Mendelian segregation and high recombination rates facilitate genetic analyses in Cryptosporidium parvum.

Very little is known about the process of meiosis in the apicomplexan parasite Cryptosporidium despite the essentiality of sex in its life cycle. Most cell lines only support asexual growth of Cryptosporidium parvum (C. parvum), but stem cell derived intestinal epithelial cells grown under air-liquid interface (ALI) conditions support the sexual cycle. To examine chromosomal dynamics during meiosis in C. parvum, we generated two transgenic lines of parasites that were fluorescently tagged with mCherry or GFP on chromosomes 1 or 5, respectively. Infection of ALI cultures or Ifngr1-/- mice with mCherry and GFP parasites resulted in cross-fertilization and the formation of "yellow" oocysts, which contain 4 haploid sporozoites that are the product of meiosis. Recombinant oocysts from the F1 generation were purified and used to infect HCT-8 cultures, and phenotypes of the progeny were observed by microscopy. All possible phenotypes predicted by independent segregation were represented equally (~25%) in the population, indicating that C. parvum chromosomes exhibit a Mendelian inheritance pattern. The most common pattern observed from the outgrowth of single oocysts included all possible parental and recombinant phenotypes derived from a single meiotic event, suggesting a high rate of crossover. To estimate the frequency of crossover, additional loci on chromosomes 1 and 5 were tagged and used to monitor intrachromosomal crosses in Ifngr1-/- mice. Both chromosomes showed a high frequency of crossover compared to other apicomplexans with map distances (i.e., 1% recombination) of 3-12 kb. Overall, a high recombination rate may explain many unique characteristics observed in Cryptosporidium spp. such as high rates of speciation, wide variation in host range, and rapid evolution of host-specific virulence factors.

Mendelian segregation and high recombination rates facilitate genetic analyses in Cryptosporidium parvum.

Very little is known about the process of meiosis in the apicomplexan parasite Cryptosporidium despite the essentiality of sex in its life cycle. Most cell lines only support asexual growth of Cryptosporidium parvum (C. parvum), but stem cell derived intestinal epithelial cells grown under air-liquid interface (ALI) conditions support the sexual cycle. To examine chromosomal dynamics during meiosis in C. parvum, we generated two transgenic lines of parasites that were fluorescently tagged with mCherry or GFP on chromosomes 1 or 5, respectively. Infection of ALI cultures or Ifngr1-/- mice with mCherry and GFP parasites produced "yellow" oocysts generated by cross-fertilization. Outcrossed oocysts from the F1 generation were purified and used to infect HCT-8 cultures, and phenotypes of the progeny were observed by microscopy. All possible phenotypes predicted by independent segregation were represented equally (~25%) in the population, indicating that C. parvum chromosomes exhibit a Mendelian inheritance pattern. Unexpectedly, the most common pattern observed from the outgrowth of single oocysts included all possible parental and recombinant phenotypes derived from a single meiotic event, suggesting a high rate of crossover. To estimate the frequency of crossover, additional loci on chromosomes 1 and 5 were tagged and used to monitor intrachromosomal crosses in Ifngr1-/- mice. Both chromosomes showed a high frequency of crossover compared to other apicomplexans with map distances (i.e., 1% recombination) of 3-12 kb. Overall, a high recombination rate may explain many unique characteristics observed in Cryptosporidium spp. such as high rates of speciation, wide variation in host range, and rapid evolution of host-specific virulence factors.

Cancer cell-intrinsic expression of MHC II in lung cancer cell lines is actively restricted by MEK/ERK signaling and epigenetic mechanisms.

BACKGROUND: Programmed death 1/programmed death ligand 1 (PD-1/PD-L1) targeted immunotherapy affords clinical benefit in ~20% of unselected patients with lung cancer. The factor(s) that determine whether a tumor responds or fails to respond to immunotherapy remains an active area of investigation. We have previously defined divergent responsiveness of two KRAS-mutant cell lines to PD-1/PD-L1 blockade using an orthotopic, immunocompetent mouse model. Responsiveness to PD-1/PD-L1 checkpoint blockade correlates with an interferon gamma (IFNγ)-inducible gene signature and major histocompatibility complex class II (MHC II) expression by cancer cells. In the current study, we aim to identify therapeutic targets that can be manipulated in order to enhance cancer-cell-specific MHC II expression. METHODS: Responsiveness to IFNγ and induction of MHC II expression was assessed after various treatment conditions in mouse and human non-small cell lung cancer (NSCLC) cell lines using mass cytometric and flow cytometric analysis. RESULTS: Single-cell analysis using mass and flow cytometry demonstrated that IFNγ consistently induced PD-L1 and MHC class I (MHC I) across multiple murine and human NSCLC cell lines. In contrast, MHC II showed highly variable induction following IFNγ treatment both between lines and within lines. In mouse models of NSCLC, MHC II induction was inversely correlated with basal levels of phosphorylated extracellular signal-regulated kinase (ERK) 1/2, suggesting potential mitogen-activated protein (MAP) kinase-dependent antagonism of MHC II expression. To test this, cell lines were subjected to varying levels of stimulation with IFNγ, and assessed for MHC II expression in the presence or absence of mitogen-activated protein kinase kinase (MEK) inhibitors. IFNγ treatment in the presence of MEK inhibitors significantly enhanced MHC II induction across multiple lung cancer lines, with minimal impact on expression of either PD-L1 or MHC I. Inhibition of histone deacetylases (HDACs) also enhanced MHC II expression to a more modest extent. Combined MEK and HDAC inhibition led to greater MHC II expression than either treatment alone. CONCLUSIONS: These studies emphasize the active inhibitory role that epigenetic and ERK signaling cascades have in restricting cancer cell-intrinsic MHC II expression in NSCLC, and suggest that combinatorial blockade of these pathways may engender new responsiveness to checkpoint therapies.

