Dynamics of circulating lymphocyte subsets changes following liver transplantation
As circulating lymphocyte subsets have been reported to be affected under physiological and pathological conditions [7–9], we selected liver transplant recipients in the absence of any postoperative complications to minimize the potential impact. A total of 78 liver transplant recipients with stable liver function were enrolled in this study. There were 65 males and 13 females with a median age of 53 years (26–68 years). 22 patients and 56 patients were diagnosed with malignant and benign diseases, respectively. The follow-up period ranged from 4 to 709 days with an average of 137 days after surgery.
First, we wanted to determine whether primary liver diseases would affect the postoperative circulating lymphocyte subsets. Primary liver diseases contained hepatitis-related cirrhosis and hepatitis-related hepatic carcinoma. Female patients were excluded as they all had benign diseases. Since male patients with hepatitis–related hepatic carcinoma were much older (58.05 ± 6.95 years v.s 50.51 ± 10.68 years, P < 0.01), male patients with benign diseases of the same age group were selected. Male patients with benign diseases (n = 32) and malignant diseases (n = 22) had similar age (55.50 ± 6.23 years v.s 58.05 ± 6.95 years, P > 0.05) and the follow-up periods (146.78 ± 172.89 d v.s 197.05 ± 175.11 d, P > 0.05). There was no statistical difference between benign diseases and malignant diseases with respect to the absolute counts and percentages of T, TCD4, TCD8, B, NK, NKT and DC (Suppl Table 1; Fig. 1).
Then, the effect of gender on circulating lymphocyte subsets was evaluated. Male patients with hepatitis-related cirrhosis and of the same age group as female patients were selected. Male patients (n = 29) and female patients (n = 13) had similar age (51.17 ± 4.85 years v.s 51.46 ± 4.58 years, P > 0.05) and the follow-up period (153.90 ± 179.67 d v.s 82.77 ± 125.29 d, P > 0.05). After comparison, there was no statistical difference between male patients and female patients with respect to the absolute counts and percentages of T, TCD4, TCD8, B, NK, NKT and DC (Suppl Table 2; Fig. 2).
Next, we wanted to check whether age played an important role in circulating lymphocyte subsets. Female patients were excluded to minimize potential affection. Subsequently, male patients were divided into the young group (< 60 years) and the elderly group (≥ 60 years). Patients from the young group (49.00 ± 8.69, n = 48) and from the elderly group (64.47 ± 2.40, n = 17) had similar percentages of malignant diseases (13/48 v.s 9/17, P > 0.05) and follow-up periods (150.85 ± 164.39 d v.s 141.06 ± 173.43 d, P > 0.05). There was no statistical difference between young patients and elderly patients with respect to the absolute counts and percentages of T, TCD4, TCD8, B, NK, NKT and DC (Suppl Table 3; Fig. 3).
After that, data from patients with different follow-up periods were analyzed. Female patients were still excluded to minimize potential affection. The follow-up periods were divided into the short-term group (< 29 d, n = 22), the middle-term group (29–180 d, n = 23) and the long-term group (> 180 d, n = 20). Patients from the three subgroups (short v.s middle v.s long) had similar age (50.18 ± 11.65 years v.s 53.57 ± 10.24 years v.s 55.65 ± 7.79 years, P > 0.05) and percentages of malignant diseases (5/22 v.s 8/23 v.s 9/20, P > 0.05). Of note, we found liver transplant recipients gained a global recovery over time. Patients from the short-term group had the lowest absolute counts of T cell subsets (T, TCD4 and TCD8; P < 0.01), NK (short v.s middle, P < 0.05; short v.s long, P < 0.01) and NKT (short v.s middle, P < 0.01; short v.s long, P < 0.01) and the lowest percentages of T cell subsets (T, TCD4 and TCD8; P < 0.01), B (P < 0.05), NK (P < 0.05) and NKT (short v.s middle, P < 0.01; short v.s long, P < 0.05) but the highest percentage of DC (short v.s middle, P < 0.05; short v.s long, P < 0.01). The rest results were similar among the subgroups (Suppl Table 4; Fig. 4).
The Shift of circulating lymphocyte subsets during acute rejection and after anti-rejection therapy
As most acute rejections reported in liver transplant recipients occur within the first year, especially the first six months [10–12], we wanted to know whether a sharp increase in the absolute counts and percentages of lymphocyte subsets was in close relation to acute rejection. Then, a total of 17 patients who experienced acute rejection episodes were enrolled. There were 15 males and 2 females with a median age of 47 years (25–69 years). 6 patients and 11 patients were diagnosed with malignant and benign diseases, respectively. The occurrence time of acute rejection ranged from 22 to 107 days with an average of 56 days after surgery. To analyze the changes of lymphocyte subsets patients with rejection were then matched by gender, age (± 3 years), primary liver diseases for transplantation (malignant or benign), and follow-up periods (± 5 d). Data from patients with acute rejection and paired controls were collected.
We found four patients had acute rejection within 28 d and 13 patients between 29–180 d (Fig. 5A), which was in accordance with the reported studies. Notably, the trough levels of tacrolimus were similar between the two groups indicating that the patients received similar immunosuppressive therapy (Fig. 5B). After comparison, we found that percentages of T, TCD4, B and NKT were higher in patients with acute rejection but there was no statistical difference concerning the absolute counts of any circulating lymphocyte subsets (Suppl Table 5, Fig. 6). Next we assessed the ability of percentages of T, TCD4, B and NKT to identify patients at risk for acute rejection. Multivariate Cox analysis showed the percentage of NKT was the strong predictor of acute rejection (Table 1). The area under the curve (AUC) of percentages of TCD4, B and NKT were 0.76, 0.73 and 0.77 on receiver operating characteristic curve analysis, respectively. The predicted probability was calculated using binary logistic that combined percentages of TCD4, B and NKT (AUC 0.89; Fig. 5C). At a cut-off value of 0.69, this new marker had a sensitivity of 70.6% and a specificity of 94.1%.
Table 1
Multivariate Cox analysis
Percentages | HR (95% CI) | P value |
TCD4 | 0.981–1.214 | 0.109 |
B | 0.962–1.749 | 0.088 |
NKT | 1.059–2.971 | 0.029 |
TCD4, CD3+CD4+T cells; B, CD19+B cells; NKT, CD3+CD56+CD16+Natural killer T cells; CI, confidence interval; HR, hazard ratio. |
Finally, in an attempt to confirm the role that percentages of circulating lymphocyte subsets played in acute rejection, these patients were sampled for a second time following anti-rejection therapy. All were first treated by adding the dosage of tacrolimus. Subsequently, 9 patients were treated with steroids due to uncontrollable rejection. All cases recovered gradually and finally had a normal liver function. The periods between before and after sampling ranged from 8 to 26 d. Data of before-after sampling were collected and compared. Notably, the results showed that percentages of T, TCD4, TCD8 and NK were lower after the treatment while the absolute counts of lymphocyte subsets remained similar between the groups (Suppl Table 6; Fig. 7).