Identification of Distinct Immune Cell Subsets Associated With Asymptomatic Infection, Disease Severity, and Viral Persistence in COVID-19 Patients.

The cell-mediated protective and pathogenic immune responses to SARS-CoV-2 infection remain largely elusive. Here we identified 76 distinct cell subsets in the PBMC samples that were associated with various clinical presentations of COVID-19 using scRNA-seq technology coupled with a deep and comprehensive analysis of unique cell surface markers and differentially expressed genes. We revealed that (TRAV1-2+CD8+)MAIT cells and (NCAM1hiCD160+)NK cells significantly enriched in the asymptomatic subjects whereas (LAG3+CD160+CD8+)NKT cells increased in the symptomatic patients. We also observed that (CD68-CSF1R-IL1BhiCD14+)classical monocytes were positively correlated with the disease severity. Moreover, (CD33-HLA-DMA-CD14+)classical monocytes and (CLEC10A-S100A9lo)pDC were associated with the viral persistence. The GO and KEGG analyses identified enriched pathways related to immune responses, inflammation, and apoptosis. These findings may enhance our understanding of the immunopathogenesis of COVID-19 and help develop novel strategies against SARS-CoV-2 infection.

Increased Expression of Tim-3 Is Associated With Depletion of NKT Cells In SARS-CoV-2 Infection.

In the ongoing coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), natural killer T (NKT) cells act as primary initiators of immune responses. However, a decrease of circulating NKT cells has been observed in COVID-19 different stages, of which the underlying mechanism remains to be elucidated. Here, by performing single-cell RNA sequencing analysis in three large cohorts of COVID-19 patients, we found that increased expression of Tim-3 promotes depletion of NKT cells during the progression stage of COVID-19, which is associated with disease severity and outcome of patients with COVID-19. Tim-3+ NKT cells also expressed high levels of CD147 and CD26, which are potential SARS-CoV-2 spike binding receptors. In the study, Tim-3+ NKT cells showed high enrichment of apoptosis, higher expression levels of mitochondrial genes and caspase genes, with a larger pseudo time value. In addition, Tim-3+ NKT cells in COVID-19 presented a stronger capacity to secrete IFN-γ, IL-4 and IL-10 compared with healthy individuals, they also demonstrated high expression of co-inhibitory receptors such as PD-1, CTLA-4, and LAG-3. Moreover, we found that IL-12 secreted by dendritic cells (DCs) was positively correlated with up-regulated expression of Tim-3 in NKT cells in COVID-19 patients. Overall, this study describes a novel mechanism by which up-regulated Tim-3 expression induced the depletion and dysfunction of NKT cells in COVID-19 patients. These findings not only have possible implications for the prediction of severity and prognosis in COVID-19 but also provide a link between NKT cells and future new therapeutic strategies in SARS-CoV-2 infection.

Distinct immune signatures discriminate between asymptomatic and presymptomatic SARS-CoV-2pos subjects.

Increasing numbers of SARS-CoV-2-positive (SARS-CoV-2pos) subjects are detected at silent SARS-CoV-2 infection stage (SSIS). Yet, SSIS represents a poorly examined time-window wherein unknown immunity patterns may contribute to the fate determination towards persistently asymptomatic or overt disease. Here, we retrieved blood samples from 19 asymptomatic and 12 presymptomatic SARS-CoV-2pos subjects, 47 age/gender-matched patients with mild or moderate COVID-19 and 27 normal subjects, and interrogated them with combined assays of 44-plex CyTOF, RNA-seq and Olink. Notably, both asymptomatic and presymptomatic subjects exhibited numerous readily detectable immunological alterations, while certain parameters including more severely decreased frequencies of CD107alow classical monocytes, intermediate monocytes, non-classical monocytes and CD62Lhi CD8+ Tnaïve cells, reduced plasma STC1 level but an increased frequency of CD4+ NKT cells combined to distinguish the latter. Intercorrelation analyses revealed a particular presymptomatic immunotype mainly manifesting as monocytic overactivation and differentiation blockage, a likely lymphocyte exhaustion and immunosuppression, yielding mechanistic insights into SSIS fate determination, which could potentially improve SARS-CoV-2 management.

Potent Neutralizing Antibodies Elicited by RBD-Fc-Based COVID-19 Vaccine Candidate Adjuvanted by the Th2-Skewing iNKT Cell Agonist.

