SARS-CoV-2-specific T cells exhibit unique features characterized by robust helper function, lack of terminal differentiation, and high proliferative potential

Convalescing COVID-19 patients mount robust T cell responses against SARS-CoV-2, suggesting an important role for T cells in viral clearance. To date, the phenotypes of SARS-CoV-2-specific T cells remain poorly defined. Using 38-parameter CyTOF, we phenotyped longitudinal specimens of SARS-CoV-2-specific CD4+ and CD8+ T cells from nine individuals who recovered from mild COVID-19. SARS-CoV-2-specific CD4+ T cells were exclusively Th1 cells, and predominantly Tcm with phenotypic features of robust helper function. SARS-CoV-2-specific CD8+ T cells were predominantly Temra cells in a state of less terminal differentiation than most Temra cells. Subsets of SARS-CoV-2-specific T cells express CD127, can homeostatically proliferate, and can persist for over two months. Our results suggest that long-lived and robust T cell immunity is generated following natural SARS-CoV-2 infection, and support an important role for SARS-CoV-2-specific T cells in host control of COVID-19.

Stromal expression of decorin, Semaphorin6D, SPARC, Sprouty1 and Tsukushi in developing prostate and decreased levels of decorin in prostate cancer.

BACKGROUND AND AIM: During prostate development, mesenchymal-epithelial interactions regulate organ growth and differentiation. In adult prostate, stromal-epithelial interactions are important for tissue homeostasis and also play a significant role in prostate cancer. In this study we have identified molecules that show a mesenchymal expression pattern in the developing prostate, and one of these showed reduced expression in prostate cancer stroma. METHODOLOGY AND PRINCIPAL FINDINGS: Five candidate molecules identified by transcript profiling of developmental prostate mesenchyme were selected using a wholemount in situ hybridisation screen and studied Decorin (Dcn), Semaphorin6D (Sema6D), SPARC/Osteonectin (SPARC), Sprouty1 (Spry-1) and Tsukushi (Tsku). Expression in rat tissues was evaluated using wholemount in situ hybridisation (postnatal day (P) 0.5) and immunohistochemistry (embryonic day (E) E17.5, E19.5; P0.5; P6; 28 & adult). Four candidates (Decorin, SPARC, Spry-1, Tsukushi) were immunolocalised in human foetal prostate (weeks 14, 16, 19) and expression of Decorin was evaluated on a human prostate cancer tissue microarray. In embryonic and perinatal rats Decorin, Semaphorin6D, SPARC, Spry-1 and Tsukushi were expressed with varying distribution patterns throughout the mesenchyme at E17.5, E19.5, P0.5 and P6.5. In P28 and adult prostates there was either a decrease in the expression (Semaphorin6D) or a switch to epithelial expression of SPARC, and Spry-1, whereas Decorin and Tsukushi were specific to mesenchyme/stroma at all ages. Expression of Decorin, SPARC, Spry-1 and Tsukushi in human foetal prostates paralleled that in rat. Decorin showed mesenchymal and stromal-specific expression at all ages and was further examined in prostate cancer, where stromal expression was significantly reduced compared with non-malignant prostate. CONCLUSION AND SIGNIFICANCE: We describe the spatio-temporal expression of Decorin, Semaphorin6D, SPARC, Spry-1 and Tsukushi in developing prostate and observed similar mesenchymal expression patterns in rat and human. Additionally, Decorin showed reduced expression in prostate cancer stroma compared to non-malignant prostate stroma.

Identification of EphrinB1 expression in prostatic mesenchyme and a role for EphB-EphrinB signalling in prostate development.

Paracrine signalling from mesenchyme to epithelium plays a key role in regulating prostate organogenesis and it is important to identify the mesenchymally expressed molecules that regulate organ growth, though currently few such molecules are known. Tyrosine kinase signalling via EphB receptors has been characterised in many developmental processes, and EphB3 mRNA expression was detected in prostate inductive mesenchyme in previous gene profiling studies. This led us to examine the expression and function of EphrinB signalling in prostate development, to determine if EphrinB ligands might function as mesenchymal paracrine regulators of prostate growth. Using PCR, wholemount in situ hybridisation, and immunohistochemistry we examined the expression of EphB receptors and EphrinB ligands in rat prostate during development to determine which showed mesenchymal expression. EphB3 and EphrinB1 transcripts and proteins were expressed in the mesenchyme of developing prostate and in female urogenital mesenchyme and smooth muscle. The function of EphrinB signalling was examined using in vitro organ culture assays of ventral prostate (VP), which were treated with EphB3-Fc and EphrinB1-Fc proteins to inhibit or augment Ephrin signalling. Addition of recombinant EphB3-Fc resulted in a significant decrease in VP organ size, while recombinant EphrinB1-Fc resulted in a significant increase in VP organ size and epithelial proliferation. Additionally, EphrinB1-Fc reduced the degree of epithelial branching in VP organs and increased ductal tip size, though without disrupting normal differentiation. We have identified expression of EphrinB1 in prostatic mesenchyme and suggest that the EphrinB signalling system acts as a regulator of prostate growth. EphrinB-EphB signalling may function as an autocrine regulator of mesenchyme and/or as a paracrine regulator of epithelia.

The bovine chemokine receptors and their mRNA abundance in mononuclear phagocytes.

