[The characterization analysis of pathogenic T cells in immune-mediated aplastic anemia mouse model]

Abstract

Objective: This study aims, in addition to characterizing pathogenic T cells trafficking to bone marrow (BM) and other organs, to establish immune-mediated AA C.B10 mouse model by DsRed mouse (B6 background) lymph nodes (LN) cells infusion after a total body irradiation (TBI) . Methods: The C.B10 mice received a 5 Gy TBI and then were infused with DsRed mouse (B6 background) LN cells at 5×10(6)/mouse via a tail vein injection. The severity of bone marrow failure (BMF) was observed by mononuclear cell count in bone marrow and peripheral blood cell count. On days 3, 6, 9, and 12, mice were sacrificed and collected BM, spleens, LN, or thymus to analyze the dynamic change and activation status of donor T cells in these organs by a flow cytometry. At day 12, the donor-derived T cells from BM, spleens, and LN were sorted to collect the DsRed(+)CD3(+)CD4(+) T cells and DsRed(+)CD3(+)CD8(+) T cells for RNA isolation and gene expression analyses by PCR array. Results: Relative to control animals that received 5 Gy TBI without LN cell infusion, AA mice developed severe BMF with dramatic decrease in total BM cells, hemoglobin, white blood cells, and platelets in peripheral blood on days 9 and 12 after the LN cell infusion. The frequencies of DsRed(+) T cells trafficking to BM, LN, and spleens increased with time. Surprisingly, although the DsRed(+) T cells in BM increased dramatically at a level much higher than those in the spleens and LN on day 12, there were very few DsRed(+) T cells in BM on days three and six, which was significantly lower than those in spleens or LN. The frequency of DsRed(+) T cells in thymus was the lowest during the whole process. On day 12, the DsRed(+)CD3(+)CD4(+) T cells of BM, LN, and spleens from AA mice were (91.38±2.10) %, (39.78±6.98) %, and (67.87±12.77) %, respectively. On the contrary, the DsRed(+)CD3(+)CD8(+)T cells of BM, LN, and spleens were (98.21±1.49) %, (94.06±4.20) %, and (96.29±1.23) %, respectively. We assessed the donor T cell phenotypes using the CD44 and CD62L markers and found that almost all of the DsRed(+)CD4(+) or DsRed(+)CD8(+) T cells in BM were effector memory T cell phenotype from day 9 to day 12. Meanwhile, transcriptome analyses showed higher expression in CD38, IFN-γ, LAG3, CSF1, SPP1, and TNFSF13B in BM DsRed(+)CD4(+) and DsRed(+)CD8(+) T cells. However, there was a lower expression in FOXP3 and CTLA4 in BM DsRed(+)CD4(+) T cells than those in spleens and LN. Conclusions: The DsRed LN cells infusion to induce BMF in CB10 mice enabled to track the donor-derived pathogenic T cells. Besides previously published findings in this model, we demonstrated that donor CD4(+) and CD8(+) T cells primarily homed to spleens and LN, expanded and differentiated, then infiltrated in BM with a terminal effector memory phenotype. The T cells infiltrated in BM showed more activation and less immunosuppression characteristics compared to those homing to spleens and LN during the BMF development.

Keywords: Anemia, aplastic; Characterization; Homing; Mouse model; Pathogenic T cells.

图1. DsRed小鼠淋巴结细胞诱导的C.B10小鼠再生障碍性贫血模型不同时间外周血WBC（A）、HGB（B）、ANC（C）、PLT（D）与股BMMNC计数（E）结果（^aP<0.05、^bP<0.001、^cP<0.0001）

AA组：再生障碍性贫血模型小鼠；TBI组：全身辐照对照组；BMMNC：骨髓单个核细胞

图2. 流式细胞术检测不同时间C.B10再生障碍性贫血模型小鼠体内DsRed⁺CD3⁺（A）和DsRed⁺CD3⁺CD8⁺（B）T细胞在不同器官的淋巴细胞内的表达

图3. 流式细胞术检测不同时间C.B10再生障碍性贫血模型小鼠体内特异性表达H60的CD8⁺细胞毒性T淋巴细胞在不同器官的表达

图4. 流式细胞术检测免疫介导的再生障碍性贫血小鼠中供鼠来源CD3⁺CD4⁺（A）和CD3⁺CD8⁺（B）T细胞CD44、CD62L表达

图5. 免疫介导的AA小鼠中供鼠来源CD4⁺（A）、CD8⁺（B）T细胞在不同淋巴组织中的活化基因表达

SP：脾脏；LN：淋巴结；BM：骨髓；CTRL：供鼠淋巴结