The development of a safe and effective COVID-19 vaccine is of paramount importance to terminate the current pandemic. An adjuvant is crucial for improving the efficacy of the subunit COVID19 vaccine. α-Galactosylceramide (αGC) is a classical iNKT cell agonist which causes the rapid production of Th1- and Th2-associated cytokines; we, therefore, expect that the Th1- or Th2-skewing analogues of αGC can better enhance the immunogenicity of the receptor-binding domain in the spike protein of SARS-CoV-2 fused with the Fc region of human IgG (RBD-Fc). Herein, we developed a universal synthetic route to the Th1-biasing (α-C-GC) and Th2-biasing (OCH and C20:2) analogues. Immunization of mice demonstrated that αGC-adjuvanted RBD-Fc elicited a more potent humoral response than that observed with Alum and enabled the sparing of antigens. Remarkably, at a low dose of the RBD-Fc protein (2 µg), the Th2-biasing agonist C20:2 induced a significantly higher titer of the neutralizing antibody than that of Alum.

Optimizing Flow Cytometric Analysis of Immune Cells in Samples Requiring Cryopreservation from Tumor-Bearing Mice.

Most shared resource flow cytometry facilities do not permit analysis of radioactive samples. We are investigating low-dose molecular targeted radionuclide therapy (MTRT) as an immunomodulator in combination with in situ tumor vaccines and need to analyze radioactive samples from MTRT-treated mice using flow cytometry. Further, the sudden shutdown of core facilities in response to the COVID-19 pandemic has created an unprecedented work stoppage. In these and other research settings, a robust and reliable means of cryopreservation of immune samples is required. We evaluated different fixation and cryopreservation protocols of disaggregated tumor cells with the aim of identifying a protocol for subsequent flow cytometry of the thawed sample, which most accurately reflects the flow cytometric analysis of the tumor immune microenvironment of a freshly disaggregated and analyzed sample. Cohorts of C57BL/6 mice bearing B78 melanoma tumors were evaluated using dual lymphoid and myeloid immunophenotyping panels involving fixation and cryopreservation at three distinct points during the workflow. Results demonstrate that freezing samples after all staining and fixation are completed most accurately matches the results from noncryopreserved equivalent samples. We observed that cryopreservation of living, unfixed cells introduces a nonuniform alteration to PD1 expression. We confirm the utility of our cryopreservation protocol by comparing tumors treated with in situ tumor vaccines, analyzing both fresh and cryopreserved tumor samples with similar results. Last, we use this cryopreservation protocol with radioactive specimens to demonstrate potentially beneficial effector cell changes to the tumor immune microenvironment following administration of a novel MTRT in a dose- and time-dependent manner.

Natural killer cells and unconventional T cells in COVID-19.

NK cells and diverse populations of unconventional T cells, such as MAIT cells, γδ T cells, invariant NKT cells, and DNTÉ‘ß cells are important early effector lymphocytes. While some of these cells, such as NK cell and MAIT cells, have well-established roles in antiviral defense, the function of other populations remains more elusive. Here, we summarize and discuss current knowledge on NK cell and unconventional T cell responses to SARS-CoV-2 infection. Also covered is the role of these cells in the pathogenesis of severe COVID-19. Understanding the early, both systemic and local (lung), effector lymphocyte response in this novel disease will likely aid ongoing efforts to combat the pandemic.

Distinct immunological signatures discriminate severe COVID-19 from non-SARS-CoV-2-driven critical pneumonia.

Immune profiling of COVID-19 patients has identified numerous alterations in both innate and adaptive immunity. However, whether those changes are specific to SARS-CoV-2 or driven by a general inflammatory response shared across severely ill pneumonia patients remains unknown. Here, we compared the immune profile of severe COVID-19 with non-SARS-CoV-2 pneumonia ICU patients using longitudinal, high-dimensional single-cell spectral cytometry and algorithm-guided analysis. COVID-19 and non-SARS-CoV-2 pneumonia both showed increased emergency myelopoiesis and displayed features of adaptive immune paralysis. However, pathological immune signatures suggestive of T cell exhaustion were exclusive to COVID-19. The integration of single-cell profiling with a predicted binding capacity of SARS-CoV-2 peptides to the patients' HLA profile further linked the COVID-19 immunopathology to impaired virus recognition. Toward clinical translation, circulating NKT cell frequency was identified as a predictive biomarker for patient outcome. Our comparative immune map serves to delineate treatment strategies to interfere with the immunopathologic cascade exclusive to severe COVID-19.

