Effect of HMBPP and peptide E7 on IL-6 production by &#x03B3;&#x03B4; 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

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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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组别	例数	30 min	5 h	12 h	24 h	36 h	48 h	72 h
对照组	9	2.10±0.24	3.48±0.41	3.25±0.50	4.34±0.27	5.40±0.28	5.47±0.28	7.23±0.42
HMBPP	9	2.97±0.41	4.45±0.34	3.61±0.31	9.86±0.45	17.98±0.62	27.25±0.43	34.49±0.88
E7	9	2.42±0.23	4.66±0.40	4.57±0.39	10.03±0.27	18.87±0.37	28.46±0.50	34.83±0.65
t值^a	?	1.85	1.84	0.61	10.51	17.90	41.95	28.33
P值^a	?	0.0826	0.0852	0.5534	<0.0001	<0.0001	<0.0001	<0.0001
t值^b	?	0.98	2.08	2.09	14.90	26.79	39.97	35.73
P值^b	?	0.3409	0.0543	0.0534	<0.0001	<0.0001	<0.0001	<0.0001

组别	例数	30 min	5 h	12 h	24 h	36 h	48 h	72 h
对照组	6	1.00	1.00	1.00	1.00	1.00	1.00	1.00
HMBPP	6	1.00±0.04	1.06±0.03	1.22±0.04	1.44±0.07	2.0±0.04	2.75±0.05	3.30±0.11
E7	6	1.02±0.05	1.02±0.04	1.24±0.05	1.39±0.09	2.00±0.07	2.83±0.06	3.60±0.09
t值^a	?	0.02	1.86	5.54	6.18	28.46	38.13	20.58
P值^a	?	0.9809	0.0931	0.0003	0.0001	<0.0001	<0.0001	<0.0001
t值^b	?	0.47	0.55	4.65	4.42	14.04	29.43	28.88
P值^b	?	0.6513	0.5935	0.0009	0.0013	<0.0001	<0.0001	<0.0001

组别	例数	5 h	12 h	24 h	36 h	48 h	72 h
对照组	11	3.75±0.34	7.86±0.31	22.28±0.94	25.65±0.97	28.48±0.58	32.66±0.91
HMBPP	11	4.78±0.40	16.99±0.86	34.71±0.87	48.78±1.23	49.07±1.24	64.37±2.04
E7	11	1.64±0.39	14.99±0.44	36.14±0.79	51.98±1.21	45.28±1.48	69.51±1.66
t值^a	?	1.95	10.01	9.73	14.73	15.07	14.21
P值^a	?	0.0653	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001
t值^b	?	1.735	13.36	11.26	16.92	10.56	19.43
P值^b	?	0.0981	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001

组别	例数	5 h	12 h	24 h	36 h	48 h	72 h
对照组	17	3.05±0.20	15.39±0.47	18.68±0.72	23.55±0.90	28.65±0.96	30.13±0.75
HMBPP	17	3.66±0.24	20.07±0.66	43.84±1.32	5.029±0.58	60.29±0.83	63.37±0.70
E7	17	3.18±0.27	21.19±0.58	40.44±1.07	48.52±0.91	60.05±0.85	64.43±1.02
t值^a	?	1.90	5.78	16.73	24.93	24.93	32.47
P值^a	?	0.0659	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001
t值^b	?	0.39	7.78	16.93	19.46	24.51	27.16
P值^b	?	0.7024	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001

组别	例数	PD-L1在mRNA水平相对表达量	CD277^＋CD14^＋ PD-L1^＋细胞比例（%）
对照组	6	1.00	3.63±0.27
HMBPP刺激组	6	3.70±0.33	12.63±0.57
E7刺激组	6	4.20±0.34	15.11±0.65
t值^a	?	8.23	9.43
P值^a	?	<0.0001	<0.0001
t值^b	?	14.26	16.39
P值^b	?	<0.0001	<0.0001

组别	例数	PD-L1	IL-6
空白对照组	5	0.96±0.02	0.96±0.03
阴性对照siRNA组	5	1.00	1.00
HMBPP＋PD-L1 siRNA转染组	5	0.44±0.04	0.53±0.03
E7＋PD-L1 siRNA转染组	5	0.52±0.04	0.55±0.01
t值^a	?	1.96	1.44
P值^a	?	0.0858	0.1893
t值^b	?	14.35	17.93
P值^b	?	<0.0001	<0.0001
t值^c	?	13.13	31.10
P值^c	?	<0.0001	<0.0001

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组别	例数	30 min	5 h	12 h	24 h	36 h	48 h	72 h
对照组	9	1.00	1.00	1.00	1.00	1.00	1.00	1.00
HMBPP	9	1.02±0.03	1.07±0.04	1.21±0.02	1.33±0.04	1.72±0.06	2.50±0.11	2.98±0.16
E7	9	1.04±0.03	1.06±0.03	1.25±0.02	1.29±0.04	1.58±0.06	2.52±0.14	3.19±0.11
t值^a	?	0.70	1.73	8.47	7.57	11.60	13.15	12.01
P值^a	?	0.4952	0.1034	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001
t值^b	?	1.12	1.80	11.42	6.83	9.78	10.94	20.51
P值^b	?	0.2785	0.0911	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001

组别	例数	30 min	5 h	12 h	24 h	36 h	48 h	72 h
对照组	9	1.00	1.00	1.00	1.00	1.00	1.00	1.00
HMBPP	9	1.02±0.03	1.07±0.04	1.21±0.02	1.33±0.04	1.72±0.06	2.50±0.11	2.98±0.16
E7	9	1.04±0.03	1.06±0.03	1.25±0.02	1.29±0.04	1.58±0.06	2.52±0.14	3.19±0.11
t值^a	?	0.70	1.73	8.47	7.57	11.60	13.15	12.01
P值^a	?	0.4952	0.1034	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001
t值^b	?	1.12	1.80	11.42	6.83	9.78	10.94	20.51
P值^b	?	0.2785	0.0911	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001

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

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