DEB-TACE与c-TACE治疗中晚期肝细胞癌的免疫微环境差异及TRADD介导Th17分化的机制

doi:10.13481/j.1671-587X.20260319

Abstract

Abstract:

Objective To investigate the differences in the local immune microenvironment after drug-eluting bead transarterial chemoembolization （DEB-TACE） and conventional transarterial chemoembolization （c-TACE） for intermediate-advanced hepatocellular carcinoma （HCC）， and to clarify the possible mechanism by which tumor necrosis factor receptor-associated death domain （TRADD） mediates the differentiation of CD4⁺T lymphocytes into T helper 17 （Th17） lymphocytes. Methods The clinical samples from 56 patients with intermediate-advanced HCC who received TACE treatment were retrospectively collected and divided into DEB-TACE group and c-TACE group， with 28 cases in each group. High-throughput RNA sequencing was used to compare the changes in gene expression profiles and infiltration abundance of immune cells in HCC tissue before and after treatment in two groups. Flow cytometry was applied to sort CD4⁺T lymphocytes and Th17 cells from HCC tissues in two groups. The expressions of TRADD， interleukin （IL）-23A， and other related molecules in the cells were detected by real-time quantitative PCR （RT-qPCR） method， Western blotting method， and immunofluorescence method. The primary CD4⁺T lymphocytes were isolated and activated in vitro， and TRADD overexpression （TRADD-OE） and knockdown （TRADD-KD） models were constructed. The proliferation viability of CD4⁺T lymphocytes was detected by methytthiazolyl diphenyl-tetra zolilim（MTT） assay； the percentages of CD161⁺CCR6⁺ in Th17 cells in various group were detected by flow cytometry； the levels of IL-17 in the cell supernatant in various groups were measured by ELISA. The expression levels of key proteins of Janus kinase/signal transducer pathway and activator of transcription（STAT） signal phosphorylated-JAK2 （p-JAK2） and phosphorylated-STAT3 （p-STAT3） proteins and its downstream transcription factors aryl hydrocarbon receptor （AHR） and RAR-related orphan receptor gamma t （RORγt） mRNA and proteins were detected by Western blotting and RT-qPCR methods. The JAK inhibitor （Ruxolitinib） was used to intervene in TRADD-OE cells， and the aforementioned Th17 differentiation and signaling pathway indicators were detected again. Results Compared with c-TACE group， the expression levels of TRADD and IL-23A in post-operation HCC tissue in DEB-TACE group were significantly increased （P<0.05）， and the infiltration abundance of central memory CD4⁺T lymphocytes and Th17 cells was significantly increased. Compared with c-TACE group， the percentage of CD4⁺ T lymphocytes in HCC tissue in DEB-TACE group was significantly increased （P<0.05）， and the expression levels of TRADD and IL-23A mRNA and protein in the sorted CD4⁺T lymphocytes and Th17 cells were significantly increased （P<0.01）. Compared with Con-OE group， the proliferation activity of CD4⁺T lymphocytes in TRADD-OE group was significantly increased （P<0.01）； the of CD161⁺CCR6⁺ Th17 cells and the level of IL-17 in the supernatant were significantly increased （P<0.01）； the expression levels of p-JAK2， p-STAT3， AHR， and RORγt mRNA and proteins in the cells were all significantly increased （P<0.01）. Compared with Con-KD group， the above indicators in TRADD-KD group were all significantly decreased （P<0.01）. After intervention with JAK inhibitor， compared with TRADD-OE group， the percentage of CD161⁺CCR6⁺ Th17 cells and the level of IL-17 in the supernatant in TRADD-OE+JAK inhibitor group were significantly decreased （P<0.01）， and the fluorescence intensities of CD161 and CCR6， as well as the expression levels of AHR and RORγt mRNA and proteins in the cells， were significantly decreased （P<0.01）. Conclusion Compared with c-TACE， DEB-TACE can more significantly remodel the local immune microenvironment of HCC and increase Th17 cell infiltration. Its mechanism may be related to the high expression of TRADD and activating the JAK/STAT signaling pathway， thereby upregulating AHR and RORγt expressions and promoting the differentiation of CD4⁺T lymphocytes into Th17 cells.

