Notch activation during early mesoderm induction modulates emergence of the T/NK cell lineage from human iPSCs.

A robust method of producing mature T cells from iPSCs is needed to realize their therapeutic potential. NOTCH1 is known to be required for the production of hematopoietic progenitor cells with T cell potential in vivo. Here we identify a critical window during mesodermal differentiation when Notch activation robustly improves access to definitive hematopoietic progenitors with T/NK cell lineage potential. Low-density progenitors on either OP9-hDLL4 feeder cells or hDLL4-coated plates favored T cell maturation into TCRab+CD3+CD8+ cells that express expected T cell markers, upregulate activation markers, and proliferate in response to T cell stimulus. Single-cell RNAseq shows Notch activation yields a 6-fold increase in multi-potent hematopoietic progenitors that follow a developmental trajectory toward T cells with clear similarity to post-natal human thymocytes. We conclude that early mesodermal Notch activation during hematopoietic differentiation is a missing stimulus with broad implications for producing hematopoietic progenitors with definitive characteristics.

Long-term analysis of antibodies elicited by SPUTNIK V: A prospective cohort study in Tucumán, Argentina.

BACKGROUND: Gam-COVID-Vac (SPUTNIK V) has been granted emergency use authorization in 70 nations and has been administered to millions worldwide. However, there are very few peer-reviewed studies describing its effects. Independent reports regarding safety and effectiveness could accelerate the final approval by the WHO. We aimed to study the long-term humoral immune response in naïve and previously infected volunteers who received SPUTNIK V. METHODS: Humoral immune responses, assayed by anti-SARS-CoV-2-spike-RBD IgG ELISA and neutralization assays, were measured in 602 healthcare workers at 0, 14, 28, 60 and 180 days after receiving SPUTNIK V between December 2020 and July 2021 in Tucumán, Argentina. FINDINGS: Seroconversion was detected in 97% of individuals after 28 days post-vaccination (dpv) (N = 405). Anti-RBD titers began to decrease after 60 dpv (N = 328), but remained detectable in 94% at 90 dpv (N = 224). At 180 dpv, anti-RDB titers persisted in 31% (N = 146). Previous infection triggered an increased immune response to the first dose and increased neutralization activity against variants of concern (VOC). Second doses in previously infected individuals further increased titers, even 90 dpv (N = 75). Basal antibody titers had more influence on post-vaccination anti-RBD responses than the time elapsed between diagnosis and vaccination (N = 274). INTERPRETATION: Data presented herein provides essential knowledge regarding the kinetics of antibodies induced by SPUTNIK V up to six months after immunization, and suggests that when considering one-dose vaccination policies for individuals with previous SARS-CoV-2 infection, serological studies to determine basal titers may be important, independent of when diagnosis occurred. FUNDING: Tucumán Public Health System (SIPROSA), Argentinean National Research Council (CONICET), National University of Tucumán (UNT).

Elevated Humoral Immune Response to SARS-CoV-2 at High Altitudes Revealed by an Anti-RBD "In-House" ELISA.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused a global pandemic with dramatic health and socioeconomic consequences. The Coronavirus Disease 2019 (COVID-19) challenges health systems to quickly respond by developing new diagnostic strategies that contribute to identify infected individuals, monitor infections, perform contact-tracing, and limit the spread of the virus. In this brief report, we developed a highly sensitive, specific, and precise "In-House" ELISA to correctly discriminate previously SARS-CoV-2-infected and non-infected individuals and study population seroprevalence. Among 758 individuals evaluated for anti-SARS-CoV-2 serology in the province of Tucumán, Argentina, we found a weak correlation between antibodies elicited against the RBD, the receptor-binding domain of the Spike protein, and the nucleocapsid (N) antigens of this virus. Additionally, we detected mild levels of anti-RBD IgG antibodies in 33.6% of individuals diagnosed with COVID-19, while only 19% showed sufficient antibody titers to be considered as plasma donors. No differences in IgG anti-RBD titers were found between women and men, neither in between different age groups ranging from 18 to 60. Surprisingly, individuals from a high altitude village displayed elevated and longer lasting anti-RBD titers compared to those from a lower altitude city. To our knowledge, this is the first report correlating altitude with increased humoral immune response against SARS-CoV-2 infection.

Organoids Model Transcriptional Hallmarks of Oncogenic KRAS Activation in Lung Epithelial Progenitor Cells.

