Understanding innate immune responses in COVID-19 is important to decipher mechanisms of host responses and interpret disease pathogenesis. Natural killer (NK) cells are innate effector lymphocytes that respond to acute viral infections but might also contribute to immunopathology. Using 28-color flow cytometry, we here reveal strong NK cell activation across distinct subsets in peripheral blood of COVID-19 patients. This pattern was mirrored in scRNA-seq signatures of NK cells in bronchoalveolar lavage from COVID-19 patients. Unsupervised high-dimensional analysis of peripheral blood NK cells furthermore identified distinct NK cell immunotypes that were linked to disease severity. Hallmarks of these immunotypes were high expression of perforin, NKG2C, and Ksp37, reflecting increased presence of adaptive NK cells in circulation of patients with severe disease. Finally, arming of CD56^bright NK cells was observed across COVID-19 disease states, driven by a defined protein-protein interaction network of inflammatory soluble factors. This study provides a detailed map of the NK cell activation landscape in COVID-19 disease.

Although NK cells are considered innate, recent studies in mice revealed the existence of a unique lineage of hepatic CD49a⁺DX5⁻ NK cells with adaptive-like features. Development of this NK cell lineage is, in contrast to conventional NK cells, dependent on T-bet but not Eomes. In this study, we describe the identification of a T-bet⁺Eomes⁻CD49a⁺ NK cell subset readily detectable in the human liver, but not in afferent or efferent hepatic venous or peripheral blood. Human intrahepatic CD49a⁺ NK cells express killer cell Ig-like receptor and NKG2C, indicative of having undergone clonal-like expansion, are CD56^bright, and express low levels of CD16, CD57, and perforin. After stimulation, CD49a⁺ NK cells express high levels of inflammatory cytokines but degranulate poorly. CD49a⁺ NK cells retain their phenotype after expansion in long-term in vitro cultures. These results demonstrate the presence of a likely human counterpart of mouse intrahepatic NK cells with adaptive-like features.

The study of human macrophages and their ontogeny is an important unresolved issue. Here, we use a humanized mouse model expressing human cytokines to dissect the development of lung macrophages from human hematopoiesis in vivo. Human CD34+ hematopoietic stem and progenitor cells (HSPCs) generated three macrophage populations, occupying separate anatomical niches in the lung. Intravascular cell labeling, cell transplantation, and fate-mapping studies established that classical CD14+ blood monocytes derived from HSPCs migrated into lung tissue and gave rise to human interstitial and alveolar macrophages. In contrast, non-classical CD16+ blood monocytes preferentially generated macrophages resident in the lung vasculature (pulmonary intravascular macrophages). Finally, single-cell RNA sequencing defined intermediate differentiation stages in human lung macrophage development from blood monocytes. This study identifies distinct developmental pathways from circulating monocytes to lung macrophages and reveals how cellular origin contributes to human macrophage identity, diversity, and localization in vivo.

Human lung tissue-resident NK cells (trNK cells) are likely to play an important role in host responses towards viral infections, inflammatory conditions and cancer. However, detailed insights into these cells are still largely lacking. Here we show, using RNA sequencing and flow cytometry-based analyses, that subsets of human lung CD69⁺CD16⁻ NK cells display hallmarks of tissue-residency, including high expression of CD49a, CD103, and ZNF683, and reduced expression of SELL, S1PR5, and KLF2/3. CD49a⁺CD16⁻ NK cells are functionally competent, and produce IFN-γ, TNF, MIP-1β, and GM-CSF. After stimulation with IL-15, they upregulate perforin, granzyme B, and Ki67 to a similar degree as CD49a⁻CD16⁻ NK cells. Comparing datasets from trNK cells in human lung and bone marrow with tissue-resident memory CD8⁺ T cells identifies core genes co-regulated either by tissue-residency, cell-type or location. Together, our data indicate that human lung trNK cells have distinct features, likely regulating their function in barrier immunity.

Objective: Rituximab is a therapeutic anti‐CD20 antibody used for in vivo depletion of B cells in proliferative and autoimmune diseases. However, the mechanisms of action are not fully understood, since not all of the therapy‐mediated effects can be explained by the depletion of antibody‐secreting cells. In addition to B cells, there is also a small population of T cells coexpressing CD20 in all individuals. This study was conducted to examine the phenotype and function of CD3+CD20+ T cells in patients with rheumatoid arthritis (RA) and healthy controls.Methods: The phenotype and apoptosis of peripheral blood mononuclear cells from healthy donors and RA patients were examined by 4‐color fluorescence‐activated cell sorting analyses. Cytokine production was determined by intracellular staining and measurement of cytokines in the supernatants. Proliferation of sorted T cell populations was analyzed using ³H‐thymidine uptake assays.Results: In healthy individuals, 0.1–6.8% of peripheral blood T cells (mean 1.6%; n = 142) coexpressed CD20, which was not significantly different from that in the peripheral blood of RA patients, in whom 0.4–2.6% of T cells (mean 1.2%; n = 27) were CD20+. During rituximab therapy, the CD20+ T cells along with the B cells were eliminated from the RA peripheral blood. Among the CD20+ T cells, 45% coexpressed CD8 and 55% coexpressed CD4. Polyclonal CD3+CD20+ cells were functionally characterized by constitutive cytokine production (i.e., interleukin‐1β and tumor necrosis factor α), a low proliferative capacity, a high activation state, and enhanced susceptibility to apoptosis.Conclusion: These findings suggest that CD20+ T cells represent a terminally differentiated cell type with immune‐regulatory and proinflammatory capacities. Depletion of CD20+ T cells may be an additional mechanism by which anti‐CD20 therapy functions in patients with RA.