Key words: Hepatocellular Carcinoma, Drug-eluting bead transarterial chemoembolization, Immune microenvironment, Tumor necrosis factor receptor-associated death domain, T helper 17 lymphocyte, Janus Kinase, Signal transducer and activator of transcription

CLC Number:

R735.7

Gengfei CAO, SHAYA·Mahati,Junpeng GU,Weizheng JI, ASIHAER·Hasimu,Weixin REN. Differences in immune microenvironment between DEB-TACE and c-TACE in treatment of hepatocellular carcinoma and mechanism of TRADD-mediated Th17 differentiation[J].Journal of Jilin University(Medicine Edition), 2026, 52(3): 764-780.

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References 35

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Gene_ID	Cell type	Fold change	P value
AIM2	Activated CD4⁺T lymphocytes	20.368 553 210	0.013 000 638
PDCD1	T follicular helper cell	15.579 615 880	0.011 096 163
CLEC5A	Regulatory T lymphocytes	14.950 457 650	0.017 072 651
ANKRD22	Th17 cell	14.514 970 750	0.016 330 799
FASLG	Natural killer cell	13.856 520 250	0.015 023 090
HOXA1	CD56 bright natural killer cell	13.207 607 280	0.005 457 394
BCL11B	CD56 bright natural killer cell	11.030 248 040	0.011 544 782
KLRC1	Natural killer cell	10.429 241 360	0.038 653 619
CDH3	CD56 bright natural killer cell	9.706 281 760	0.015 512 624
CD70	Type 1 T helper cell	9.309 774 469	0.000 586 023
L1CAM	Regulatory T lymphocytes	9.298 367 003	0.006 524 248

Gene_ID	Cell type	Fold change	P value
ABHD3	Central memory CD4⁺T lymphocytes	1.299 519 371	0.031 902 364
ANK1	Th17 cell	15.934 043 830	5.156 56E-05
ANKRD22	Th17 cell	6.135 125 138	0.023 938 76
BZW2	Central memory CD4⁺T lymphocytes	1.475 780 042	0.012 213 002
CAMTA1	Th17 cell	1.103 822 788	0.026 864 438
CCDC65	Th17 cell	1.407 963 750	0.047 138 832
CD40	Th17 cell	1.621 074 362	0.017 409 637
CD63	Central memory CD4⁺T lymphocytes	1.306 966 557	0.000 565 941
ELMO2	Central memory CD4⁺T lymphocytes	1.442 394 442	0.025 307 557
IFT80	Th17 cell	1.486 992 324	0.023 311 561
IL17RA	Th17 cell	2.020 523 713	0.012 006 794
IL23A	Th17 cell	1.870 852 777	0.011 222 365
ITGB1	Central memory CD4⁺T lymphocytes	1.129 685 920	0.047 012 047
LSP1	Central memory CD4⁺T lymphocytes	2.381 922 808	0.030 755 375
SFXN3	Central memory CD4⁺T lymphocytes	1.909 800 611	0.002 226 840
STX4	Central memory CD4⁺T lymphocytes	1.432 307 982	0.005 207 203
TNIP2	Th17 cell	1.535 863 268	0.008 326 440
TRADD	Central memory CD4⁺T lymphocytes	1.701 717 402	0.007 064 042

Group	Cell type	TRADD mRNA	IL-23A mRNA	LSP1 mRNA	ITGB1 mRNA
c-TACE	CD4⁺T	0.83±0.07	0.58±0.02	0.94±0.05	1.04±0.05
	Th17	0.81±0.02	0.57±0.01	0.97±0.02	0.98±0.01
DEB-TACE	CD4⁺T	1.00±0.03^*	1.00±0.02^*	1.00±0.03	1.00±0.02
	Th17	1.01±0.06^*	0.99±0.05^*	0.98±0.05	0.97±0.04

Group	AHR mRNA	RORγt mRNA
Con-OE	1.00±0.06	1.00±0.10
Con-KD	1.08±0.10	1.05±0.10
TRADD-OE	2.98±0.01^*	1.52±0.06^*
TRADD-KD	0.63±0.05^△	0.88±0.12^△

Group	AHR mRNA	RORγt mRNA
Con-OE	1.01±0.11	1.01±0.13
TRADD-OE	1.56±0.23	1.64±0.13
Con-OE+JAK inhibitor	0.69±0.06^*	0.65±0.13^*
TRADD-OE+JAK inhibitor	1.25±0.12^△	1.13±0.11^△

Differences in immune microenvironment between DEB-TACE and c-TACE in treatment of hepatocellular carcinoma and mechanism of TRADD-mediated Th17 differentiation

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