Rituximab Down-Regulate Th17 Cell Differentiation in Diffuse Large b-Cell Lymphoma Patients
Authors: Zhu Zhigang 
Published: October 2019

Abstract
Background:
It has been reported in patients with rheumatoid arthritis that the application of rituximab may reduce percentage of Th17 cells in peripheral blood, and showed better clinical results. However, the anti-tumor effect of Th17 cells is still not clear. RCHOP regimen is the first-line regimen for diffuse large B-cell lymphoma. This study examined the levels of the Th17 percentages and IL-17 , IL-21 , IL-23 and TGF-β levels in serum of patients with DLBCL before and after receiving R-CHOP or CHOP chemotherapy. In addition, peripheral blood mononuclear cells (PBMNCs) co-cultured with rituximab in vitro was used to further explore rituximab role on regulation of Th17 cells for patients with DLBCL.
Methods:
All patients were diagnosised by pathology, in which immunohistochemical analysis showed positive CD20. All DLBCL patients and healthy volunteers were excluded autoimmune disease, infection, second tumors and other haematologic diseases.
Firsly, patients were assigned to 5 groups, 31 cases in the newly diagnosised group,21 cases in CHOP-CR group and 25 cases in RCHOP-CR group, 10 cases in CHOP-NonCR group and 4 cases in RCHOP-NonCR group, respectively. The percentage of Th17 cells in the PBMNCs of each group was tested by flow cytometry; the related cytokines IL-17, IL-21, IL-23 and TGF-β in serum were measured by enzyme-linked immunosorbent assay (ELISA).
In addition, we collected peripheral blood (PB) from both 20 cases of newly diagosised DLBCL patients and healthy volunteers, PBMCs were separated and divided into four equal parts, and then they were assigned to 4 subgroups according to different culture conditions: blank control (subgroup A), rituximab subgroup (subgroup B), rituximab and serum (from patients or control respectively) subgroup (subgroup C) and polarization subgroup (subgroup D) (supplementation with IL- 6 and TGF-β). After cultured in vitro, the percentage of Th17 cells of each subgroup was tested by flow cytometry; IL- 17 in the supernatant was measured by ELISA.
Results:
Firstly , DLBCL patients before and after chemotherapy showed as follows.
1 The percentage of Th17 cells(0.67±0.21) and the levels (pg/ml) of IL-17AIL-21AIL-23(5.929±1.342A130.632±17.945A51.681±9.808 ) in the newly diagosised group and the CHOP-CR group (1.07±0.37, 6.526±0.538, 132.119±7.700, 50.245±7.668) were both significantly lower than that in the control group (2.53±0.63, 8.435±2.031, 149.265±12.316, 55.303±7.778) (p<0.05). The level of TGF-β (pg/ml) in newly diagosised group (370.615±98.444) was significantly higher than that in the control group (311.895±73.365) (p<0.05).
2 The percentage of Th17 cells(2.38±0.59)and the levels of IL-17,IL-21,IL-23 (pg/ml ) in the RCHOP-CR group (7.724±0.780, 148.412±7.355, 55.668±7.532) were significantly higher than that in the newly diagosised patients (5.929±1.342, 130.632±17.945, 51.681±9.808) and in the CHOP-CR group (1.07±0.37, 6.526±0.538, 132.119±7.700, 50.245±7.668 ) (p<0.05). The level of TGF-β (pg/ml) in the RCHOP-CR group (283.904±59.223) was significantly lower than that in the CHOP-CR group (341.481±95.597) (p<0.05).
In addition, The experimental culture in vitro showed results as follows.
1 In the control group,the percentage of Th17 cells (17.12 ± 4.90) and the levels (pg/ml) of IL- 17 (45.735 ± 10.012) in subgroup D were significantly higher than that in subgroup AABAC(p<0.05). The subgroup A, B and C were no statistical difference with each other (p>0.05).
2 The percentage of Th17 cells (0.69 ± 0.24) and the level of IL-17(pg/ml) in the DLBCL subgroup A(6.012 ± 1.312)were significantly lower than that in the control subgroup A (2.43 ± 0.61, 8.217 ± 1.681) (p<0.05).
3 In the DLBCL group,after culture supplemented with rituximab,the percentage of Th17 cells (2.34 ± 0.48, 2.31 ± 0.53, 16.92 ± 4.81) and the level of IL- 17 (pg/ml ) (7.944 ± 1.538, 7.957 ± 1.533, 44.417 ± 9.881) in subgroup B,C,D respectively were all significantly higher than that in subgroup A. In addition, the percentage of Th17 cells and the level of IL- 17 in subgroup D was significantly higher than that in subgroup B and C, respectively (p<0.05), but there was no statistical difference between subgroup B and C (p>0.05).
Conclusion :
Th17 cells might be negatively correlated with the DLBCL development. Rituximab could up -regulate Th17 cells differentiation in PB of DLBCL patients, and it is related with the effect of chemotherapy.

Disclosures
No relevant conflicts of interest to declare.