Epithelial damage and tissue γδ T cells promote a unique tumor-protective IgE response

IgE is an ancient and conserved immunoglobulin isotype with potent immunological function. Nevertheless, the regulation of IgE responses remains an enigma, and evidence of a role for IgE in host defense is limited. Here we report that topical exposure to a common environmental DNA-damaging xenobiotic initiated stress surveillance by γδTCR+ intraepithelial lymphocytes that resulted in class switching to IgE in B cells and the accumulation of autoreactive IgE. High-throughput antibody sequencing revealed that γδ T cells shaped the IgE repertoire by supporting specific variable-diversity-joining (VDJ) rearrangements with unique characteristics of the complementarity-determining region CDRH3. This endogenous IgE response, via the IgE receptor FcεRI, provided protection against epithelial carcinogenesis, and expression of the gene encoding FcεRI in human squamous-cell carcinoma correlated with good disease prognosis. These data indicate a joint role for immunosurveillance by T cells and by B cells in epithelial tissues and suggest that IgE is part of the host defense against epithelial damage and tumor development

Transcriptional bursts explain autosomal random monoallelic expression and affect allelic imbalance.

Transcriptional bursts render substantial biological noise in cellular transcriptomes. Here, we investigated the theoretical extent of allelic expression resulting from transcriptional bursting and how it compared to the amount biallelic, monoallelic and allele-biased expression observed in single-cell RNA-sequencing (scRNA-seq) data. We found that transcriptional bursting can explain the allelic expression patterns observed in single cells, including the frequent observations of autosomal monoallelic gene expression. Importantly, we identified that the burst frequency largely determined the fraction of cells with monoallelic expression, whereas the burst size had little effect on monoallelic observations. The high consistency between the bursting model predictions and scRNA-seq observations made it possible to assess the heterogeneity of a group of cells as their deviation in allelic observations from the expected. Finally, both burst frequency and size contributed to allelic imbalance observations and reinforced that studies of allelic imbalance can be confounded from the inherent noise in transcriptional bursting. Altogether, we demonstrate that allele-level transcriptional bursting renders widespread, although predictable, amounts of monoallelic and biallelic expression in single cells and cell populations

Molecular and spatial landmarks of early mouse skin development

A wealth of specialized cell populations within the skin facilitates its hair-producing, protective, sensory, and thermoregulatory functions. How the vast cell-type diversity and tissue architecture develops is largely unexplored. Here, with single-cell transcriptomics, spatial cell-type assignment, and cell-lineage tracing, we deconstruct early embryonic mouse skin during the key transitions from seemingly uniform developmental precursor states to a multilayered, multilineage epithelium, and complex dermal identity. We identify the spatiotemporal emergence of hair-follicle-inducing, muscle-supportive, and fascia-forming fibroblasts. We also demonstrate the formation of the panniculus carnosus muscle (PCM), sprouting blood vessels without pericyte coverage, and the earliest residence of mast and dendritic immune cells in skin. Finally, we identify an unexpected epithelial heterogeneity within the early single-layered epidermis and a signaling-rich periderm layer. Overall, this cellular and molecular blueprint of early skin development-which can be explored at https://kasperlab.org/tools-establishes histological landmarks and highlights unprecedented dynamic interactions among skin cells.Peer reviewe

FOXP3, MHC class II, age at presentation and rescue therapy are independent prognostic factors in canine B cell lymphoma.

<p><u>Abbreviations</u>: TTR, time to remission; PFS, progression-free survival; OS, overall survival; CI, confidence interval; FOXP3⁺ (% CD5⁺CD4⁺), frequency of FOXP3⁺ cells within the sequential CD5⁺ and CD4⁺ gates; FOXP3⁺ (%), frequency of FOXP3⁺ cells within the total harvested population; median ratio =  ratio of median remission or survival time (TTR/PFS/OS) of the sub-group of dogs with a value of the variable greater than the median of the group to that of the sub-group of dogs with a value of the variable less than or equal to the median of the group.</p

Staining antibodies, isotype controls and fluorochromes.

<p><u>Abbreviations</u>: FITC, fluorescein isothiocyanate; PE, phycoerythrin; APC, allophycocyanin.</p

Canine B cell lymphoma shows no deficit of cytotoxic T cells relative to thymic Tregs.

<p>When expressed as a proportion of all cells aspirated from the lymph node, the frequency of CD8⁺ T cells (A,B) was decreased in BCL (and MCT), again attributed to neoplastic effacement (p = 0.0005). The frequency of CD8⁺ cells within the CD5⁺ T cell population also showed differences between groups (C; p = 0.04). The ratio of CD8⁺:FOXP3⁺ cells (D) was not significantly different between groups (p = 0.06), though dogs with BCL showed the six highest ratios of the cohort, and a trend for higher values in the BCL group was observed. Indeed, the ratio of CD8⁺:FOXP3⁺Helios⁺ cells (E) was higher in the BCL than RH cases (p = 0.04); only two data points for the MCT (3.48; 3.95) were available for this variable and were not therefore represented. Representative dot plots for all the groups are shown alongside their respective isotype controls, followed by box-and-whisker plots summarising the data. (For key to symbols, refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105027#pone-0105027-g002" target="_blank">Figure 2</a>).</p

FOXP3⁺ Treg frequency is no higher in canine B cell lymphoma than control samples.

