JAK-STAT signaling maintains homeostasis in T cells and macrophages.

Immune cells need to sustain a state of constant alertness over a lifetime. Yet, little is known about the regulatory processes that control the fluent and fragile balance that is called homeostasis. Here we demonstrate that JAK-STAT signaling, beyond its role in immune responses, is a major regulator of immune cell homeostasis. We investigated JAK-STAT-mediated transcription and chromatin accessibility across 12 mouse models, including knockouts of all STAT transcription factors and of the TYK2 kinase. Baseline JAK-STAT signaling was detected in CD8+ T cells and macrophages of unperturbed mice-but abrogated in the knockouts and in unstimulated immune cells deprived of their normal tissue context. We observed diverse gene-regulatory programs, including effects of STAT2 and IRF9 that were independent of STAT1. In summary, our large-scale dataset and integrative analysis of JAK-STAT mutant and wild-type mice uncovered a crucial role of JAK-STAT signaling in unstimulated immune cells, where it contributes to a poised epigenetic and transcriptional state and helps prepare these cells for rapid response to immune stimuli.

Fibroblasts are a site of murine cytomegalovirus lytic replication and Stat1-dependent latent persistence in vivo.

To date, no herpesvirus has been shown to latently persist in fibroblastic cells. Here, we show that murine cytomegalovirus, a ß-herpesvirus, persists for the long term and across organs in PDGFRα-positive fibroblastic cells, with similar or higher genome loads than in the previously known sites of murine cytomegalovirus latency. Whereas murine cytomegalovirus gene transcription in PDGFRα-positive fibroblastic cells is almost completely silenced at 5 months post-infection, these cells give rise to reactivated virus ex vivo, arguing that they support latent murine cytomegalovirus infection. Notably, PDGFRα-positive fibroblastic cells also support productive virus replication during primary murine cytomegalovirus infection. Mechanistically, Stat1-deficiency promotes lytic infection but abolishes latent persistence of murine cytomegalovirus in PDGFRα-positive fibroblastic cells in vivo. In sum, fibroblastic cells have a dual role as a site of lytic murine cytomegalovirus replication and a reservoir of latent murine cytomegalovirus in vivo and STAT1 is required for murine cytomegalovirus latent persistence in vivo.

The small and large intestine contain related mesenchymal subsets that derive from embryonic Gli1+ precursors.

The intestinal lamina propria contains a diverse network of fibroblasts that provide key support functions to cells within their local environment. Despite this, our understanding of the diversity, location and ontogeny of fibroblasts within and along the length of the intestine remains incomplete. Here we show that the small and large intestinal lamina propria contain similar fibroblast subsets that locate in specific anatomical niches. Nevertheless, we find that the transcriptional profile of similar fibroblast subsets differs markedly between the small intestine and colon suggesting region specific functions. We perform in vivo transplantation and lineage-tracing experiments to demonstrate that adult intestinal fibroblast subsets, smooth muscle cells and pericytes derive from Gli1-expressing precursors present in embryonic day 12.5 intestine. Trajectory analysis of single cell RNA-seq datasets of E12.5 and adult mesenchymal cells suggest that adult smooth muscle cells and fibroblasts derive from distinct embryonic intermediates and that adult fibroblast subsets develop in a linear trajectory from CD81+ fibroblasts. Finally, we provide evidence that colonic subepithelial PDGFRαhi fibroblasts comprise several functionally distinct populations that originate from an Fgfr2-expressing fibroblast intermediate. Our results provide insights into intestinal stromal cell diversity, location, function, and ontogeny, with implications for intestinal development and homeostasis.

Postnatal expansion of mesenteric lymph node stromal cells towards reticular and CD34+ stromal cell subsets.

Gut-draining mesenteric lymph nodes (LN) provide the framework to shape intestinal adaptive immune responses. Based on the transcriptional signatures established by our previous work, the composition and immunomodulatory function of LN stromal cells (SC) vary according to location. Here, we describe the single-cell composition and development of the SC compartment within mesenteric LNs derived from postnatal to aged mice. We identify CD34+ SC and fibroblastic reticular stromal cell (FRC) progenitors as putative progenitors, both supplying the typical rapid postnatal mesenteric LN expansion. We further establish the location-specific chromatin accessibility and DNA methylation landscape of non-endothelial SCs and identify a microbiota-independent core epigenomic signature, showing characteristic differences between SCs from mesenteric and skin-draining peripheral LNs. The epigenomic landscape of SCs points to dynamic expression of Irf3 along the differentiation trajectories of FRCs. Accordingly, a mesenchymal stem cell line acquires a Cxcl9+ FRC molecular phenotype upon lentiviral overexpression of Irf3, and the relevance of Irf3 for SC biology is further underscored by the diminished proportion of Ccl19+ and Cxcl9+ FRCs in LNs of Irf3-/- mice. Together, our data constitute a comprehensive transcriptional and epigenomic map of mesenteric LNSC development in early life and dissect location-specific, microbiota-independent properties of non-endothelial SCs.

