Highlights d SARS-CoV-2-infected RMs mimic signatures of inflammation seen in COVID-19 patients d Baricitinib suppresses production of pro-inflammatory cytokines in lung macrophages d Baricitinib limits recruitment of neutrophils to the lung and NETosis d Baricitinib preserves innate antiviral and SARS-CoV-2specific T cell responses

Chronic infectious diseases have a substantial impact on the human B cell compartment including a notable expansion of B cells here termed atypical B cells (ABCs). Using unbiased single-cell RNA sequencing (scRNA-seq), we uncovered and characterized heterogeneities in naïve B cell, classical memory B cells, and ABC subsets. We showed remarkably similar transcriptional profiles for ABC clusters in malaria, HIV, and autoimmune diseases and demonstrated that interferon-γ drove the expansion of ABCs in malaria. These observations suggest that ABCs represent a separate B cell lineage with a common inducer that further diversifies and acquires disease-specific characteristics and functions. In malaria, we identified ABC subsets based on isotype expression that differed in expansion in African children and in B cell receptor repertoire characteristics. Of particular interest, IgD+IgMlo and IgD−IgG+ ABCs acquired a high antigen affinity threshold for activation, suggesting that ABCs may limit autoimmune responses to low-affinity self-antigens in chronic malaria.

A combination of vaccination approaches will likely be necessary to fully control the SARS-CoV-2 pandemic. Here, we show that modified vaccinia Ankara (MVA) vectors expressing membrane anchored pre-fusion stabilized spike (MVA/S), but not secreted S1, induced strong neutralizing antibody responses against SARS-CoV-2 in mice. In macaques, the MVA/S vaccination induced strong neutralizing antibodies and CD8 + T cell responses, and showed protection from SARS-CoV-2 infection and virus replication in the lung as early as day 2 following intranasal or intratracheal challenge. Single-cell RNA sequencing analysis of lung cells at day 4 post-infection revealed that MVA/S vaccination also protected macaques from infection-induced inflammation and B cell abnormalities, and lowered induction of interferon stimulated genes. These results demonstrate that MVA/S vaccination induces both neutralizing antibodies and CD8 + T cells in the blood and lung and serves as a potential vaccine candidate against SARS-CoV-2.

T lymphocytes are critical for effective immunity, and the ability to study their behavior in vitro can facilitate major insights into their development, function, and fate. However, the composition of human plasma differs from conventional media, and we hypothesized that such differences could impact immune cell physiology. Here, we showed that relative to the medium typically used to culture lymphocytes (RPMI), a physiologic medium (human plasma-like medium; HPLM) induced markedly different transcriptional responses in human primary T cells and in addition, improved their activation upon antigen stimulation. We found that this medium-dependent effect on T cell activation is linked to Ca 2+ , which is six-fold higher in HPLM than in RPMI. Thus, a medium that more closely resembles human plasma has striking effects on T cell biology, further demonstrates that medium composition can profoundly affect experimental results, and broadly suggests that physiologic media may offer a valuable way to study cultured immune cells.

The COVID-19 pandemic remains a global health crisis, yet, the immunopathological mechanisms driving the development of severe disease remain poorly defined. Here, we utilize a rhesus macaque (RM) model of SARS-CoV-2 infection to delineate perturbations in the innate immune system during acute infection using an integrated systems analysis. We found that SARS-CoV-2 initiated a rapid infiltration (two days post infection) of plasmacytoid dendritic cells into the lower airway, commensurate with IFNA production, natural killer cell activation, and induction of interferon-stimulated genes. At this early interval, we also observed a significant increase of blood CD14-CD16+ monocytes. To dissect the contribution of lung myeloid subsets to airway inflammation, we generated a novel compendium of RM-specific lung macrophage gene expression using a combination of sc-RNA-Seq data and bulk RNA-Seq of purified populations under steady state conditions. Using these tools, we generated a longitudinal sc-RNA-seq dataset of airway cells in SARS-CoV-2-infected RMs. We identified that SARS-CoV-2 infection elicited a rapid recruitment of two subsets of macrophages into the airway: a C206+MRC1- population resembling murine interstitial macrophages, and a TREM2+ population consistent with CCR2+ infiltrating monocytes, into the alveolar space. These subsets were the predominant source of inflammatory cytokines, accounting for ~75% of IL6 and TNF production, and >90% of IL10 production, whereas the contribution of CD206+MRC+ alveolar macrophages was significantly lower. Treatment of SARS-CoV-2 infected RMs with baricitinib (Olumiant®), a novel JAK1/2 inhibitor that recently received Emergency Use Authorization for the treatment of hospitalized COVID-19 patients, was remarkably effective in eliminating the influx of infiltrating, non-alveolar macrophages in the alveolar space, with a concomitant reduction of inflammatory cytokines. This study has delineated the major subsets of lung macrophages driving inflammatory and anti-inflammatory cytokine production within the alveolar space during SARS-CoV-2 infection.

