3.2 CXCR6 + CD8 + T Cells show difference in Patients with PBC (early-stage PBC and late-stage PBC)
We enrolled a total of 118 PBC patientsbased on elevated cholestatic liver enzymes and typical histological features in liver biopsy specimens. According to the Ludwig classification [10], patients were categorized in two groups:early-stage PBC (E-PBC, 62 cases) and late-stage PBC (L-PBC, 56 cases). Significant statistical differences were observed between the two groups in markers such as ALT, AST, G, A, AKP, GGT, TBI, DBI, and TBA (P < 0.05). Similarly, there were significant statistical differences in blood lipids, including LDL and Lipa (P < 0.05). However, there were no statistically significant differences between the two groups in terms of gender, age, WBC, Hb, PLT, CHOL, TG, high-density lipoprotein (HDL), ferritin, antinuclear antibodies (ANA), and anti-mitochondrial antibodies (AMA2, AMA4, AMA9) (P > 0.05) (Table 1). It is noteworthy that immunohistochemical analysis of hepatic CXCR6 + CD8 + T cells in the portal vein area revealed a significant increase in their numbers in both early-stage and late-stage PBC patients compared to normal livers (Fig. 2A, B, C). Furthermore, compared to E-PBC patients (n = 21), L-PBC patients (n = 25) exhibited a marked increase in the number of CXCR6 + CD8 + T cells, with a significant statistical difference in the expression of this specific T cell subset between the two groups (z=-5.667, P = 0.000), as detailed in Table 1. This suggests a strong correlation between CXCR6 + CD8 + T cells and the severity of PBC, potentially implicating them in the progression of the disease..
The clinical manifestations of PBC typically include elevated GGT and AKP levels, and severe cases may also present with elevated ALT and AST levels. To explore the relationship between CXCR6 + CD8 + T cells and liver inflammation, Spearman correlation analysis was conducted, revealing a positive correlation between CXCR6 and AKP (r = 0.3150, P = 0.003) and GGT (r = 0.6839, P < 0.000) levels. However, the correlation with ALT (r = 0.1097, P = 0.4679) and AST (r = 0.1771, P = 0.2391) was minimal. These results indicate a stronger correlation between the abundance of this specific T cell subset and elevated AKP and GGT levels, suggesting their involvement in bile duct injury processes, leading to significant increases in AKP and GGT (Fig. 2D, E, F, and G). In conclusion, CXCR6 + CD8 + T cells isolated from patients with late-stage PBC patients exhibited inflammatory activity, suggesting their potential role in the pathological process of PBC.
Table 1
Summarizes the main clinical and biochemical characteristics of the 118 patients with E-PBC and L-PBC
|
E-PBC(n = 62)
|
L-PBC(n = 56)
|
t/Χ2/z
|
P
|
Age(year)
|
53.76 ± 8.84
|
53.05 ± 11.83
|
t = 0.369
|
0.713
|
Sex
|
Male
|
9(14.5)
|
8(14.3)
|
Χ2 = 0.001
|
0.972
|
female
|
53(85.5)
|
48(85.7)
|
WBC(×10^9/L)
|
4.80 ± 1.76
|
5.01 ± 1.75
|
t=-0.613
|
0.541
|
Hb(g/L)
|
123.98 ± 16.20
|
121.69 ± 14.57
|
t = 0.707
|
0.481
|
PLT(×10^9/L )
|
173.31 ± 77.22
|
169.62 ± 71.22
|
t = 0.245
|
0.807
|
ALT(U/L)
|
43(26, 104.5)
|
97(45, 141)
|
z=-2.760
|
0.006
|
AST(U/L)
|
40(26, 78)
|
66(43, 120)
|
z=-3.956
|
0.000
|
A(g/L)
|
40.04 ± 4.48
|
38.20 ± 4.44
|
t = 2.243
|
0.027
|
G(g/L)
|
27.40 ± 5.53
|
30.50 ± 6.70
|
t=-2.751
|
0.007
|