Mutant KRAS is a common driver in epithelial cancers. Nevertheless, molecular changes occurring early after activation of oncogenic KRAS in epithelial cells remain poorly understood. We compared transcriptional changes at single-cell resolution after KRAS activation in four sample sets. In addition to patient samples and genetically engineered mouse models, we developed organoid systems from primary mouse and human induced pluripotent stem cell-derived lung epithelial cells to model early-stage lung adenocarcinoma. In all four settings, alveolar epithelial progenitor (AT2) cells expressing oncogenic KRAS had reduced expression of mature lineage identity genes. These findings demonstrate the utility of our in vitro organoid approaches for uncovering the early consequences of oncogenic KRAS expression. This resource provides an extensive collection of datasets and describes organoid tools to study the transcriptional and proteomic changes that distinguish normal epithelial progenitor cells from early-stage lung cancer, facilitating the search for targets for KRAS-driven tumors.

Generation of mesenchyme free intestinal organoids from human induced pluripotent stem cells.

Efficient generation of human induced pluripotent stem cell (hiPSC)-derived human intestinal organoids (HIOs) would facilitate the development of in vitro models for a variety of diseases that affect the gastrointestinal tract, such as inflammatory bowel disease or Cystic Fibrosis. Here, we report a directed differentiation protocol for the generation of mesenchyme-free HIOs that can be primed towards more colonic or proximal intestinal lineages in serum-free defined conditions. Using a CDX2eGFP iPSC knock-in reporter line to track the emergence of hindgut progenitors, we follow the kinetics of CDX2 expression throughout directed differentiation, enabling the purification of intestinal progenitors and robust generation of mesenchyme-free organoids expressing characteristic markers of small intestinal or colonic epithelium. We employ HIOs generated in this way to measure CFTR function using cystic fibrosis patient-derived iPSC lines before and after correction of the CFTR mutation, demonstrating their future potential for disease modeling and therapeutic screening applications.

Transcriptional Immunoprofiling at the Tick-Virus-Host Interface during Early Stages of Tick-Borne Encephalitis Virus Transmission.

Emerging and re-emerging diseases transmitted by blood feeding arthropods are significant global public health problems. Ticks transmit the greatest variety of pathogenic microorganisms of any blood feeding arthropod. Infectious agents transmitted by ticks are delivered to the vertebrate host together with saliva at the bite site. Tick salivary glands produce complex cocktails of bioactive molecules that facilitate blood feeding and pathogen transmission by modulating host hemostasis, pain/itch responses, wound healing, and both innate and adaptive immunity. In this study, we utilized Illumina Next Generation Sequencing to characterize the transcriptional immunoprofile of cutaneous immune responses to Ixodes ricinus transmitted tick-borne encephalitis virus (TBEV). A comparative immune gene expression analysis of TBEV-infected and uninfected tick feeding sites was performed. Our analysis reveals that ticks create an inflammatory environment at the bite site during the first 3 h of feeding, and significant differences in host responses were observed between TBEV-infected and uninfected tick feeding. Gene-expression analysis reveals modulation of inflammatory genes after 1 and 3 h of TBEV-infected tick feeding. Transcriptional levels of genes specific to chemokines and cytokines indicated a neutrophil-dominated immune response. Immunohistochemistry of the tick feeding site revealed that mononuclear phagocytes and fibroblasts are the primary target cells for TBEV infection and did not detect TBEV antigens in neutrophils. Together, the transcriptional and immunohistochemistry results suggest that early cutaneous host responses to TBEV-infected tick feeding are more inflammatory than expected and highlight the importance of inflammatory chemokine and cytokine pathways in tick-borne flavivirus transmission.

Murine cutaneous responses to the rocky mountain spotted fever vector, Dermacentor andersoni, feeding.

Tick salivary glands produce complex cocktails of bioactive molecules that facilitate blood feeding and pathogen transmission by modulating host hemostasis, pain/itch responses, wound healing, and both innate and adaptive immunity. In this study, cutaneous responses at Dermacentor andersoni bite-sites were analyzed using Affymetrix mouse genome arrays and histopathology at 12, 48, 96 and 120 h post- infestation (hpi) during primary infestations and 120 hpi during secondary infestations. The microarray data suggests: (1) chemotaxis of neutrophils, monocytes, and other cell types; (2) production and scavenging of reactive oxygen species; and, (3) keratin- based wound healing responses. Histological analysis supported the microarray findings. At 12 hpi, a mild inflammatory infiltrate was present in the dermis, especially concentrated at the junction between dermal connective tissue and underlying adipose tissue. A small lesion was located immediately under the hypostome and likely represents the feeding "pool." Surprisingly, at 48 hpi, the number of inflammatory cells had not increased from 12 hpi, perhaps mirroring the reduction in gene expression seen at this time point. The feeding lesion is very well defined, and extravasated erythrocytes are readily evident around the hypostome. By 96 hpi, the inflammatory infiltrate has increased dramatically and the feeding lesion appears to have moved deeper into the dermis. At 120 hpi, most of the changes at 96 hpi are intensified. The infiltrate is very dense, the epidermis is markedly thickened, the feeding lesion is poorly defined and the dermal tissue near the hypostome appears to be loosing its normal architecture. In conclusion, during D. andersoni feeding infiltration of inflammatory cells increases across time concurrent with significant changes in the epidermal and dermal compartments near the feeding tick. The importance of changes in the epidermal layer in the host response to ticks is not known, however, it is possible the host attempts to "slough off" the tick by greatly increasing epithelial cell replication.

