Rapid and accurate SARS-CoV-2 diagnostic testing is essential for controlling the ongoing COVID-19 pandemic. The current gold standard for COVID-19 diagnosis is real-time RT-PCR detection of SARS-CoV-2 from nasopharyngeal swabs. Low sensitivity, exposure risks to healthcare workers, and global shortages of swabs and personal protective equipment, however, necessitate the validation of new diagnostic approaches. Saliva is a promising candidate for SARS-CoV-2 diagnostics because (1) collection is minimally invasive and can reliably be self-administered and (2) saliva has exhibited comparable sensitivity to nasopharyngeal swabs in detection of other respiratory pathogens, including endemic human coronaviruses, in previous studies. To validate the use of saliva for SARS-CoV-2 detection, we tested nasopharyngeal and saliva samples from confirmed COVID-19 patients and self-collected samples from healthcare workers on COVID-19 wards. When we compared SARS-CoV-2 detection from patient-matched nasopharyngeal and saliva samples, we found that saliva yielded greater detection sensitivity and NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.consistency throughout the course of infection. Furthermore, we report less variability in self-sample collection of saliva. Taken together, our findings demonstrate that saliva is a viable and more sensitive alternative to nasopharyngeal swabs and could enable at-home self-administered sample collection for accurate large-scale SARS-CoV-2 testing.

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Interleukin-9 is a T cell cytokine that acts through a γC-family receptor on target cells. We determined that T cells from mice deficient in the TH17 pathway genes ROR-γ and IL-23R produced abundant IL-9, and observed significant growth inhibition of B16F10 melanoma tumor in these mice. IL-9 blocking antibodies reversed this tumor growth inhibition, and enhanced tumor growth in normal mice. IL9R−/− mice showed accelerated tumor growth, while administration of rIL-9 to tumor bearing mice inhibited tumor growth. Adoptive transfer of tumor antigen-specific TH9 cells blocked tumor growth; this was reversed by anti-IL-9. Exogenous rIL-9 inhibited tumor growth in Rag1−/− mice, but not in mast cell deficient mice, suggesting a T cell independent process. Finally, we found TH9 cells in normal human skin and blood, and low IL-9 production from melanoma tumor infiltrating lymphocytes. These results suggest a role for IL-9 in tumor immunity, and suggest therapeutic strategies.

Central memory T (TCM) cells in lymph nodes (LN) and resident memory T (TRM) cells in peripheral tissues play distinct roles in protective immunity1-5. Both are generated after primary infections, but the clonal origin of TRM and TCM cells is unclear. To address this question, mice were immunized through the skin with either a protein antigen, a chemical hapten, or a non-replicating poxvirus. We then analyzed antigen activated T cells from different tissues using high-throughput sequencing (HTS) of the gene (Tcrbv) encoding T cell receptor gene β chain CDR3 region to simultaneously track thousands of unique T cells6. For every abundant TRM clone generated in the skin, an abundant TCM clone bearing the identical TCR was present in lymph nodes (LN). Thus antigen reactive skin TRM and LN TCM clones were derived from a common naive T cell precursor after skin immunization, generating overlapping TCR repertoires. Although they bore the same TCR, TRM mediated rapid contact hypersensitivity (CHS)7 responses in mice, whereas TCM mediated delayed and attenuated responses. Studies in human subjects confirmed the generation of skin TRM in allergic contact dermatitis. Thus, immunization through skin simultaneously generates skin TRM and LN TCM in similar numbers from the same naïve T cells.

Although memory T cells within barrier tissues can persist as permanent residents, at least some exchange with blood. The extent to which this occurs is unclear. Here we show that memory CD4+ T cells in mouse skin are in equilibrium with the circulation at steady state. These cells are dispersed throughout the inter-follicular regions of the dermis and form clusters with antigen presenting cells around hair follicles. After infection or administration of a contact sensitizing agent, there is a sustained increase in skin CD4+ T-cell content, which is confined to the clusters, with a concomitant CCL5-dependent increase in CD4+ T-cell recruitment. Skin CCL5 is derived from CD11b+ cells and CD8+ T cells, with the elimination of the latter decreasing CD4+ T-cell numbers. These results reveal a complex pattern of tissue-retention and equilibration for CD4+ memory T cells in skin, which is altered by infection and inflammation history.

The recent spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exemplifies the critical need for accurate and rapid diagnostic assays to prompt clinical and public health interventions. Currently, several quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assays are being used by clinical, research, and public health laboratories. However, it is currently unclear if results from different tests are comparable. Our goal was to evaluate the primer-probe sets used in four common diagnostic assays available on the World Health Organization (WHO) website. To facilitate this effort, we generated RNA transcripts to be used as assay standards and distributed them to other laboratories for internal validation. We then used these (1) RNA transcript standards, (2) full-length SARS-CoV-2 RNA, and (3) pre-COVID-19 nasopharyngeal swabs, and (4) clinical samples from COVID-19 patients to determine analytical efficiency and sensitivity of the qRT-PCR primer-probe sets. We show that all primer-probe sets can be used to detect SARS-CoV-2, but there are clear differences in the ability to differentiate between true negatives and positives with low amounts of virus. We found that several primer-probe sets cross-react with SARS-CoV-2-negative nasopharyngeal swabs. However, background cross-reactivity by the 2019-nCoV_N2 set issued by the US Centers for Disease Control and Prevention did not interfere with outcomes of the combined "N1" and "N2" assay when testing COVID-19 clinical samples. Our findings characterize the limitations of currently used primer-probe sets and can assist other laboratories in selecting appropriate assays for the detection of SARS-CoV-2.