AKP(U/L)
|
134(104, 179)
|
280(173, 353)
|
z=-4.692
|
0.000
|
GGT(U/L)
|
116(62, 218)
|
330(134, 675)
|
z=-4.223
|
0.000
|
TBI(µmol/L)
|
11.86(7.88, 19.83)
|
21.61(13.73, 49.63)
|
z=-3.614
|
0.000
|
DBil(µmol/L)
|
2.6(1.72,6.35)
|
7.9(4.15, 32.64)
|
z=-4.403
|
0.000
|
TBA(umol/L)
|
16.2(7.55, 32.65)
|
28.2(12.3, 96.9)
|
z=-3.422
|
0.001
|
CHOL(mmol/L)
|
4.57(3.61, 5.29)
|
5.06(3.99, 6.27)
|
z=-1.719
|
0.086
|
TG(mmol/L)
|
1.4(0.95, 1.64)
|
1.33(0.85, 2.09)
|
z=-0.927
|
0.345
|
HDL(mmol/L)
|
1.48(1.14, 1.67)
|
1.50(1.28, 1.99)
|
z=-1.268
|
0.205
|
LDL(mmol/L)
|
2.38(1.79, 2.84)
|
2.85(2.03, 3.52)
|
z=-2.038
|
0.042
|
Lipa(mg/dL)
|
60(24, 94)
|
41(24, 94)
|
z=-1.774
|
0.076
|
Ferritin(µg/L)
|
100.5(47.4, 164.85)
|
131.9(62.7, 243.4)
|
z=-0.546
|
0.585
|
The number of CXCR6 + CD8 + T cell in liver(n/HPF )
|
56(14, 93.5)*༈n = 21༉
|
139(82, 181.5)*༈n = 25༉
|
z=-3.849
|
0.000
|
ANA quantification
|
320(100, 320)
|
320(320, 400)
|
Z=-0.481
|
0.631
|
AMA2
|
Neg
|
28(45.2)
|
18(32.1)
|
Χ2 = 2.096
|
0.148
|
Pos
|
34(54.8)
|
38(67.9)
|
AMA4
|
Neg
|
44(83)
|
38(88.4)
|
Χ2 = 0.546
|
0.460
|
Pos
|
9(17)
|
5(11.6)
|
AMA9
|
Neg
|
47(88.7)
|
41(95.3)
|
Χ2 = 1.671
|
0.434
|
Pos
|
5(9.4)
|
2(4.7)
|
P values refer to comparisons between E-PBC and L-PBC patients
PBC primary biliary cholangitis,, ALP alkaline phosphatase, GGT gamma-glutamyl transpeptidase, ALT alanine aminotransferase, AST aspartate aminotransferase, ALB albumin, TBIL total bilirubin, PLT platelet count,AMA-M2 ,4,9,anti-mitochondrial M2,4 and 9 antibody.,TG triglyceride,TBA total bile acids,CHOL cholesterol,HDL high-density lipoprotein, LDL Low-density lipoprotein, Lipa lipoprotein a
3.3 CXCR6 + CD8 + T Cells Isolated From Patients With L-PBC Show a Higher Activity of Inflammation and Fibrosis Degree
Due to the differential infiltration of CXCR6 + CD8 + T cells across various stages of PBC, our aim was to elucidate the relationship between these specialized T cells and the degree of liver inflammation and fibrosis. Employing HE and Masson's staining techniques, we observed a positive correlation between CXCR6 + CD8 + T cells and liver inflammation and fibrosis (Fig. 3A). Statistical analysis revealed significant differences in the abundance of CXCR6-positive cells between different states of PBC (E-PBC and L-PBC) (Fig. 3B, P = 0.000), as well as significant statistical differences between different grades of inflammation (G grades) and fibrosis (S grades) (with H = 27.119, P = 0.000 and H = 12.147, P = 0.007, respectively). These findings strongly suggest that the involvement of CXCR6 + CD8 + T cells in the processes of inflammation and fibrosis in PBC.
3.4 The elevation of CXCR6 + CD8 + T cells is considered one of the factors contributing to poor response in PBC.
The aforementioned results demonstrate a significant correlation between the quantity of CXCR6 + CD8 + T cells and the levels of inflammation, liver fibrosis, and the disease progression of PBC. Here, the relationship between these T cells and the response to UDCA treatment was explored. According to the AASLD guidance [11], patients undergoing standard UDCA treatment were monitored for six months. A total of 64 patients were included in the follow-up, and categorized into responder and non-responder groups, with 31 patients identified as responders and 33 as non-responders.
