly up-regulated [(3.70±0.33) and (4.20±0.34) vs 1.00]. When the expression of PD-L1 was knocked down, the expression of IL-6 in γδ T cells was significantly decreased [(0.53±0.03) and (0.55±0.01) vs 1.00]. All the above differences were statistically significant (all P<0.05).

Conclusion

HMBPP and peptide E7 up-regulate PD-L1 expression in CD277⁺ CD14⁺ mononuclear-macrophages, which are involved in stimulating co-cultured γδ T cells to produce more IL-6. This finding provides a basis for the research of the anti-tuberculosis immune mechanism of γδ T cells and the immunotherapy of tuberculosis patients.

Key words: γδ T cells, CD14⁺ CD277⁺ monocyte-macrophages, Interleukin-6, Programmed cell death ligand-1

Zhu Chang, Zhixiong Mei, Yimin Fang, Jiao Wang, Mingliu Huang, Xiaomin Lai. Effect of HMBPP and peptide E7 on IL-6 production by γδ T cells in pleural fluid of tuberculosis patients and umbilical cord blood[J]. Chinese Journal of Clinicians(Electronic Edition), 2020, 14(08): 592-598.

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References 26

1	Li Y, Wang X, Teng D, et al. Identification of the Ligands of TCRγδ by Screening the Immune Repertoire of γδT Cells From Patients With Tuberculosis [J]. Front Immunol, 2019, 10: 2282.
2	WHO. Global Tuberculosis Report 2019 [R]. Geaeva: WHO, 2019.
3	Allison TJ, Winter CC, Fournié JJ, et al. Structure of a human γδ T-cell antigen receptor [J]. Nature, 2001, 411(6839): 820-824.
4	Salim M, Knowles TJ, Baker AT, et al. BTN3A1 discriminates γδ T cell phosphoantigens from nonantigenic small molecules via a conformational sensor in its B30. 2 domain [J]. ACS Chem Biol, 2017, 12(10): 2631-2643.
5	王姣, 方毅敏, 梅志雄, 等. CD277^＋/-的备选抗原递呈细胞在γδ T细胞识别不同抗原中的限制性递呈作用 [J]. 热带医学杂志, 2018, 18(5): 555-560.
6	Ha SH, Choi H, Park JY, et al. Mycobacterium tuberculosis-Secreted Protein, ESAT-6, Inhibits Lipopolysaccharide-Induced MMP-9 Expression and Inflammation Through NF-κB and MAPK Signaling in RAW 264.7 Macrophage Cells [J]. Inflammation, 2020, 43(1): 54-65.
7	Li B, Rossman MD, Imir T, et al. Disease-specific changes in gammadelta T cell repertoire and function in patients with pulmonary tuberculosis [J]. J Immunol, 1996, 157(9): 4222-4229.
8	Compte E, Pontarotti P, Collette Y, et al. Frontline: characterization of BT3 molecules belonging to the B7 family expressed on immune cells [J]. Eur J Immunol, 2004, 34(8): 2089-2099.
9	Qaqish A, Huang D, Chen CY, et al. Adoptive transfer of phosphoantigen-specific γδ T cell subset attenuates Mycobacterium tuberculosis infection in nonhuman primates [J]. J Immunol, 2017, 198(12): 4753-4763.
10	Villani FNA, Da Costa Rocha MO, Nunes MD, et al. Trypanosoma cruzi-Induced Activation of Functionally Distinct αβ and γδ CD4⁻ CD8⁻ T Cells in Individuals with Polar Forms of Chagas′ Disease [J]. Infect Immun, 2010, 78(10): 4421-4430.
11	Antonelli LR, Dutra WO, Oliveira RR, et al. Disparate immunoregulatory potentials for double-negative (CD4⁻ CD8⁻) αβ and γδ T cells from human patients with cutaneous leishmaniasis [J]. Infect Immun, 2006, 74(11): 6317-6323.
12	Rita Casetti, Angelo Martino. The plasticity of gamma delta T cells: innate immunity, antigen presentation and new immunotherapy [J]. Cell Mol Immunol, 2008, 5(3): 161-170.
13	Gioia C, Agrati C, Goletti D, et al. Different Cytokine Production and Effector/Memory Dynamics of αβ^＋ or γδ^＋ T-Cell Subsets in the Peripheral Blood of Patients with Active Pulmonary Tuberculosis [J]. Int J Immunopathol Pharmacol, 2003, 16(3): 247-252.
14	Ravimohan S, Maenetje P, Auld SC, et al. A Common NLRC4 Gene Variant Associates With Inflammation and Pulmonary Function in Human Immunodeficiency Virus and Tuberculosis [J]. Clin Infect Dis, 2019, 22: ciz898.
15	Manabe YC, Andrade BDB, Gupte N, et al. A Parsimonious Host Inflammatory Biomarker Signature Predicts Incident TB and Mortality in Advanced HIV [J]. Clin Infect Dis, 2019, 25: ciz1147.
16	Kishimoto T. The biology of interleukin-6 [J]. Blood, 1989, 74(1): 1-10.
17	Kishimoto T. Interleukin-6: from basic science to medicine-40 years in immunology [J]. Annu Rev Immunol, 2005, 23: 1-21.
18	Akira S, Taga T, Kishimoto T. Interleukin-6 in biology and medicine [J]. Adv Immunol, 1993, 54: 1-78.
19	Kang S, Tanaka T, Kishimoto T. Therapeutic uses of anti-interleukin-6 receptor antibody [J]. Int Immunol, 2015, 27(1): 21-29.
20	Tanaka T, Narazaki M, Kishimoto T. Therapeutic targeting of the interleukin-6 receptor [J]. Annu Rev Pharmacol Toxicol, 2012, 52: 199-219.
21	Tanaka T, Narazaki M, Kishimoto T. IL-6 in inflammation, immunity, and disease [J]. Cold Spring Harb Perspect Biol, 2014, 6(10): a016295.
22	Garbers C, Heink S, Korn T, et al. Interleukin-6: designing specific therapeutics for a complex cytokine [J]. Nat Rev Drug Discov, 2018, 17(6): 395-412.
23	Dong H, Zhu G, Tamada K, et al. B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion [J]. Nat Med, 1999, 5(12): 1365-1369.
24	Liang SC, Greenwald RJ, Latchman YE, et al. PD-L1 and PD-L2 have distinct roles in regulating host immunity to cutaneous leishmaniasis [J]. Eur J Immunol, 2006, 36(1): 58-64.
25	Ye ZJ, Zhou Q, Yin W, et al. Interleukin 22-producing CD4^＋ T cells in malignant pleural effusion [J]. Cancer lett, 2012, 326(1): 23-32.
26	Bian B, Fanale D, Dusetti N, et al. Prognostic significance of circulating PD-1, PD-L1, pan-BTN3As, BTN3A1 and BTLA in patients with pancreatic adenocarcinoma [J]. Oncoimmunology, 2019, 8(4): e1561120.