High-Dimensional Analysis of Postsplenectomy Peripheral Immune Cell Changes.

Although the consequences of splenectomy are well understood in mice, much less is known about the immunologic changes that occur following splenectomy in humans. We sought to characterize the circulating immune cell populations of patients before and after elective splenectomy to determine if these changes are related to postsplenectomy survival outcomes. Retrospective clinical information was collected from 95 patients undergoing elective splenectomy compared with 91 patients undergoing pancreaticoduodenectomy (Whipple procedure). We further analyzed peripheral blood from five patients in the splenectomy group, collected before and after surgery, using single-cell cytometry by time-of-flight mass spectrometry. We compared pre- and postsplenectomy data to characterize both the major and minor immune cell populations in significantly greater detail. Compared with patients undergoing a Whipple procedure, splenectomized patients had significant and long-lasting elevated counts of lymphocytes, monocytes, and basophils. Cytometry by time-of-flight mass spectroscopy analysis demonstrated that the elevated lymphocytes primarily consisted of naive CD4+ T cells and a population of activated CD25+CD56+CD4+ T cells, whereas the elevated monocyte counts were mainly mature, activated monocytes. We also observed a significant increase in the expression of the chemokine receptors CCR6 and CCR4 on several cellular populations. Taken together, these data indicate that significant immunological changes take place following splenectomy. Whereas other groups have compared splenectomized patients to healthy controls, this study compared patients undergoing elective splenectomy to those undergoing a similar major abdominal surgery. Overall, we found that splenectomy results in significant long-lasting changes in circulating immune cell populations and function.

Sphingosine-1-Phosphate Lyase Inhibition Alters the S1P Gradient and Ameliorates Crohn's-Like Ileitis by Suppressing Thymocyte Maturation.

BACKGROUND: Lymphocytes recirculate from tissues to blood following the sphingosine-1-phosphate (S1P) gradient (low in tissues, high in blood), maintained by synthetic and degradative enzymes, among which the S1P lyase (SPL) irreversibly degrades S1P. The role of SPL in the intestine, both during homeostasis and IBD, is poorly understood. We hypothesized that modulation of tissue S1P levels might be advantageous over S1P receptor (S1PR) agonists (eg, fingolimod, ozanimod, etrasimod), as without S1PR engagement there might be less likelihood of potential off-target effects. METHODS: First we examined SPL mRNA transcripts and SPL localization in tissues by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. The in vivo effects of the SPL inhibitors 4-deoxypyridoxine hydrochloride (30 mg/L) and 2-acetyl-4 (tetrahydroxybutyl)imidazole (50 mg/L) were assessed through their oral administration to adult TNF∆ARE mice, which spontaneously develop Crohn's-like chronic ileitis. The effect of SPL inhibition on circulating and tissue lymphocytes, transcriptional regulation of proinflammatory cytokines, and on the histological severity of ileitis was additionally examined. Tissue S1P levels were determined by liquid chromatography-mass spectrometry. Mechanistically, the potential effects of high S1P tissue levels on intestinal leukocyte apoptosis were assessed via terminal deoxynucleotidyl transferase dUTP nick end-labeling assay and annexin 5 staining. Finally, we examined the ability of T cells to home to the intestine, along with the effects of SPL inhibition on cellular subsets within immune compartments via flow and mass cytometry. RESULTS: S1P lyase was ubiquitously expressed. In the gut, immunohistochemistry predominantly localized it to small intestinal epithelia, although the lamina propria leukocyte fraction had higher mRNA transcripts. Inhibition of SPL markedly increased local intestinal S1P levels, induced peripheral lymphopenia, downregulated proinflammatory cytokines, and attenuated chronic ileitis in mice. SPL inhibition reduced T and myeloid cells in secondary lymphoid tissues and the intestine and decreased naïve T-cell recruitment. The anti-inflammatory activity of SPL inhibition was not mediated by leukocyte apoptosis, nor by interference with the homing of lymphocytes to the intestine, and was independent of its peripheral lymphopenic effect. However, SPL inhibition promoted thymic atrophy and depleted late immature T cells (CD4+CD8+ double positive), with accumulation of mature CD4+CD8- and CD4-CD8+ single-positive cells. CONCLUSIONS: Inhibition of the S1P lyase alters the S1P gradient and attenuates chronic ileitis via central immunosuppression. SPL inhibition could represent a potential way to tame an overactive immune response during IBD and other T-cell-mediated chronic inflammatory diseases.