Single-cell transcriptional profiling of splenic fibroblasts reveals subset-specific innate immune signatures in homeostasis and during viral infection.

Our understanding of the composition and functions of splenic stromal cells remains incomplete. Here, based on analysis of over 20,000 single cell transcriptomes of splenic fibroblasts, we characterized the phenotypic and functional heterogeneity of these cells in healthy state and during virus infection. We describe eleven transcriptionally distinct fibroblastic cell clusters, reassuring known subsets and revealing yet unascertained heterogeneity amongst fibroblasts occupying diverse splenic niches. We further identify striking differences in innate immune signatures of distinct stromal compartments in vivo. Compared to other fibroblasts and to endothelial cells, Ly6C+ fibroblasts of the red pulp were selectively endowed with enhanced interferon-stimulated gene expression in homeostasis, upon systemic interferon stimulation and during virus infection in vivo. Collectively, we provide an updated map of fibroblastic cell diversity in the spleen that suggests a specialized innate immune function for splenic red pulp fibroblasts.

Cytomegalovirus inhibition of extrinsic apoptosis determines fitness and resistance to cytotoxic CD8 T cells.

Viral immune evasion is currently understood to focus on deflecting CD8 T cell recognition of infected cells by disrupting antigen presentation pathways. We evaluated viral interference with the ultimate step in cytotoxic T cell function, the death of infected cells. The viral inhibitor of caspase-8 activation (vICA) conserved in human cytomegalovirus (HCMV) and murine CMV (MCMV) prevents the activation of caspase-8 and proapoptotic signaling. We demonstrate the key role of vICA from either virus, in deflecting antigen-specific CD8 T cell-killing of infected cells. vICA-deficient mutants, lacking either UL36 or M36, exhibit greater susceptibility to CD8 T cell control than mutants lacking the set of immunoevasins known to disrupt antigen presentation via MHC class I. This difference is evident during infection in the natural mouse host infected with MCMV, in settings where virus-specific CD8 T cells are adoptively transferred. Finally, we identify the molecular mechanism through which vICA acts, demonstrating the central contribution of caspase-8 signaling at a point of convergence of death receptor-induced apoptosis and perforin/granzyme-dependent cytotoxicity.

Life-long control of cytomegalovirus (CMV) by T resident memory cells in the adipose tissue results in inflammation and hyperglycemia.

Cytomegalovirus (CMV) is a ubiquitous herpesvirus infecting most of the world's population. CMV has been rigorously investigated for its impact on lifelong immunity and potential complications arising from lifelong infection. A rigorous adaptive immune response mounts during progression of CMV infection from acute to latent states. CD8 T cells, in large part, drive this response and have very clearly been demonstrated to take up residence in the salivary gland and lungs of infected mice during latency. However, the role of tissue resident CD8 T cells as an ongoing defense mechanism against CMV has not been studied in other anatomical locations. Therefore, we sought to identify additional locations of anti-CMV T cell residency and the physiological consequences of such a response. Through RT-qPCR we found that mouse CMV (mCMV) infected the visceral adipose tissue and that this resulted in an expansion of leukocytes in situ. We further found, through flow cytometry, that adipose tissue became enriched in cytotoxic CD8 T cells that are specific for mCMV antigens from day 7 post infection through the lifespan of an infected animal (> 450 days post infection) and that carry markers of tissue residence. Furthermore, we found that inflammatory cytokines are elevated alongside the expansion of CD8 T cells. Finally, we show a correlation between the inflammatory state of adipose tissue in response to mCMV infection and the development of hyperglycemia in mice. Overall, this study identifies adipose tissue as a location of viral infection leading to a sustained and lifelong adaptive immune response mediated by CD8 T cells that correlates with hyperglycemia. These data potentially provide a mechanistic link between metabolic syndrome and chronic infection.