Early immunologic events at the tick-host interface.

Ixodes species ticks are competent vectors of tick-borne viruses including tick-borne encephalitis and Powassan encephalitis. Tick saliva has been shown to facilitate and enhance viral infection. This likely occurs by saliva-mediated modulation of host responses into patterns favorable for viral infection and dissemination. Because of the rapid kinetics of tick-borne viral transmission, this modulation must occur as early as tick attachment and initiation of feeding. In this study, cutaneous bite-site lesions were analyzed using Affymetrix mouse genome 430A 2.0 arrays and histopathology at 1, 3, 6, and 12 hours after uninfected Ixodes scapularis nymphal tick attachment. At 1 and 3 hrs after attachment, the gene expression profile is markedly different than at later time points. Upregulated gene ontology term clusters enriched at 1 and 3 hrs were related to post-translational modification. At 6 and 12 hrs, cytoskeletal rearrangements, DNA replication/cell division, inflammation, and chemotaxis were prominent clusters. At 6 and 12 hrs, extracellular matrix, signaling, and DNA binding clusters were downregulated. Histopathological analysis shows minimal inflammation at 1 and 3 hrs but an appreciable neutrophil infiltrate at 6 and 12 hrs. In addition, putative hyperemia, localized necrosis, and increased ECM deposition were identified. Putting the gene expression and histopathology analysis together suggests early tick feeding is characterized by modulation of host responses in resident cells that merges into a nascent, neutrophil-driven immune response by 12 hrs post-attachment.

Transcriptional profiling of the murine cutaneous response during initial and subsequent infestations with Ixodes scapularis nymphs.

BACKGROUND: Ixodes scapularis ticks are hematophagous arthropods capable of transmitting many infectious agents to humans. The process of blood feeding is an extended and continuous interplay between tick and host responses. While this process has been studied extensively in vitro, no global understanding of the host response to ticks has emerged. METHODS: To address this issue, we used PCR-arrays to measure skin-specific expression of 233 discrete genes at 8 time points during primary and secondary infestations of mice with pathogen-free I. scapularis nymphs. Selected results were then validated at the mRNA and protein levels by additional real-time PCR and bioplex assay. RESULTS: Primary infestation was characterized by the late induction of an innate immune response. Lectin pattern recognition receptors, cytokines, and chemokines were upregulated consistent with increased neutrophil and macrophage migration. Gene ontology and pathway analyses of downregulated genes suggested inhibition of gene transcription and Th17 immunity. During the secondary infestation, additional genes were modulated suggesting a broader involvement of immune cells including CD8 and CD4 positive T lymphocytes. The cytokine response showed a mixed Th1/Th2 profile with a potential for T regulatory cell activity. Key gene ontology clusters observed during the secondary infestation were cell migration and activation. Matrix metalloproteinases were upregulated, apoptosis-related genes were differentially modulated, and immunoreceptor signaling molecules were upregulated. In contrast, transcripts related to mitogenic, WNT, Hedgehog, and stress pathways were downregulated. CONCLUSIONS: Our results support a model of tick feeding where lectin pattern recognition receptors orchestrate an innate inflammatory response during primary infestation that primes a mixed Th1/Th2 response upon secondary exposure. Tick feeding inhibits gene transcription and Th17 immunity. Salivary molecules may also inhibit upregulation of mitogenic, WNT, Hedgehog, and stress pathways and enhance the activity of T regulatory cells, production of IL-10, and suppressors of cytokine signaling molecules (SOCS). This study provides the first comprehensive transcriptional analysis of the murine host response at the I. scapularis bite site and suggests both a potential model of the host cutaneous response and candidate genes for further description and investigation.