COVID-19, caused by SARS-CoV-2, has recently affected over 300,000 people and killed more than 10,000. The manner in which the key immune cell subsets change and their states during the course of COVID-19 remain unclear. Here, we applied single-cell technology to comprehensively characterize transcriptional changes in peripheral blood mononuclear cells during the recovery stage of COVID-19. Compared with healthy controls, in patients in the early recovery stage (ERS) of COVID-19, T cells decreased remarkably, whereas monocytes increased. A detailed analysis of the monocytes revealed that there was an increased ratio of classical CD14 ++ monocytes with high inflammatory gene expression as well as a greater abundance of CD14 ++ IL1B + monocytes in the ERS. CD4 + and CD8 + T cells decreased significantly and expressed high levels of inflammatory genes in the ERS. Among the B cells, the plasma cells increased remarkably, whereas the naïve B cells decreased. Our study identified several novel B cell-receptor (BCR) changes, such as IGHV3-23 and IGHV3-7, and confirmed isotypes (IGHV3-15, IGHV3-30, and IGKV3-11) previously used for virus vaccine development. The strongest pairing frequencies, IGHV3-23-IGHJ4, indicated a monoclonal state associated with SARS-CoV-2 specificity. Furthermore, integrated analysis predicted that IL-1β and M-CSF may be novel candidate target genes for inflammatory storm and that TNFSF13, IL-18, IL-2 and IL-4 may be beneficial for the recovery of COVID-19 patients. Our study provides the first evidence of an inflammatory immune signature in the ERS, suggesting that COVID-19 patients are still vulnerable after hospital discharge. Our identification of novel BCR signaling may lead to the development of vaccines and antibodies for the treatment of COVID-19. Software and AlgorithmsPrism 8 https://www.graphpad.com/scientificsoftware/ prism/ Cell Ranger 3.1.0 https://support.10xgenomics.com Loupe Browser 4.0.0 https://support.10xgenomics.com Loupe V(D)J Browser 3.0.0 https://support.10xgenomics.com R 3.6.1 https://www.r-project.org/ Seurat V3 [37] https://satijalab.org/ Monocle3 [38] https://cole-trapnell-lab.github.io/monocle3 GO analysis [39] https://www.metascape.org Patients 10 COVID-19 patients diagnosed with by real-time fluorescent RT-PCR were collected in the Wuhan Hankou Hospital China. Patients were divided into early-recovery stage (ERS) group and late-recovery stage (LRS) group according to the days from first negative nucleic acid transfer date to blood sampling date. We defined the RES group of five cases as the date of nucleic acid turningnegative to blood sampling is less than seven days and LRS group of five cases as is more than fourteen days. The 10 patients consisted of five males and five females and ranged from ages 40 to 70 years old, with a median of 50 years old. The demographic characteristics of these patients are provided in Fig. S1. A written informed consent was regularly obtained from all patients. The study was approved by the Ethics Committee of Wuhan Hankou Hospital, ...

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Alpha-fetoprotein (AFP)-secreting hepatocellular carcinoma (HCC), which accounts for ~75% of HCCs, is more aggressive with a worse prognosis than those without AFP production. The mechanism through which the interaction between tumors and the microenvironment leads to distinct phenotypes is not yet clear. Therefore, our study aims to identify the characteristic features and potential treatment targets of AFP-negative HCC (ANHC) and AFP-positive HCC (APHC). We utilized single-cell RNA sequencing to analyze 6 ANHC, 6 APHC, and 4 adjacent normal tissues. Integrated multi-omics analysis together with survival analysis were also performed. Further validation was conducted via cytometry time-of-flight on 30 HCCs and multiplex immunohistochemistry on additional 59 HCCs. Our data showed that the genes related to antigen processing and interferon-γ response were abundant in tumor cells of APHC. Meanwhile, APHC was associated with multifaceted immune distortion, including exhaustion of diverse T cell subpopulations, and the accumulation of tumor-associated macrophages (TAMs). Notably, TAM-SPP1+ was highly enriched in APHC, as was its receptor CD44 on T cells and tumor cells. Targeting the Spp1-Cd44 axis restored T cell function in vitro and significantly reduced tumor burden when treated with either anti-Spp1 or anti-Cd44 antibody alone or in combination with anti-Pd-1 antibody in the mouse model. Furthermore, elevated IL6 and TGF-β1 signaling contributed to the enrichment of TAM-SPP1+ in APHC. In conclusion, this study uncovered a highly suppressive microenvironment in APHC and highlighted the role of TAM-SPP1+ in regulating the immune microenvironment, thereby revealing the SPP1-CD44 axis as a promising target for achieving a more favorable immune response in APHC treatment.