Retinoic Acid Signaling in Thymic Epithelial Cells Regulates Thymopoiesis.

Despite the essential role of thymic epithelial cells (TEC) in T cell development, the signals regulating TEC differentiation and homeostasis remain incompletely understood. In this study, we show a key in vivo role for the vitamin A metabolite, retinoic acid (RA), in TEC homeostasis. In the absence of RA signaling in TEC, cortical TEC (cTEC) and CD80loMHC class IIlo medullary TEC displayed subset-specific alterations in gene expression, which in cTEC included genes involved in epithelial proliferation, development, and differentiation. Mice whose TEC were unable to respond to RA showed increased cTEC proliferation, an accumulation of stem cell Ag-1hi cTEC, and, in early life, a decrease in medullary TEC numbers. These alterations resulted in reduced thymic cellularity in early life, a reduction in CD4 single-positive and CD8 single-positive numbers in both young and adult mice, and enhanced peripheral CD8+ T cell survival upon TCR stimulation. Collectively, our results identify RA as a regulator of TEC homeostasis that is essential for TEC function and normal thymopoiesis.

Immune Protection by a Cytomegalovirus Vaccine Vector Expressing a Single Low-Avidity Epitope.

Experimental CMV-based vaccine vectors expressing a single MHC class I-restricted high-avidity epitope provided strong, T cell-dependent protection against viruses or tumors. In this study we tested the low-avidity epitope KCSRNRQYL, and show that a mouse CMV (MCMV) vector provides complete immune control of recombinant vaccinia virus expressing the same epitope if KCSRNRQYL is expressed within the immediate-early MCMV gene ie2 The same epitope expressed within the early M45 gene provided no protection, although MCMV vectors expressing the high-avidity epitope SSIEFARL induced protective immunity irrespective of gene expression context. Immune protection was matched by Ag-induced, long-term expansion of effector memory CD8 T cells, regardless of epitope avidity. We explained this pattern by observing regularities in Ag competition, where responses to high-avidity epitopes outcompeted weaker ones expressed later in the replicative cycle of the virus. Conversely, robust and early expression of a low-avidity epitope compensated its weak intrinsic antigenicity, resulting in strong and sustained immunity and immune protection.

IRF8 Transcription-Factor-Dependent Classical Dendritic Cells Are Essential for Intestinal T Cell Homeostasis.

The role of dendritic cells (DCs) in intestinal immune homeostasis remains incompletely defined. Here we show that mice lacking IRF8 transcription-factor-dependent DCs had reduced numbers of T cells in the small intestine (SI), but not large intestine (LI), including an almost complete absence of SI CD8αß(+) and CD4(+)CD8αα(+) T cells; the latter requiring ß8 integrin expression by migratory IRF8 dependent CD103(+)CD11b(-) DCs. SI homing receptor induction was impaired during T cell priming in mesenteric lymph nodes (MLN), which correlated with a reduction in aldehyde dehydrogenase activity by SI-derived MLN DCs, and inefficient T cell localization to the SI. These mice also lacked intestinal T helper 1 (Th1) cells, and failed to support Th1 cell differentiation in MLN and mount Th1 cell responses to Trichuris muris infection. Collectively these results highlight multiple non-redundant roles for IRF8 dependent DCs in the maintenance of intestinal T cell homeostasis.

Context-Dependent Development of Lymphoid Stroma from Adult CD34(+) Adventitial Progenitors.

Despite the key role of primary and secondary lymphoid organ stroma in immunity, our understanding of the heterogeneity and ontogeny of these cells remains limited. Here, we identify a functionally distinct subset of BP3(-)PDPN(+)PDGFRß(+)/α(+)CD34(+) stromal adventitial cells in both lymph nodes (LNs) and thymus that is located within the vascular niche surrounding PDPN(-)PDGFRß(+)/α(-)Esam-1(+)ITGA7(+) pericytes. CD34(+) adventitial cells developed in late embryonic thymus and in postnatal LNs and in the thymus originated, along with pericytes, from a common anlage-seeding progenitor population. Using lymphoid organ re-aggregate grafts, we demonstrate that adult CD34(+) adventitial cells are capable of differentiating into multiple lymphoid stroma-like subsets including pericyte-, FRC-, MRC-, and FDC-like cells, the development of which was lymphoid environment-dependent. These findings extend the current understanding of lymphoid mesenchymal cell heterogeneity and highlight a role of the CD34(+) adventitia as a potential ubiquitous source of lymphoid stromal precursors in postnatal tissues.

