The Effect And Molecular Mechanism Of Kras In Acute Graft-versus-Host Disease

Background:Allogeneic hematopoietic cell transplantation(allo-HSCT)is a curative treatment for patients with hematologic malignancies.While acute graft-versus-host disease(aGVHD),which is the most serious complication after allo-HSCT limits its application and success.aGVHD is initiated by donor T cells that recognize recipient histoincompatible antigens.T cells play very important roles in the development of aGVHD.Ras proteins are membrane-associated binary molecular swiches which cycle between active guanosine triphosphate(GTP)-bound and inactive guanosine diphosphate(GDP)-bound states.Stimulation of T-cell antigen receptors(TCR)results in Ras activation.In mature T cells,activation of Ras typically leads to recruitment and activation of RAF,inducing(via MEK/ERK pathway)many cellular responses critical for lymphocyte function.There are four highly homologous members,kras(K-ras4A,K-ras4B),Nras,and Hras in Mammals,each Ras isoforms has their own specificity,but little is known about how kras play roles in T cell development and function.Our previous study have shown that kras deficiency has no effect on early T cell development.However,whether kras deficiency impairs the proliferation and function of CD4+and CD8+T cells and whether kras play roles in the occurence of aGVHD are still unknown.In this study,we would study how kras protein and its downstream signaling pathways play roles in the pathogenesis of aGVHD.Methods:1、Establishment of Kras Knockout mouse model(VavCreKrasfl/fl):exon 1 of Kras was flanked with two LoxP sites,and the generated brasfl/fl mice were crossed to VavCre mice to generate VavCreKrasfl/fl mice.In this model,Kras expression is deleted in the entire hematopoietic system.2、MHC-mismatched aGVHD mouse model:BALB/c recipients were lethally irradiated(900 rad).T cells which isolated and purified from donor spleens plus Ragl deficient BM were transferred to recipients by tail vein injection on day 0.Recipients were monitored everyday for survival and aGVHD severity were assessed based on the clinical GVHD score system.3、GVL mouse model:A201uc B cell leukemia/lymphoma cells were given to lethally irradiated(900 rad)BALB/c recipients by peritoneal injection,and then T cells from donors plus Ragl deficient BM were transferred to recipients at the same day.Recipients were monitored everyday for survival.4、T cell proliferation,differentiation,activation,cytokine secretion,and the Kras signaling pathway protein expression were tested by using Mixed lymphocyte reaction,Flow Cytometry,Western Blot,and RNA-seq.Results:1、In aGVHD mouse model,Kras-deprived donor T cells inhibit aGVHD but did not impair GVL.2、Kras depletion affects T cells proliferation in mixed lymphocyte reaction.3、In aGVHD mouse model,Kras does not affect alloactivation of T cells in vivo but affects cytokine production of T cells.4、In aGVHD mouse model,Kras deprived alloreactive T cells have blunted Ras/MEK/Erk activation.Conclusion:In aGVHD mouse model,Kras-deprived donor T cells have decreased proliferation,cytokine production and blunted Kras signaling pathway activation,by which inhibit aGVHD development.However it preserves GVL.This study may provide a promising strategy to control GVHD wile maintaining GVT effects after allo-HSCT.