Analysis of the Long-Term Impact on Cellular Immunity in COVID-19-Recovered Individuals Reveals a Profound NKT Cell Impairment.

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) affected over 120 million people and killed over 2.7 million individuals by March 2021. While acute and intermediate interactions between SARS-CoV-2 and the immune system have been studied extensively, long-term impacts on the cellular immune system remain to be analyzed. Here, we comprehensively characterized immunological changes in peripheral blood mononuclear cells in 49 COVID-19-convalescent individuals (CI) in comparison to 27 matched SARS-CoV-2-unexposed individuals (UI). Despite recovery from the disease for more than 2 months, CI showed significant decreases in frequencies of invariant NKT and NKT-like cells compared to UI. Concomitant with the decrease in NKT-like cells, an increase in the percentage of annexin V and 7-aminoactinomycin D (7-AAD) double-positive NKT-like cells was detected, suggesting that the reduction in NKT-like cells results from cell death months after recovery. Significant increases in regulatory T cell frequencies and TIM-3 expression on CD4 and CD8 T cells were also observed in CI, while the cytotoxic potential of T cells and NKT-like cells, defined by granzyme B (GzmB) expression, was significantly diminished. However, both CD4 and CD8 T cells of CI showed increased Ki67 expression and were fully able to proliferate and produce effector cytokines upon T cell receptor (TCR) stimulation. Collectively, we provide a comprehensive characterization of immune signatures in patients recovering from SARS-CoV-2 infection, suggesting that the cellular immune system of COVID-19 patients is still under a sustained influence even months after the recovery from disease.IMPORTANCE Wuhan was the very first city hit by SARS-CoV-2. Accordingly, the patients who experienced the longest phase of convalescence following COVID-19 reside here. This enabled us to investigate the "immunological scar" left by SARS-CoV-2 on cellular immunity after recovery from the disease. In this study, we characterized the long-term impact of SARS-CoV-2 infection on the immune system and provide a comprehensive picture of cellular immunity of a convalescent COVID-19 patient cohort with the longest recovery time. We revealed that the cellular immune system of COVID-19 patients is still under a sustained influence even months after the recovery from disease; in particular, a profound NKT cell impairment was found in the convalescent phase of COVID-19.

Immune responses and pathogenesis in persistently PCR-positive patients with SARS-CoV-2 infection.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 emerged in China in December 2019 and then rapidly spread worldwide. Why COVID-19 patients with the same clinical condition have different outcomes remains unclear. This study aimed to examine the differences in the phenotype and functions of major populations of immune cells between COVID-19 patients with same severity but different outcomes. Four common type adult inpatients with laboratory confirmed COVID-19 from Beijing YouAn Hospital, Capital Medical University were included in this study. The patients were divided into two groups based on whether or not COVID-19 polymerase chain reaction (PCR)-negative conversion occurred within 3 weeks. Peripheral blood samples were collected to compare the differences in the phenotype and functions of major populations of immune cells between the two groups of patients. The result shows that the proportions of CD3+ CD8+ CD38+ HLA-DR+ CD27- effector T killer cells generally declined, whereas that of CD3+ CD4+ CD8+ double-positive T cells (DPTs) increased in the persistently PCR-positive patients. In summary, considering the imbalance between effector T killer cells/CD3+CD4+CD8+ DPTs was a possible key factor for PCR-negative conversion in patients with COVID-19.

COVID-19 and Asthma: Reflection During the Pandemic.

Coronavirus disease 2019 (COVID-19) is a global pandemic infectious disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), and abnormal, overactivated innate immunity and "cytokine storms" have been proposed as potential pathological mechanisms for rapid COVID-19 progression. Theoretically, asthmatic patients should have increased susceptibility and severity for SARS-CoV-2 infection due to a deficient antiviral immune response and the tendency for exacerbation elicited by common respiratory viruses. However, existing studies have not shown an expected prevalence of asthmatic individuals among COVID-19 patients. Certain aspects of type 2 immune response, including type 2 cytokines (IL-4, IL-13, etc.) and accumulation of eosinophils, might provide potential protective effects against COVID-19. Furthermore, conventional therapeutics for asthma, including inhaled corticosteroids, allergen immunotherapy (AIT), and anti-IgE monoclonal antibody, might also reduce the risks of asthmatics suffering infection of the virus through alleviating inflammation or enhancing antiviral defense. The interactions between COVID-19 and asthma deserve further attention and clarification.