<p>The frequency of FOXP3⁺ cells in fine needle aspirates harvested from the lymph nodes of dogs with B cell lymphoma (BCL), T cell lymphoma (TCL), reactive hyperplasia (RH) and mast cell tumors (MCT) is shown, expressed as a proportion of all cells (A, B, showing the effect of neoplastic effacement of normal lymphoid tissue; p = 0.00005), gated CD5⁺ cells (C, showing the resident T cells; p = 0.41), or sequentially gated CD5⁺ and then CD4⁺ T cells (D, showing specifically the resident CD4⁺ T cells; p = 0.74). Representative dot plots are shown for each of the groups, followed by box-and-whisker plots summarising the data. In each of the dot plots, the respective isotype control stain is shown above the specific stain. In this and subsequent figures, red symbols correspond to data from dogs ‘pre-treated’ with corticosteroids or any chemotherapeutic drug within three weeks of sampling. In the case of RH cases, square symbols (□) represent data from dogs with dermatitis; diamonds (◊) represent data from dogs with distant neoplasia; triangles (Δ) represent data from dogs with immune-mediated disease; and inverted triangles (▾) represent data from dogs with systemic infection. The expression of FOXP3 by PBMCs (E; p = 0.21) or by the CD5⁺ (F; p = 0.53) or specific CD5⁺CD4⁺ (G; p = 0.15) T cells within PBMCs similarly showed no differences between groups.</p

Canine T cell lymphoma is associated with high-frequency Helios expression.

<p>Helios expression by cells harvested by fine needle aspiration from the lymph nodes of dogs with B cell lymphoma (BCL), T cell lymphoma (TCL), reactive hyperplasia (RH) and mast cell tumors (MCT) was examined as a marker for thymic Tregs, but the unexpectedly high frequency of Helios⁺ events in a few B cell and several T cell lymphoma cases suggested neoplastic expression of this marker. Median frequency of Helios expression was significantly different between the groups (p = 0.00003). Representative dot plots for BCL and TCL cases, with their respective isotype control stains, is shown (A), followed by box-and-whisker plots summarising the data (B). (For key to symbols, refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105027#pone-0105027-g002" target="_blank">Figure 2</a>).</p

Expression of T cell FOXP3 and Helios, and tumor cell MHC class II, all impact prognosis in dogs with B cell lymphoma.

<p>The frequency of FOXP3⁺ cells within the ‘All’ gate (A), or within a cascaded All→CD5⁺→CD4⁺ gate (B), influenced survival times. Overall survival times were shorter in those dogs with frequencies of FOXP3⁺ T cells higher than the median value (A: p = 0.02); a similar pattern in progression-free survival (PFS) was observed (B: p = 0.01). The frequency of Helios⁺ cells within the ‘All’ gate also influenced PFS (C: p = 0.01). The intensity of MHC class II expression by the lymphoma cells, expressed as the ratio of geometric mean fluorescence intensity of MHC II⁺:MHC II⁻ cells within the CD5⁻ subset of the ‘All’ gate, was also of prognostic significance: time to remission was longer in those cases with less MHC class II expression per cell (D: p = 0.02).</p

Clinical, cytological and treatment details of dogs in the study.

<p><u>Abbreviations</u>: Me, entire male; Mn, neutered male; Fe, entire female; Fn, neutered female; WHO, World Health Organization; N/A, not applicable; DLBCL, diffuse large B cell lymphoma; PTCL, peripheral T cell lymphoma; CHOP, cyclophosphamide, doxorubicin, vincristine, prednisolone protocol; COP, cyclophosphamide, vincristine, prednisolone protocol; Pred, prednisolone alone; NSAIDS, non-steroidal anti-inflammatory drugs.</p><p><u>Notes</u>: ¹: Age when presenting signs were first observed. ²: Body condition score was assessed by different clinicians on either a 5 or 9 point scale: ‘under-conditioned’ was defined by a score of 1–2/5 or 1–3/9; ‘optimal’ was defined by a score of 2.5–3/5 or 4–5/9; while ‘over-conditioned’ was defined by a score of 3.5–5/5 or 6–9/9. ³: Serum or plasma calcium concentration was not measured in one of the T cell lymphoma cases prior to initiation of therapy. ⁴: The initial treatment protocol is listed. ‘Other’ treatments included cytarabine, L-asparaginase and lomustine (B cell lymphoma) and L-asparaginase, lomustine, prednisolone, masitinib and chlorambucil (T cell lymphoma).</p