BACKGROUND: The chemokine and chemokine receptor families play critical roles in both the healthy and diseased organism mediating the migration of cells. The chemokine system is complex in that multiple chemokines can bind to one chemokine receptor and vice versa. Although chemokine receptors have been well characterised in humans, the chemokine receptor repertoire of cattle is not well characterised and many sequences are yet to be experimentally validated. RESULTS: We have identified and sequenced bovine homologs to all identified functional human chemokine receptors. The bovine chemokine receptors show high levels of similarity to their human counterparts and similar genome arrangements. We have also characterised an additional bovine chemokine receptor, not present in the available genome sequence of humans or the more closely related pigs or horses. This receptor shows the highest level of similarity to CCR1 but shows significant differences in regions of the protein that are likely to be involved in ligand binding and signalling. We have also examined the mRNA abundance levels of all identified bovine chemokine receptors in mononuclear phagocytic cells. Considerable differences were observed in the mRNA abundance levels of the receptors, and interestingly the identified novel chemokine receptor showed differing levels of mRNA abundance to its closest homolog CCR1. The chemokine receptor repertoire was shown to differ between monocytes, macrophages and dendritic cells. This may reflect the differing roles of these cells in the immune response and may have functional consequences for the trafficking of these cells in vivo. CONCLUSIONS: In summary, we have provided the first characterisation of the complete bovine chemokine receptor gene repertoire including a gene that is potentially unique to cattle. Further study of this receptor and its ligands may reveal a specific role of this receptor in cattle. The availability of the bovine chemokine receptor sequences will allow further characterisation of the function of these genes and will confer wide-reaching benefits to the study of this important aspect of the bovine immune response.

A role for notch signaling in stromal survival and differentiation during prostate development.

Notch1 signaling is involved in epithelial growth and differentiation of prostate epithelia, and we have examined the role that notch signaling plays in the stroma of the developing prostate. We initially observed expression of delta-like 1 (Dlk1) and Notch2 in gene profiling studies of prostatic mesenchyme, and anticipated that they might be expressed in a key subset of inductive mesenchyme. Using quantitative RT-PCR, Northern blotting, and whole mount in situ hybridization, we confirmed that both Dlk1 and Notch2 mRNAs showed a restricted expression pattern within subsets of the stroma during prostate development. Localization of Dlk1 and Notch2 proteins mirrored the transcript expression, and showed both distinct and overlapping expression patterns within the stroma. Dlk1 and Notch2 were coexpressed in condensed inductive mesenchyme of the ventral mesenchymal pad (VMP), and were partially colocalized in the smooth muscle (SM) layer of the urethral stroma. In addition, Dlk1 was not expressed in SM adjacent to the VMP in female urethra. The function of notch signaling was examined using organ cultures of prostate rudiments and a small molecule inhibitor of notch receptor activity. Inhibition of notch signaling led to a loss of stromal tissue in both prostate and female VMP cultures, suggesting that this pathway was required for stromal survival. Inhibition of notch signaling also led to changes in both epithelial and stromal differentiation, which was evident in altered distributions of SM alpha-actin and p63 in prostates grown in vitro. The effects of notch signaling upon the stroma were only evident in the presence of testosterone, in contrast to effects upon epithelial differentiation.

Transcriptional profiling of inductive mesenchyme to identify molecules involved in prostate development and disease.

BACKGROUND: The mesenchymal compartment plays a key role in organogenesis, and cells within the mesenchyme/stroma are a source of potent molecules that control epithelia during development and tumorigenesis. We used serial analysis of gene expression (SAGE) to profile a key subset of prostatic mesenchyme that regulates prostate development and is enriched for growth-regulatory molecules. RESULTS: SAGE libraries were constructed from prostatic inductive mesenchyme and from the complete prostatic rudiment (including inductive mesenchyme, epithelium, and smooth muscle). By comparing these two SAGE libraries, we generated a list of 219 transcripts that were enriched or specific to inductive mesenchyme and that may act as mesenchymal regulators of organogenesis and tumorigenesis. We identified Scube1 as enriched in inductive mesenchyme from the list of 219 transcripts; also, quantitative RT-PCR and whole-mount in situ hybridization revealed Scube1 to exhibit a highly restricted expression pattern. The expression of Scube1 in a subset of mesenchymal cells suggests a role in prostatic induction and branching morphogenesis. Additionally, Scube1 transcripts were expressed in prostate cancer stromal cells, and were less abundant in cancer associated fibroblasts relative to matched normal prostate fibroblasts. CONCLUSION: The use of a precisely defined subset of cells and a back-comparison approach allowed us to identify rare mRNAs that could be overlooked using other approaches. We propose that Scube1 encodes a novel stromal molecule that is involved in prostate development and tumorigenesis.

Characterisation of bovine inducible nitric oxide synthase.

Inducible nitric oxide (iNOS) is an enzyme that catalyzes the production of the reactive nitrogen intermediate nitric oxide (NO). NO is an important signalling molecule, released by numerous cells, that acts in many tissues to regulate a diverse range of physiological and biological processes, including neurotransmission, immune defence and the regulation of cell death (apoptosis). NO plays a major role in the killing of intracellular pathogens as part of the innate immune response. iNOS is known to be induced by a number of stimuli including cytokines as well as pathogens and their components. As yet, a full-length bovine iNOS sequence has only been predicted from the genome, although partial sequences from cDNA are available. Here, we have identified a 3471bp transcript for bovine iNOS, isolated from RNA from bovine alveolar macrophages stimulated with the intracellular pathogen Mycobacterium bovis. When translated this gives a protein of 1156 amino acids. Bovine iNOS shows a high degree of similarity to iNOS from other species, and also shares a common protein domain structure.