供体CD_4^+ CD_25^+ T细胞及调控基因FOXP_3在急性移植物抗宿主病中的作用

The role of CD_4^+ CD_(25)^+ T cell and FOXP_3 in host acute graft rejection

目的探讨供体 CD_4^+ CD_(25)^+ T 细胞亚群、FOXP_3调控基因的表达与受者移植物抗宿主病(GVHD)的相关性。方法 (1)30例异基因造血干细胞移植(allo-HSCT),采用免疫荧光标记和流式细胞术检测并比较供体粒细胞集落刺激因子(G-CSF)动员前外周血、动员后采集物 CD_4^+ CD_(25)^+ T 细胞亚群比例,随访异基因移植后 GVHD 的发生率和严重程度。(2)应用 RT-PCR 技术检测供体 FOXP_3基因表达情况,分析其与 GVHD、疾病复发的相关性。结果 (1)所有患者均获造血重建,粒细胞绝对数(ANC)≥0.5×10~9/L 的中位时间为14(12～15)d,PLT≥20×10~9/L 为18(15～25)d。30例allo-HSCT,中位随访时间12.8(8～16)个月,Ⅰ～Ⅳ度急性 GVHD 分别为3、4、3、5例。慢性 GVHD 6例。(2)供体 G-CSF 动员前外周血、动员后采集物 CD_4^+ CD_(25)^+ T 细胞亚群分别为(2.67±0.38)%、(5.01±1.33)%,两者相比差异无统计学意义(P>0.05)。(3)移植后无急性 GVHD 组、Ⅰ～Ⅱ度急性 GVHD 组、Ⅲ～Ⅳ度急性 GVHD 组供体 CD_4^+ CD_(25)^+ T 细胞亚群分别为(5.05±1.34)%、(4.17±1.73)%、(1.98±1.10)%。其中Ⅰ～Ⅱ度急性 GVHD 组与Ⅲ～Ⅳ度急性 GVHD 组相比差异有统计学意义(P=0.04),无急性 GVHD 组与Ⅲ～Ⅳ度急性 GVHD 组相比差异有统计学意义(P=0.002)。(4)30例 allo-HSCT,7例 FOXP_3基因表达阳性,5/7例移植后无急性 GVHD,其中3例移植后复发,另2/7例移植后Ⅰ度急性 GVHD,Ⅱ～Ⅳ度急性 GVHD 患者 FOXP_3均不表达。结论 (1)供体CD_4^+ CD_(25)^+ T 细胞亚群比例与受者急性 GVHD 的发生具有一定的相关性,提高供体 CD_4^+ CD_(25)^+ T 细胞数量有望减低移植后急性 GVHD 发生率。(2)供体移植物 FOXP_3基因表达阳性,与移植后有无严重急性 GVHD 发生存在一定相关性。

Objective To explore the relationship among CD4^＋ CD25^＋ regulatory T cell, the expression of FOXP3 mRNA levels of donor bone marrow （ BM ） and graft versus host disease （ GVHD ） in allogeneic hematopoietic stem cell transplantation （allo-HSCT）. Methods （1）Thirty patients undergoing allo-HSCT were enrolled in this study. The levels of donor CD4^＋ CD25^＋ regulatory T cell were compared in the patients with GVHD and those without GVHD with immnofluorescence and flowcytometry. （2）The donor BM expressions of FOXP3 mRNA levels were analyzed by using reverse transcriptase polymerase chain reaction （RT-PCR）. Results （ 1 ） All patients achieved engraftment. The mediantime for ANC ≥0. 5 × 10^9/L and PLT ≥ 20 ×10^9/L were attained at day 14（range, 12-15）and day 18 （range, 15-25） respectively. With a median follow-up of 12. 8 （8-16） months, acute GVHD （aGVHD） occurred in 15 of the 30 patients （50.0%） with grade Ⅱ -Ⅳ aGVHD （40. 0% ）. Chronic GVHD developed in 6 （extensive 2, limited 4） out of the 30 patients （20.0%）. （2）The levels of donor CD4^＋ CD25^＋ regulatory T cell pre-and post-mobilization were （2. 67 ± 0. 38 ） % and （5.01 ± 1.33 ） % respectively ; no difference was observed （ P 〉 0. 05 ）. （ 3 ） Patients with Ⅰ - Ⅱ aGVHD demonstrated higher donor-type CD4^＋ CD25^＋ immune regulatory T cell level than those with Ⅲ-Ⅳ aGVHD. Patients with Ⅲ-Ⅳ aGVHD showed much lower donor-type CD4^＋CD25^＋ immune regulatory T cell level than those without GVHD. No difference was observed between the patients with Ⅰ - Ⅱ aGVHD and those without aGVHD. （4）The transcripts of FOXP3 donor BM were detected in seven donors for the thirty recipients who received allo-HSCT. Among the seven patients who received stem cells from these donors five had no GVHD after transplantation and the remaining two developed Ⅰ aGVHD. Donor BM FOXP3 transcripts were never detected in patients with Ⅱ-Ⅳ aGVHD. Conclusions （ 1 ） Donor-type CD4^＋ CD25^＋ immune regulatory T cell level was relatied to recipient Ⅰ-Ⅳ aGVHD. Up-grade donor-type CD4^＋ CD25^＋ immune regulatory T cell level could reduce the incidence of aGVHD. （2） Donor BM FOXP3 transcripts were detected in patients without serious aGVHD after transplantation.