IFNγ-Dependent Tissue-Immune Homeostasis Is Co-opted in the Tumor Microenvironment

Homeostatic programs balance immune protection and self-tolerance. Such mechanisms likely impact autoimmunity and tumor formation, respectively. How homeostasis is maintained and impacts tumor surveillance is unknown. Here, we find that different immune mononuclear phagocytes share a conserved steady-state program during differentiation and entry into healthy tissue. IFNγ is necessary and sufficient to induce this program, revealing a key instructive role. Remarkably, homeostatic and IFNγ-dependent programs enrich across primary human tumors, including melanoma, and stratify survival. Single-cell RNA sequencing (RNA-seq) reveals enrichment of homeostatic modules in monocytes and DCs from human metastatic melanoma. Suppressor-of-cytokine-2 (SOCS2) protein, a conserved program transcript, is expressed by mononuclear phagocytes infiltrating primary melanoma and is induced by IFNγ. SOCS2 limits adaptive anti-tumoral immunity and DC-based priming of T cells in vivo, indicating a critical regulatory role. These findings link immune homeostasis to key determinants of anti-tumoral immunity and escape, revealing co-opting of tissue-specific immune development in the tumor microenvironment. Keywords: dendritic cells; homeostasis; differentiation; IFNγ; tumor microenvironment; melanoma tolerance; immunotherapy; suppressor-of-cytokine-signaling 2 (SOCS2); tissue mononuclear phagocyte

Linking single-cell measurements of mass, growth rate, and gene expression

Mass and growth rate are highly integrative measures of cell physiology not discernable via genomic measurements. Here, we introduce a microfluidic platform enabling direct measurement of single-cell mass and growth rate upstream of highly multiplexed single-cell profiling such as single-cell RNA sequencing. We resolve transcriptional signatures associated with single-cell mass and growth rate in L1210 and FL5.12 cell lines and activated CD8+ T cells. Further, we demonstrate a framework using these linked measurements to characterize biophysical heterogeneity in a patient-derived glioblastoma cell line with and without drug treatment. Our results highlight the value of coupled phenotypic metrics in guiding single-cell genomics.status: publishe

Linking single-cell measurements of mass, growth rate, and gene expression

Mass and growth rate are highly integrative measures of cell physiology not discernable via genomic measurements. Here, we introduce a microfluidic platform enabling direct measurement of single-cell mass and growth rate upstream of highly multiplexed single-cell profiling such as single-cell RNA sequencing. We resolve transcriptional signatures associated with single-cell mass and growth rate in L1210 and FL5.12 cell lines and activated CD8+ T cells. Further, we demonstrate a framework using these linked measurements to characterize biophysical heterogeneity in a patient-derived glioblastoma cell line with and without drug treatment. Our results highlight the value of coupled phenotypic metrics in guiding single-cell genomics. Keywords: Single-cell RNA-Seq, Mass, Growth, Serial suspended microchannel resonator, Multi-omics, Single cell, T cell activation, Glioblastoma, GBM, Drug response, Microfluidics, Biophysical propertie

MR1-Restricted MAIT Cells From The Human Lung Mucosal Surface Have Distinct Phenotypic, Functional, and Transcriptomic Features That Are Preserved in HIV Infection

Mucosal associated invariant T (MAIT) cells are a class of innate-like T cells that utilize a semi-invariant αβ T cell receptor to recognize small molecule ligands produced by bacteria and fungi. Despite growing evidence that immune cells at mucosal surfaces are often phenotypically and functionally distinct from those in the peripheral circulation, knowledge about the characteristics of MAIT cells at the lung mucosal surface, the site of exposure to respiratory pathogens, is limited. HIV infection has been shown to have a profound effect on the number and function of MAIT cells in the peripheral blood, but its effect on lung mucosal MAIT cells is unknown. We examined the phenotypic, functional, and transcriptomic features of major histocompatibility complex (MHC) class I-related (MR1)-restricted MAIT cells from the peripheral blood and bronchoalveolar compartments of otherwise healthy individuals with latent Mycobacterium tuberculosis (Mtb) infection who were either HIV uninfected or HIV infected. Peripheral blood MAIT cells consistently co-expressed typical MAIT cell surface markers CD161 and CD26 in HIV-negative individuals, while paired bronchoalveolar MAIT cells displayed heterogenous expression of these markers. Bronchoalveolar MAIT cells produced lower levels of pro-inflammatory cytokine IFN-γ and expressed higher levels of co-inhibitory markers PD-1 and TIM-3 than peripheral MAIT cells. HIV infection resulted in decreased frequencies and pro-inflammatory function of peripheral blood MAIT cells, while in the bronchoalveolar compartment MAIT cell frequency was decreased but phenotype and function were not significantly altered. Single-cell transcriptomic analysis demonstrated greater heterogeneity among bronchoalveolar compared to peripheral blood MAIT cells and suggested a distinct subset in the bronchoalveolar compartment. The transcriptional features of this bronchoalveolar subset were associated with MAIT cell tissue repair functions. In summary, we found previously undescribed phenotypic and transcriptional heterogeneity of bronchoalveolar MAIT cells in HIV-negative people. In HIV infection, we found numeric depletion of MAIT cells in both anatomical compartments but preservation of the novel phenotypic and transcriptional features of bronchoalveolar MAIT cells.</jats:p

