Hepatocellular carcinoma and macrophage interaction induced tumor immunosuppression via Treg requires TLR4 signaling

Table 1 Clinical and laboratory characteristics of the 60 hepatocellular carcinoma patients

Items	Results
Age (yr) median (range)	53.5 (17-77)
Gender (male/female)	51/9
Virus (HBV/HCV)	48/4
TNM stage (I/II/III/IV)	19/24/17/0
AFP (μg/L), median (range)	350 (1.8-127 278)
Blood neutrophil (%)	62.29 ± 10.96
Blood monocyte (%)	7.21 ± 1.69
Hemoglobin concentration (g/L)	124.47 ± 17.04

TNM: Tumor-node-metastasis; HBV: Hepatitis B virus; HCV: Hepatitis C virus; AFP: α-fetoprotein.

Table 2 Primer sequences and conditions used for reverse transcription-polymerase chain reaction

Gene	Sequence	Product size (bp)	Annealing temperature (°C)
mTLR4	5′-GCTTTCACCTCTGCCTTCAC-3′	259	57
	3′-AGGCGATACAATTCCACCTG-5′
HuTLR4	5′-GAAATGGAGGCACCCCTTC-3′	506	52
	3′-GAATATTCCTTTGCATAGGT-5′
CCL22	5′-AAGACAGTATCTGCTGCCAGG-3′	141	57
	3′-GATCGGCACAGATATCTCGG-5′
IL-10	5′-GGTTGCCAAGCCTTATCGGA-3′	190	60
	3′-ACCTGCTCCACTGCCTTGCT-5′
TNF-α	5′-CATCTTCTCAAAATTCGAGTGACAA-3′	175	58
	3′-TGGGAGTAGACAAGGTACAACCC-5′
β-actin	5′-TCACCCACACTGTGCCCATCTACGA-3′	300	50
	3′-GATAACCGTTGCTCGCCAAGGCTAC-5′

TNF: Tumor necrosis factor; IL: Interleukin; TLR: Toll-like receptor.

Table 3 Correlation between the proportion of CD4+CD25^highFOXP3+ regulatory T cells in peripheral blood and clinicopathological characteristics of hepatocellular carcinoma

Items	n	Treg (%)	P values
Sex			0.201
Male	51	68.95 ± 20.71
Female	9	59.37 ± 18.97
UICC/TNM stage			0.781
I	19	66.41 ± 20.28
II-III	41	68.02 ± 20.97
AFP (μg/L)			< 0.001
≤ 20	23	53.13 ± 17.96
> 20	37	76.45 ± 16.84
HBsAg			0.611
Positive	48	68.20 ± 19.69
Negative	12	64.78 ± 24.66
Margin			0.981
Clear	29	67.45 ± 19.74
Unclear	31	67.57 ± 21.69
Capsule			0.058
Complete	28	62.14 ± 21.92
Incomplete	32	72.22 ± 18.43
Tumor diameter (cm)			0.245
≤ 5	23	59.84 ± 19.06
6-9	18	75.94 ± 18.43
≥ 10	19	68.81 ± 21.84
Tumor number			0.009
Single	52	63.83 ± 20.55
≥ 2	8	86.41 ± 6.18
Cirrhosis			0.881
Presence	23	68.02 ± 20.42
Absence	37	67.19 ± 20.98
Tumor differentiation			0.152
WD	11	56.61 ± 24.98
MD	7	69.60 ± 20.45
PD	42	70.02 ± 18.59

MD: Moderately differentiated; WD: Well differentiated; PD: Poorly differentiated; AFP: α-fetoprotein; Tregs: Regulatory T cells; TNM: Tumor-node-metastasis; UICC: Union for International Cancer Control.