基因名称	引物序列
IL-6	上游引物	5′-AAAGAGGCACTGGCAGAAAA-3′
IL-6	下游引物	5′-AGCTCTGGCTTGTTCCTCAC-3′
PD-L1	上游引物	5′-GGACAAGCAGTGACCATCAAG-3′
PD-L1	下游引物	5′- CCCAGAATTACCAAGTGAGTCCT-3′
ACTB	上游引物	5′-TGAAGGTAGTTTCGTGGATGC-3′
ACTB	下游引物	5′-TCCCTGGAGAAGAGCTACGA-3′

基因名称	引物序列
IL-6	上游引物	5′-AAAGAGGCACTGGCAGAAAA-3′
IL-6	下游引物	5′-AGCTCTGGCTTGTTCCTCAC-3′
PD-L1	上游引物	5′-GGACAAGCAGTGACCATCAAG-3′
PD-L1	下游引物	5′- CCCAGAATTACCAAGTGAGTCCT-3′
ACTB	上游引物	5′-TGAAGGTAGTTTCGTGGATGC-3′
ACTB	下游引物	5′-TCCCTGGAGAAGAGCTACGA-3′

名称	序列
siPD-L1	正义链	5′-CCACCAAUUCCAAGAGAGATT-3′
siPD-L1	反义链	5′-UCUCUCUUGGAAUUGGUGGTT-3′
NC	正义链	5′-UUCUCCGAACGUGUCACGUTT-3′
NC	反义链	5′-ACGUGACACGUUCGGAGAATT-3′

名称	序列
siPD-L1	正义链	5′-CCACCAAUUCCAAGAGAGATT-3′
siPD-L1	反义链	5′-UCUCUCUUGGAAUUGGUGGTT-3′
NC	正义链	5′-UUCUCCGAACGUGUCACGUTT-3′
NC	反义链	5′-ACGUGACACGUUCGGAGAATT-3′

组别	例数	30 min	5 h	12 h	24 h	36 h	48 h	72 h
对照组	6	2.38±0.36	3.35±0.23	3.80±0.26	5.02±0.35	5.17±0.36	5.82±0.35	7.41±0.49
HMBPP	6	3.36±0.37	3.87±0.25	4.88±0.45	7.67±0.50	9.29±0.55	19.94±0.85	23.76±0.90
E7	6	2.82±0.15	4.31±0.39	4.84±0.50	7.73±0.39	8.35±0.49	21.60±1.57	26.03±1.27
t值^a	?	1.89	1.53	2.05	4.34	6.31	15.44	14.75
P值^a	?	0.0877	0.1568	0.0676	0.0015	<0.0001	<0.0001	<0.0001
t值^b	?	1.11	2.12	1.80	5.21	5.26	9.80	13.69
P值^b	?	0.2915	0.0597	0.1019	0.0004	<0.0001	<0.0001	<0.0001

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