Through single-factor analysis, notable statistical variances were observed between the two groups in various biochemical indicators, including ALT, AST, G, AKP, GGT, TBI, Dbi, and TBA, as well as Hb, HDL, and LDL (p < 0.05) (Table 2). Employing rank sum tests, it was discerned that the levels of specialized CXCR6 + CD8 + T cells in immunohistochemistry exhibited significant disparities between responders and non-responders (Z= -3.057, p = 0.002) (Fig. 4). The count of hepatic CXCR6 + CD8 + T cells per HPF was notably higher in UDCA responders (n = 5) compared to UDCA non-responders (n = 5), indicating an association between CXCR6 + CD8 + T cells and responsiveness to UDCA treatment.
Table 2
Summarization of the main clinical and biochemical characteristics of the 64 patients in UDCA non-responders compared with UDCA responders
|
UDCA responders(n = 31 )
|
UDCA non-responders (n = 33)
|
t/Χ2/z
|
P
|
Age(year)
|
51.55 ± 11.30
|
53.39 ± 10.00
|
t=-0.393
|
0.491
|
Sex
|
Male
|
4(12.9)
|
3(9.1)
|
0.238
|
0.625
|
Female
|
27(87.1)
|
30(90.9)
|
WBC(10^9/L)
|
5.10 ± 1.65
|
4.99 ± 1.86
|
t = 0.223
|
0.825
|
Hb(g/L)
|
129.4 ± 16.70
|
116.64 ± 14.92
|
t = 2.942
|
0.005
|
PLT(10^9/L )
|
165.39 ± 81.90
|
177.75 ± 78.06
|
t=-0.568
|
0.572
|
ALT(U/L)
|
26.5(18,49.25)
|
77(43,110)
|
z=-3.279
|
0.001
|
AST(U/L)
|
28(24,42.5)
|
61(43,88)
|
z=-4.824
|
0.000
|
A(g/L)
|
40.56 ± 4.54
|
38.31 ± 3.94
|
t = 0.575
|
0.038
|
G(g/L)
|
27.38 ± 5.00
|
31.11 ± 5.56
|
t=-2.817
|
0.006
|
AKP(U/L)
|
115.5(82,135)
|
330(210,405)
|
z=-6.872
|
0.000
|
GGT(U/L)
|
68(38.75,113.75)
|
360(155,675)
|
z=-5.179
|
0.000
|
TBI(µmol/L)
|
9.50(5.90,12.4)
|
22.64(12.75,49.63)
|
z=-2.935
|
0.003
|
DBil(µmol/L)
|
1.72(1.18,2.64)
|
8.39(3.82,32.64)
|
z=-3.849
|
0.000
|
TBA(umol/L)
|
13.65(8,27.45)
|
48.3(20.2,97.9)
|
z=-3.649
|
0.000
|
CHOL(mmol/L)
|
4.53(3.52,5.20)
|
5.98(5.06,8.22)
|
z=-3.337
|
0.001
|
TG(mmol/L)
|
1.32(1.02,2.21)
|
1.56(0.85,2.18)
|
z=-0.613
|
0.540
|
HDL(mmol/L)
|
1.33(1.15,1.58)
|
1.94(1.57,2.29)
|
z=-3.705
|
0.000
|
LDL(mmol/L)
|
2.49(1.73,3.14)
|
2.94(2.19,3.61)
|
z=-2.647
|
0.008
|
Lipa(mg/dL)
|
91(47.5,188.25)
|
41(14,94)
|
z=-0.975
|
0.330
|
Ferritin(µg/L)
|
60.5(27.65,132.15)
|
88.7(42.8,177.9)
|
z=-1.365
|
1.172
|
The number of CXCR6 + CD8 + T cell in liver (n/HPF )
|
76(18,132)(n = 5)
|
149(80.25,191.25)(n = 5)
|
z=-2.245
|
0.0025
|
Paris I and Paris II criteria were utilized in late-stage (stage III–IV) and early-stage (stage I–II) PBC patients, respectively, to identify responders to UDCA. The data are presented as medians with interquartile ranges.
Abbreviations: PBC: Primary Biliary Cholangitis;ALP: Alkaline Phosphatase;GGT: Gamma-Glutamyl Transpeptidase;ALT: Alanine Aminotransferase;AST: Aspartate Aminotransferase;ALB: Albumin;TBIL: Total Bilirubin;PLT: Platelet;TG: Triglyceride;TBA: Total Bile Acids;CHOL: Cholesterol;HDL: High-Density Lipoprotein;LDL: Low-Density Lipoprotein;Lipa: Lipoprotein A