Early Phases of COVID-19 Are Characterized by a Reduction in Lymphocyte Populations and the Presence of Atypical Monocytes.

Background: Severe acute respiratory syndrome coronavirus 2 is a recently discovered pathogen responsible of coronavirus disease 2019 (COVID-19). The immunological changes associated with this infection are largely unknown. Methods: We evaluated the peripheral blood mononuclear cells profile of 63 patients with COVID-19 at diagnosis. We also assessed the presence of association with inflammatory biomarkers and the 28-day mortality. Results: Lymphocytopenia was present in 51 of 63 (80.9%) patients, with a median value of 720 lymphocytes/µl (IQR 520-1,135). This reduction was mirrored also on CD8+ (128 cells/µl, IQR 55-215), natural killer (67 cells/µl, IQR 35-158) and natural killer T (31 cells/µl, IQR 11-78) cells. Monocytes were preserved in total number but displayed among them a subpopulation with a higher forward and side scatter properties, composed mainly of cells with a reduced expression of both CD14 and HLA-DR. Patients who died in the 28 days from admission (N=10, 15.9%), when compared to those who did not, displayed lower mean values of CD3+ (337.4 cells/µl vs 585.9 cells/µl; p=0.028) and CD4+ cells (232.2 cells/µl vs 381.1 cells/µl; p=0.042) and an higher percentage of CD8+/CD38+/HLA-DR+ lymphocytes (13.5% vs 7.6%; p=0.026). Discussion: The early phases of COVID-19 are characterized by lymphocytopenia, predominance of Th2-like lymphocytes and monocytes with altered immune profile, which include atypical mononuclear cells.

Phenotypical and functional alteration of unconventional T cells in severe COVID-19 patients.

COVID-19 includes lung infection ranging from mild pneumonia to life-threatening acute respiratory distress syndrome (ARDS). Dysregulated host immune response in the lung is a key feature in ARDS pathophysiology. However, cellular actors involved in COVID-19-driven ARDS are poorly understood. Here, in blood and airways of severe COVID-19 patients, we serially analyzed unconventional T cells, a heterogeneous class of T lymphocytes (MAIT, γδT, and iNKT cells) with potent antimicrobial and regulatory functions. Circulating unconventional T cells of COVID-19 patients presented with a profound and persistent phenotypic alteration. In the airways, highly activated unconventional T cells were detected, suggesting a potential contribution in the regulation of local inflammation. Finally, expression of the CD69 activation marker on blood iNKT and MAIT cells of COVID-19 patients on admission was predictive of clinical course and disease severity. Thus, COVID-19 patients present with an altered unconventional T cell biology, and further investigations will be required to precisely assess their functions during SARS-CoV-2-driven ARDS.

Defining the CD39/CD73 Axis in SARS-CoV-2 Infection: The CD73- Phenotype Identifies Polyfunctional Cytotoxic Lymphocytes.

The ectonucleotidases CD39 and CD73 regulate immune responses by balancing extracellular ATP and adenosine in inflammation and are likely to be involved in the pathophysiology of COVID-19. Here, we analyzed CD39 and CD73 on different lymphocyte populations in a small cohort of COVID-19 patients and in healthy individuals. We describe a significantly lower level of expression of CD73 on cytotoxic lymphocyte populations, including CD8+ T, natural killer T (NKT), and natural killer (NK) cells, during COVID-19. Interestingly, the decrease of CD73 on CD8+ T cells and NKT cells correlated with serum ferritin levels. Furthermore, we observed distinct functional differences between the CD73+ and CD73- subsets of CD8+ T cells and NKT cells with regard to cytokine/toxin secretion. In COVID-19 patients, the majority of the CD73-CD8+ T cells were capable of secreting granzyme B, perforin, tumor necrosis factor (TNF-α) or interferon-gamma (IFN-γ). To conclude, in this first study of CD39 and CD73 expression of lymphocytes in COVID-19, we show that CD8+ T cells and NKT cells lacking CD73 possess a significantly higher cytotoxic effector functionality compared to their CD73+ counterparts. Future studies should investigate differences of cellular CD39 and CD73 expression in patients at different disease stages and their potential as prognostic markers or targets for immunomodulatory therapies.