High-Dimensional Characterization of IL-10 Production and IL-10-Dependent Regulation during Primary Gammaherpesvirus Infection.

IL-10 is a potent immunomodulatory cytokine produced by multiple cell types to restrain immune activation. Many herpesviruses use the IL-10 pathway to facilitate infection, but how endogenous IL-10 is regulated during primary infection in vivo remains poorly characterized. In this study, we infected mice with murine gammaherpesvirus 68 (γHV68) and analyzed the production and genetic contribution of IL-10 by mass cytometry (cytometry by time-of-flight) analysis. γHV68 infection elicited a breadth of effector CD4 T cells in the lungs of acutely infected mice, including a highly activated effector subset that coexpressed IFN-γ, TNF-α, and IL-10. By using IL-10 GFP transcriptional reporter mice, we identified that IL-10 was primarily expressed within CD4 T cells during acute infection in the lungs. IL10gfp-expressing CD4 T cells were highly proliferative and characterized by the expression of multiple coinhibitory receptors, including PD-1 and LAG-3. When we analyzed acute γHV68 infection of IL-10-deficient mice, we found that IL-10 limits the frequency of both myeloid and effector CD4 T cell subsets in the infected lung, with minimal changes at a distant mucosal site. These data emphasize the unique insights that high-dimensional analysis can afford in investigating antiviral immunity and provide new insights into the breadth, phenotype, and function of IL-10-expressing effector CD4 T cells during acute virus infection.

Multidimensional analysis of Gammaherpesvirus RNA expression reveals unexpected heterogeneity of gene expression.

Virus-host interactions are frequently studied in bulk cell populations, obscuring cell-to-cell variation. Here we investigate endogenous herpesvirus gene expression at the single-cell level, combining a sensitive and robust fluorescent in situ hybridization platform with multiparameter flow cytometry, to study the expression of gammaherpesvirus non-coding RNAs (ncRNAs) during lytic replication, latent infection and reactivation in vitro. This method allowed robust detection of viral ncRNAs of murine gammaherpesvirus 68 (γHV68), Kaposi's sarcoma associated herpesvirus and Epstein-Barr virus, revealing variable expression at the single-cell level. By quantifying the inter-relationship of viral ncRNA, viral mRNA, viral protein and host mRNA regulation during γHV68 infection, we find heterogeneous and asynchronous gene expression during latency and reactivation, with reactivation from latency identified by a distinct gene expression profile within rare cells. Further, during lytic replication with γHV68, we find many cells have limited viral gene expression, with only a fraction of cells showing robust gene expression, dynamic RNA localization, and progressive infection. Lytic viral gene expression was enhanced in primary fibroblasts and by conditions associated with enhanced viral replication, with multiple subpopulations of cells present in even highly permissive infection conditions. These findings, powered by single-cell analysis integrated with automated clustering algorithms, suggest inefficient or abortive γHV infection in many cells, and identify substantial heterogeneity in viral gene expression at the single-cell level.

Tumor-intrinsic response to IFNγ shapes the tumor microenvironment and anti-PD-1 response in NSCLC.

Targeting PD-1/PD-L1 is only effective in ∼20% of lung cancer patients, but determinants of this response are poorly defined. We previously observed differential responses of two murine K-Ras-mutant lung cancer cell lines to anti-PD-1 therapy: CMT167 tumors were eliminated, whereas Lewis Lung Carcinoma (LLC) tumors were resistant. The goal of this study was to define mechanism(s) mediating this difference. RNA sequencing analysis of cancer cells recovered from lung tumors revealed that CMT167 cells induced an IFNγ signature that was blunted in LLC cells. Silencing Ifngr1 in CMT167 resulted in tumors resistant to IFNγ and anti-PD-1 therapy. Conversely, LLC cells had high basal expression of SOCS1, an inhibitor of IFNγ. Silencing Socs1 increased response to IFNγ in vitro and sensitized tumors to anti-PD-1. This was associated with a reshaped tumor microenvironment, characterized by enhanced T cell infiltration and enrichment of PD-L1hi myeloid cells. These studies demonstrate that targeted enhancement of tumor-intrinsic IFNγ signaling can induce a cascade of changes associated with increased therapeutic vulnerability.

