Aberrant pathogenic GM-CSF+ T cells and inflammatory CD14+CD16+ monocytes in severe pulmonary syndrome patients of a new coronavirus

Yonggang Zhou; Binqing Fu; Xiaohu Zheng; Dongsheng Wang; Changcheng Zhao; Yingjie qi; Rui Sun; Zhigang Tian; Xiaoling Xu; Haiming Wei

In patients infected with SARS-CoV, it has been reported that the severity of pulmonary 55 immune injury correlated with extensive infiltration of neutrophils and macrophages in the 56 lungs 13,14 , accompanied with increased numbers of neutrophils and monocytes and lower 57 CD8 + and CD4 + T cell counts in the peripheral blood samples [15] [16] [17] The number of T cells also significantly decreased from both ICU and non-ICU patients. The To demonstrate the status of these aberrant altered T cells, several lymphoid antigens have 76 been analyzed on T cells. These CD4 + T cells in patients infected with 2019-nCoV have 77 higher expression of CD69, CD38, and CD44 compared with healthy controls (Fig.1a, b) , 78 indicating their activated status. OX40 have been reported to play a major role in promoting 79 clonal expansion and inducing production of several cytokines in T cells 19 . In patients 80 infected with 2019-nCoV, OX40 expression increased remarkably on CD4 + T cells, especially 81 in severe ICU patients (Fig.1a, b) . CD8 + T cells in patients infected with 2019-nCoV also 82 showed activated phenotype with higher expression of CD69, CD38 and CD44 (Fig.1c, d) . 83 41BB (CD137; TNFRS9) is an activation-induced co-stimulatory molecule, which is 84 important to priming immune responses of cytotoxic CD8 + T cells 20 . In ICU patients infected 85 with 2019-nCoV, the expression of 41BB increased significantly compared to healthy controls 86 (Fig.1c, d) . It has been reported that co-expression of Tim-3 and PD-1 may represent a subset 87 of T cells with more severe exhaustion in virus infections 21, 22 . It is worth noting that much 88 3 higher percentage of co-expression Tim3 + PD-1 + T subset exist both in CD4 + and CD8 + T cells 89 from patients of 2019-nCoV ( Fig.1e- To further identify the key pathogenic cytokines and the main source of these cytokines, 93 interferon- (IFN), TNF-, granulocyte-macrophage colony-stimulating factor (GM-CSF) 94 and IL-6 have been selected to analyzed through intracellular cytokine staining, for these 95 inflammatory mediators have been proven to be critical as the primary cause of inflammatory 96 cytokine storm in patients infected with SARS-CoV or MERS-CoV 23,24 . Without 97 re-stimulation with PMA or incubation with monensin, high percentage of GM-CSF + and 98 IL-6 + expressions could been found in CD4 + T cells from patients infected with 2019-nCoV in 99 both ICU and non-ICU patients compared to healthy controls (Fig.2a, c) . ICU patients with 100 more severe pneumonia showed correlated higher percentage of GM-CSF + and IL-6 + CD4 + T 101 cells (Fig.2a, GM-CSF has been recently been implicated in the pathogenesis of inflammatory and 114 autoimmune diseases, in a mechanism that controls diverse pathogenic capabilities of 115 inflammatory myeloid cells. Among these myeloid cells, monocyte is the pathogenic GM-CSF 116 responsive cells that require GM-CSF to initiate tissue damage in both mouse and human 26,27 . 117 To identify whether inflammatory monocyte exist in patients infected 2019-nCoV, phenotype 118 and subpopulation of monocytes have been analysis. There was little CD14 + CD16 + 119 inflammatory monocyte subset in healthy controls. By contrast, significant higher percentage 120 of CD14 + CD16 + inflammatory monocyte exist in peripheral blood of patient infected 121 2019-nCoV. The percentage of CD14 + CD16 + monocyte was much higher in severe pulmonary 122 syndrome patients from ICU (Fig.3a, c)