Construction And Application Of Single-cell Atlas Of Peripheral Blood Mononuclear Cells

The immune system is the first line of defense against pathogens and diseases.Immune alterations are the cause of some diseases.For example,hyperactive immunnity makes immune cell attack normal and healthy tissue,while weak immunostatus may closely linked to tumor growth and poor outcome.Immune cells are the core of immune system,and after maturation,they travel throughout human body via blood circulation and lymphatic circulation,which connect lymphatic organs and lymphatic tissues into a functional integration.Herein,It’s efficient to reflect immune status through monitoring immune cell alteration,which would be important to adopt timely intervention to stop or slow down the onset and development of diseases when abnormalities occurred.Flow cytometry and Mass cytometry have been an important tool to analyse peripheral blood immune cells and clarify the pathogenesis of diseases.Due to the limitations of the technical principles,these two techniques collect smallscale information.what’s more,the quality of the data depends on the specificity of antibodies,and only up to a hundred parameters can be analyzed simultaneously.Single-cell RNA sequencing(scRNA-seq)is an emerging technology to obtain the transcriptional states of cells at the single-cell level,greatly deepening our understanding of cell types in complex tissues as well as helping us to identify new cell types or states.With the development of single cell isolation technology,it has gone from sequencing dozens of cells to now being able to capture and isolate nearly a million cells simultaneously.Along with transcriptome information,we can also obtain information on the abundance of cell surface proteins using antibody labeling technology,greatly enriching our understanding of cell heterogeneity and cell status in target tissues.Studying the alteration and variety of Peripheral blood mononuclear cell(PBMC)under pathological conditions is one of the important areas of single cell transcriptome sequencing application.With the help of scRNA-seq,we can not only monitor the changes in peripheral immune system,but also analyze the changes of peripheral immune cells before and after treatment to optimize the therapeutic programs and to get a better prognosis.A large amount of single-cell transcriptomic data of PBMC have been produced,but the annotation results of cells vary widely among researchers.Those messy cell annotations have seriously hindered the cross-talks and progresses of related investigations.To address this issue,we collected nearly 3 million single-cell data and constructed a complete reference set of PBMC immune cell annotations using uniform data filtering criteria as well as hierarchical analysis.We identified multiple new cell types(states)by transcriptome expression features,and finally classified PBMC into 76 subgroups,and used this to construct a support vector machine classifier with a classification accuracy of 0.94.By reanalyzing the validation datasets,we further verified the applicability of the classifier in practice,and the cell type annotations obtained using the classifier can accurately replicate the conclusions draw in the original article and even retrieve more information,indicating that the classifier we constructed can greatly simplify the analysis process and provide more comprehensive information on the immune system status.Corona Virus Disease 2019(COVID-19)outbroke in December 2019 with the pathogen Severe Acute Respiratory Syndrome Coronavirus 2(SARS-CoV-2)and was defined as a pandemic by the WHO in March 2020,which has caused millions of deaths to date,resulting in a severe social burden and economic loss worldwide.Vaccine development,as well as vaccination against COVID-19,is underway at an unprecedented pace.The country is primarily using a novel inactivated coronavirus vaccine,with more than 2.22 billion doses have been innoculated as of October 2021,and is preparing to start a booster vaccination program.Vaccination is traditionally thought to promote specific antibody formation primarily through humoral immunity,but the effect of vaccines on the immune state is unknown.To investigate this issue,we recruited 11 volunteers and collected their peripheral blood and PBMC before and after vaccination.we divided the 11 volunteers into two groups,the first group received the second dose on day 14 after the first vaccination,the second group was given the second dose on day 28 after the first inoculation,and peripheral blood was collected on days 7,14,28,42,56,and 90 after the first inoculation for IgM/IgG assay,and neutralizing antibodies were measured for samples on days 28,42,56,and 90.On day 14 after completion of the two doses vaccinations,the positive IgG test rate reached 100%in both groups of volunteers,but then began to decline.It is noteworthy that the 100%positive IgG test rate in the second group of volunteers(28 days between 2 inoculation)lasted until day 56,and not only that,the neutralizing antibody results also reflected that the neutralizing antibody titers in the second group of volunteers were higher than those in the first group,indicating that a 28-day interval between inoculation could provide a better humoral immune response.Subsequently,we performed scRNA-seq of PBMC from volunteers before vaccination and 28 days after the first inoculation,and we found that although there were differences in humoral immune responses between the two groups,there was a high similarity in the changes of immune states.Further analyzing the differentially expressed genes before and after vaccination,we found that a large number of chemokines and pro-inflammatory factors was up-regulated after vaccination,while genes for type I interferon response,associated with antiviral,were down-regulated,and these changes shared a common trend across nearly all immune cell types.Through gene regulatory network analysis,we identified transcription factors such as STAT1,STAT2,NKFB2 and RELB as possible master regulators responsible for these changes and found that these regulators were closely associated in type I interferon response and T cell activation and differentiation.These results above suggest that the innate immune function of the organism may be suppressed and the adaptive immune function is enhanced after COVID-19 vaccination.In this study,we integrated and analyzed a large amount of single-cell transcriptome data,constructed human peripheral immune cells atlas through multilevel cellular annotation,and elucidated the effects of inactivated vaccines of COVID19 on the peripheral immune state of normal humans using single-cell transcriptome analysis,which fully demonstrated the applicability of the peripheral immune cells analysis under different immune conditions and provided a new idea for future precision medicine.