Targeting DDR2 enhances tumor response to anti-PD-1 immunotherapy.

While a fraction of cancer patients treated with anti-PD-1 show durable therapeutic responses, most remain unresponsive, highlighting the need to better understand and improve these therapies. Using an in vivo screening approach with a customized shRNA pooled library, we identified DDR2 as a leading target for the enhancement of response to anti-PD-1 immunotherapy. Using isogenic in vivo murine models across five different tumor histologies-bladder, breast, colon, sarcoma, and melanoma-we show that DDR2 depletion increases sensitivity to anti-PD-1 treatment compared to monotherapy. Combination treatment of tumor-bearing mice with anti-PD-1 and dasatinib, a tyrosine kinase inhibitor of DDR2, led to tumor load reduction. RNA-seq and CyTOF analysis revealed higher CD8+ T cell populations in tumors with DDR2 depletion and those treated with dasatinib when either was combined with anti-PD-1 treatment. Our work provides strong scientific rationale for targeting DDR2 in combination with PD-1 inhibitors.

Host-associated bacterial community succession during amphibian development.

Amphibians undergo significant developmental changes during their life cycle, as they typically move from a primarily aquatic environment to a more terrestrial one. Amphibian skin is a mucosal tissue that assembles communities of symbiotic microbiota. However, it is currently not well understood as to where amphibians acquire their skin symbionts, and whether the sources of microbial symbionts change throughout development. In this study, we utilized data collected from four wild boreal toad populations (Anaxyrus boreas); specifically, we sampled the skin bacterial communities during toad development, including eggs, tadpoles, subadults and adults as well as environmental sources of bacteria (water, aquatic sediment and soil). Using 16S rRNA marker gene profiling coupled with SourceTracker, we show that while primary environmental sources remained constant throughout the life cycle, secondary sources of boreal toad symbionts significantly changed with development. We found that toad skin communities changed predictably across development and that two developmental disturbance events (egg hatching and metamorphosis) dictated major changes. Toad skin communities assembled to alternative stable states following each of these developmental disturbances. Using the predicted average rRNA operon copy number of the communities at each life stage, we showed how the skin bacterial communities undergo a successional pattern whereby "fast-growing" (copiotroph) generalist bacteria dominate first before "slow-growing" (oligotroph) specialized bacteria take over. Our study highlights how host-associated bacterial community assembly is tightly coupled to host development and that host-associated communities demonstrate successional patterns akin to those observed in free-living bacteria as well as macrofaunal communities.

A Beginner's Guide to Analyzing and Visualizing Mass Cytometry Data.

Mass cytometry has revolutionized the study of cellular and phenotypic diversity, significantly expanding the number of phenotypic and functional characteristics that can be measured at the single-cell level. This high-dimensional analysis platform has necessitated the development of new data analysis approaches. Many of these algorithms circumvent traditional approaches used in flow cytometric analysis, fundamentally changing the way these data are analyzed and interpreted. For the beginner, however, the large number of algorithms that have been developed, as well as the lack of consensus on best practices for analyzing these data, raise multiple questions: Which algorithm is the best for analyzing a dataset? How do different algorithms compare? How can one move beyond data visualization to gain new biological insights? In this article, we describe our experiences as recent adopters of mass cytometry. By analyzing a single dataset using five cytometry by time-of-flight analysis platforms (viSNE, SPADE, X-shift, PhenoGraph, and Citrus), we identify important considerations and challenges that users should be aware of when using these different methods and common and unique insights that can be revealed by these different methods. By providing annotated workflow and figures, these analyses present a practical guide for investigators analyzing high-dimensional datasets. In total, these analyses emphasize the benefits of integrating multiple cytometry by time-of-flight analysis algorithms to gain complementary insights into these high-dimensional datasets.

Follicular regulatory T cells impair follicular T helper cells in HIV and SIV infection.

Human and simian immunodeficiency viruses (HIV and SIV) exploit follicular lymphoid regions by establishing high levels of viral replication and dysregulating humoral immunity. Follicular regulatory T cells (TFR) are a recently characterized subset of lymphocytes that influence the germinal centre response through interactions with follicular helper T cells (TFH). Here, utilizing both human and rhesus macaque models, we show the impact of HIV and SIV infection on TFR number and function. We find that TFR proportionately and numerically expand during infection through mechanisms involving viral entry and replication, TGF-ß signalling, low apoptosis rates and the presence of regulatory dendritic cells. Further, TFR exhibit elevated regulatory phenotypes and impair TFH functions during HIV infection. Thus, TFR contribute to inefficient germinal centre responses and inhibit HIV and SIV clearance.