LYG1 Deficiency Attenuates the Severity of Acute Graft-Versus-Host Disease via Skewing Allogeneic T Cells Polarization Towards Treg Cells
Authors: Zeyin Liang
Miao Yan
Zhengyang Song
Yongjin Shi
Qingya Wang
Bo Tang
Yang Zhang
Yujun Dong
Yuan Li
Chenchen Qin
Zhengyu Yu
Hanyun Ren
Shengchao Miao
Huihui Liu
Journal: Frontiers in Immunology (2021)
Discipline: IMMUNOLOGY
Keywords: LYG1; Th1 cells; Treg cells; aGVHD; allogeneic CD4+ T cells
Affiliations: Department of Hematology, Peking University First Hospital, Beijing, China.

Abstract:  Acute graft-versus-host disease (aGVHD) is a lethal complication after allogeneic hematopoietic stem cell transplantation. The mechanism involves the recognition of host antigens by donor-derived T cells which induces augmented response of alloreactive T cells. In this study, we characterized the role of a previously identified novel classical secretory protein with antitumor function-LYG1 (Lysozyme G-like 1), in aGVHD. LYG1 deficiency reduced the activation of CD4+ T cells and Th1 ratio, but increased Treg ratio in vitro by MLR assay. By using major MHC mismatched aGVHD model, LYG1 deficiency in donor T cells or CD4+ T cells attenuated aGVHD severity, inhibited CD4+ T cells activation and IFN-γ expression, promoted FoxP3 expression, suppressed CXCL9 and CXCL10 expression, restrained allogeneic CD4+ T cells infiltrating in target organs. The function of LYG1 in aGVHD was also confirmed using haploidentical transplant model. Furthermore, administration of recombinant human LYG1 protein intraperitoneally aggravated aGVHD by promoting IFN-γ production and inhibiting FoxP3 expression. The effect of rhLYG1 could partially be abrogated with the absence of IFN-γ. Furthermore, LYG1 deficiency in donor T cells preserved graft-versus-tumor response. In summary, our results indicate LYG1 regulates aGVHD by the alloreactivity of CD4+ T cells and the balance of Th1 and Treg differentiation of allogeneic CD4+ T cells, targeting LYG1 maybe a novel therapeutic strategy for preventing aGVHD. 

Retracted: No

PubPeer comments:
 Dear authors, Fig.3F contains a duplicated FACS plot. I would further advise to check if Fig.3G and H have the same compensation as IFN appears overcompensated in Lyg1- -. ![file] , 
 Thank you for your advice. We have revised the data on Fig3F and are contacting the editor to modify it. The FACS parameters, including voltage and compensation for Fig3G and 3H were the same. The compensation of (PE-IFNg minus PE CY7-CD4) for Fig3G had a little higher, but not influence the result. The compensation of (PE-IFNg minus BV510-CD8) for Fig3H was 0, so there was not overcompensation for Fig3H. We have adjusted it and attached the FACS dot plots and contour plots for Fig3G and 3H. , 
 #1 Thank you for your advice. We have revised the data on Fig3F and are contacting the editor to modify it. The FACS parameters, including voltage and compensation for Fig3G and 3H were the same. The compensation of (PE-IFNg minus PE CY7-CD4) for Fig3G had a little higher, but not influence the result. The compensation of (PE-IFNg minus BV510-CD8) for Fig3H was 0, so there was not overcompensation for Fig3H. We have adjusted it and attached the FACS dot plots and contour plots for Fig3G and 3H. , 
 #3 Thank you for your response. My concerns regarding the compensation of IFNg were actually about the comparison of LYG1+ + and LYG1u2013 u2013 in Fig.3G and H, respectively. Do both have the same compensation values? Overcompensation of IFNg could actually lead to the difference you see and show in the bar graphs of the respective figures, therefore it is important to assure equal conditions during analyses. ![file] , 
 Thank you for your advice. We do confirm that the FACS conditions including voltages and compensations, for all groups including LYG1+ +, LYG1- - and negative controls in Fig3G and H, were actually all the same. Both of the compensation values (PE-IFNg minus BV510-CD8) of LYG1+ + and LYG1- - in Fig3H were 0, so there was not overcompensation for Fig3H. Both of the compensation values (PE-IFNg minus PE CY7-CD4) of LYG1+ + and LYG1- - in Fig3G were 0.24%. We adjusted the compensation values (PE-IFNg minus PE CY7-CD4) , the percentages of IFNg positive cells gated on CD4+ T cells were 56% of LYG1+ + and 26% of LYG1- -, which didn t impact our conclusions. The attached images showed the percentages of IFNg positive cells gated on CD3+ T cells and the medians for adjusting this compensations. ![file] ![file] , 
 #5 Thank you for your response. I am not really sure what you mean by  adjusting compensation values ? WT and KO should have the same compensation matrix, especially since they were measured the same day. Further, you mention that you measured CD8 in BV510, however, the FACS plot shows that it was measured in the V450 channel, which would be suited to detect Bv421? It would be great if you could upload the two FACS files of Fig.3H for review, maybe I just don t get your point and looking at a FACS file might make things easier to understand. , 
 #6 Thank you for your suggestions. 1.tWe do confirm that we measured all groups including LYG1+ +, LYG1- - and negative controls in Fig3G and 3H at the same time on the same day. The FACS conditions for measuring and analysis, including voltages and compensations, for all groups including LYG1+ +, LYG1- - and negative controls in Fig3G and 3H, were actually all the same. 2.tWe are sorry we have made a mistake, we measured CD8 in the V450 channel. So it is BV450 (BV421t), not BV510. 3.tThe compensation of (PE-IFNg minus PE CY7-CD4) for Fig3G had a little higher (0.24%), so in the last response we adjusted compensation values of PE-IFNg minus PE CY7-CD4 to 0% for LYG1+ + and LYG1- - group meanwhile, you can see the result didn t impact our conclusions. 4.tIf further help is required, please send me your email address, we would like to provide the two FACS files of Fig.3G and 3H. ,