L'identité professionnelle des enseignants en formation professionnelle ayant débuté à enseigner après plusieurs années d'expérience dans un métier : portrait d'un processus

Le nouveau contexte québécois en éducation a mis de l'avant la professionnalisation du métier de l'enseignant. Les enseignants de la formation professionnelle (F.P.) n'y ont pas échappé et, dorénavant, ils doivent agir et être considérés comme des enseignants à part entière tels que ceux du régulier, soit des professionnels de l'enseignement. Or, le processus de professionnalisation d'un individu est très complexe et passe par la réussite de plusieurs facteurs. L'un d'eux est qu'il réussisse à développer son identité face à la profession. Pour l'enseignant, il est primordial qu'il arrive à se représenter la profession enseignante et lui-même comme enseignant. Tout cela a déjà été largement discuté par plusieurs chercheurs dont Zeichner et Gore (1990), Nault (1993), Mukamurera et Uwamariya (2005) et Riopel (2006) pour ne nommer que ceux-là. Ainsi, la réussite du processus de professionnalisation passe par une insertion réussie dans cette même profession. Cependant, comme le souligne Balleux (2006), la plupart des enseignants ?uvrant en formation professionnelle entrent dans leur nouvelle carrière d'enseignant après une vie professionnelle généralement bien entamée. Aussi, ils commencent rarement leur carrière suite à une formation universitaire. Il y a donc une différence marquée entre les enseignants du régulier et ceux de la formation professionnelle au niveau du processus à l'intérieur duquel ils développent leur identité enseignante

Additional file 11: Figure S10. of Multiplexed, targeted profiling of single-cell proteomes and transcriptomes in a single reaction

Change in average, standard deviation, and frequency of expression. Density traces (each with their own arbitrary units) for change in mean, standard deviation, and frequency of expression are shown for genes quantified as both RNA (a) and protein (b). Each row depicts a time point transition (24 – 0 hr, 48 – 24 hr, 48 – 0 hr for the top, middle, and bottom rows, respectively) for every gene with at least two cells above detection in every time point for both RNA and protein (19 genes). The ticks display individual measurements from each time point transition. (PDF 508 kb

Additional file 8: Figure S7. of Multiplexed, targeted profiling of single-cell proteomes and transcriptomes in a single reaction

Standard RNA probe curves using ERCC Spike-Ins. Two-fold dilutions of ERCC Spike-Ins were backloaded into the C1 IFC and processed according to the PEA/STA protocol. Shown here are the STA measurements with the y-axis values representing ∆Ct values from only lysis buffer or a threshold value of 24 if undetected in lysis buffer alone. Plots are ordered by decreasing concentration in the ERCC mix with bad fits arising around ERCC 14 (which corresponds to ~121 molecules loaded into the top dilution, Additional file 1: Table S10 and S11). Each data point plotted is the average of eight separate capture sites in the C1 IFC with error bars showing the standard error of the mean. Gray (green) dashes show the level above which the probability for a detection event being real is p = 0.01 (0.05). Points used for fitting the red trend line are colored blue. (PDF 1135 kb

Optofluidic real-time cell sorter for longitudinal CTC studies in mouse models of cancer

Circulating tumor cells (CTCs) play a fundamental role in cancer progression. However, in mice, limited blood volume and the rarity of CTCs in the bloodstream preclude longitudinal, in-depth studies of these cells using existing liquid biopsy techniques. Here, we present an optofluidic system that continuously collects fluorescently labeled CTCs from a genetically engineered mouse model (GEMM) for several hours per day over multiple days or weeks. The system is based on a microfluidic cell sorting chip connected serially to an unanesthetized mouse via an implanted arteriovenous shunt. Pneumatically controlled microfluidic valves capture CTCs as they flow through the device, and CTC-depleted blood is returned back to the mouse via the shunt. To demonstrate the utility of our system, we profile CTCs isolated longitudinally from animals over 4 days of treatment with the BET inhibitor JQ1 using single-cell RNA sequencing (scRNA-Seq) and show that our approach eliminates potential biases driven by intermouse heterogeneity that can occur when CTCs are collected across different mice. The CTC isolation and sorting technology presented here provides a research tool to help reveal details of how CTCs evolve over time, allowing studies to credential changes in CTCs as biomarkers of drug response and facilitating future studies to understand the role of CTCs in metastasis.National Institutes of Health (Grant 1R01-CA184956, Grant 5U24AI118672, Grant 1U54CA217377, Grant 1R33CA202820, Grant 2U19AI089992, Grant 1R01HL134539, Grant 2RM1HG006193 and Grant 2P01AI039671)National Institutes of Health (Award 1DP2GM119419)National Cancer Institute (Grant P30-CA14051