T lymphocytes are critical for effective immunity and the ability to study their behavior in synthetic media in vitro facilitates major discoveries in their development, function, and fate.However, the composition of human plasma differs from synthetic media and we hypothesized that these differences could have important effects on cell physiology. We therefore compared T lymphocyte activation in human plasma-like medium (HPLM) to RPMI supplemented with dialyzed FBS (RPMI dFBS ) and found that it entrained markedly different transcriptional responses. We also found that the concentration of calcium in RPMI dFBS is six-fold lower than HPLM causing altered T cell activation which could be reversed by calcium addition. Thus, investigators should be cognizant of differences between commonly used media formulations and HPLM which is based on the in vivo plasma environment as these could profoundly affect their experimental results. Physiologic media may be a valuable new way to study immune cells in culture.3

Persistent exposure to antigen leads to T cell exhaustion and immunologic dysfunction. We examined the immune exhaustion markers TIGIT and PD-1 in HIV-infected and healthy individuals and the relationship with cytotoxic CD8 + T lymphocyte (CTL) activity. Frequencies of TIGIT but not PD-1 positively correlated with CTL activity in HIV-aviremic and healthy individuals; however, there was no correlation in HIV-viremic individuals. Transcriptome analyses revealed upregulation of genes associated with antiviral immunity in TIGIT + versus TIGIT -CD8 + T cells. Our data suggest that TIGIT +CD8 + T cells do not necessarily represent a state of immune exhaustion and maintain an intrinsic cytotoxicity in HIV-infected individuals.

Background Babesia rossi is a leading cause of morbidity and mortality among the canine population of sub-Saharan Africa, but pathogenesis remains poorly understood. Previous studies of B. rossi infection were derived from clinical cases, in which neither the onset of infection nor the infectious inoculum was known. Here, we performed controlled B. rossi inoculations in canines and evaluated disease progression through clinical tests and whole blood transcriptomic profiling. Results Two subjects were administered a low inoculum (104 parasites) while three received a high (108 parasites). Subjects were monitored for 8 consecutive days; anti-parasite treatment with diminazene aceturate was administered on day 4. Blood was drawn prior to inoculation as well as every experimental day for assessment of clinical parameters and transcriptomic profiles. The model recapitulated natural disease manifestations including anemia, acidosis, inflammation and behavioral changes. Rate of disease onset and clinical severity were proportional to the inoculum. To analyze the temporal dynamics of the transcriptomic host response, we sequenced mRNA extracted from whole blood drawn on days 0, 1, 3, 4, 6, and 8. Differential gene expression, hierarchical clustering, and pathway enrichment analyses identified genes and pathways involved in response to hemolysis, metabolic changes, and several arms of the immune response including innate immunity, adaptive immunity, and response to viral infection. Conclusions This work comprehensively characterizes the clinical and transcriptomic progression of B. rossi infection in canines, thus establishing a large mammalian model of severe hemoprotozoal disease to facilitate the study of host-parasite biology and in which to test novel anti-disease therapeutics. The knowledge gained from the study of B. rossi in canines will not only improve our understanding of this emerging infectious disease threat in domestic dogs, but also provide insight into the pathobiology of human diseases caused by Babesia and Plasmodium species.