CCRL1/ACKR4 is expressed in key thymic microenvironments but is dispensable for T lymphopoiesis at steady state in adult mice.

Thymus colonisation and thymocyte positioning are regulated by interactions between CCR7 and CCR9, and their respective ligands, CCL19/CCL21 and CCL25. The ligands of CCR7 and CCR9 also interact with the atypical receptor CCRL1 (also known as ACKR4), which is expressed in the thymus and has recently been reported to play an important role in normal αßT-cell development. Here, we show that CCRL1 is expressed within the thymic cortex, predominantly by MHC-II(low) CD40(-) cortical thymic epithelial cells and at the subcapsular zone by a population of podoplanin(+) thymic epithelial cells in mice. Interestingly, CCRL1 is also expressed by stromal cells which surround the pericytes of vessels at the corticomedullary junction, the site for progenitor cell entry and mature thymocyte egress from the thymus. We show that CCRL1 suppresses thymocyte progenitor entry into the thymus, however, the thymus size and cellularity are the same in adult WT and CCRL1(-/-) mice. Moreover, CCRL1(-/-) mice have no major perturbations in T-cell populations at different stages of thymic differentiation and development, and have a similar rate of thymocyte migration into the blood. Collectively, our findings argue against a major role for CCRL1 in normal thymus development and function.

Mesenchymal cells regulate retinoic acid receptor-dependent cortical thymic epithelial cell homeostasis.

The vitamin A metabolite and transcriptional modulator retinoic acid (RA) is recognized as an important regulator of epithelial cell homeostasis in several tissues. Despite the known importance of the epithelial compartment of the thymus in T cell development and selection, the potential role of RA in the regulation of thymic cortical and medullary epithelial cell homeostasis has yet to be addressed. In this study, using fetal thymus organ cultures, we demonstrate that endogenous RA signaling promotes thymic epithelial cell (TEC) cell-cycle exit and restricts TEC cellularity preferentially in the cortical TEC compartment. Combined gene expression, biochemical, and functional analyses identified mesenchymal cells as the major source of RA in the embryonic thymus. In reaggregate culture experiments, thymic mesenchyme was required for RA-dependent regulation of TEC expansion, highlighting the importance of mesenchyme-derived RA in modulating TEC turnover. The RA-generating potential of mesenchymal cells was selectively maintained within a discrete Ly51(int)gp38(+) subset of Ly51(+) mesenchyme in the adult thymus, suggesting a continual role for mesenchymal cell-derived RA in postnatal TEC homeostasis. These findings identify RA signaling as a novel mechanism by which thymic mesenchyme influences TEC development.

The sensing of environmental stimuli by follicular dendritic cells promotes immunoglobulin A generation in the gut.

In the Peyer's patches (PPs), germinal centers (GCs) are chronically induced by bacteria and are the major sites for generation of gut immunoglobulin A (IgA) immune responses. Whether follicular dendritic cells (FDCs) within the GCs directly contribute to the IgA production in PPs is unknown. We showed here that direct stimulation of FDCs by bacterial products and retinoic acid synergistically enhanced the expression of the chemokine CXCL13, the survival factor BAFF, and molecules that facilitate the secretion and activation of the cytokine TGF-beta1. A reduced production of these molecules by PP FDCs associated with deficiencies in the Toll-like receptor pathway or vitamin A resulted in decreased numbers of GC B cells and defective generation of IgA(+) B cells within PP GCs. Our data indicate that PP FDCs are conditioned by environmental stimuli to express key factors for B cell migration, survival, and preferential generation of IgA in gut.

[Role of Cre-loxP-mediated conditional gene targeting in understanding the function of Notch receptors]. / Udzial techniki warunkowej inaktywacji genów opartej na systemie Cre-loxP w post pie wiedzy na temat roli receptorów Notch.

The Notch family of cell surface receptors and their ligands constitute an evolutionarily conserved signaling pathway that is used by invertebrates and vertebrates to regulate a broad spectrum of cell specification events through local cell interactions. After ligand binding Notch receptor undergoes proteolytic processing ultimately liberating the cytoplasmic domain of the Notch receptor which translocates to the nucleus and activates target genes. In all animal models tested, mutations in Notch genes invariably resulted in developmental abnormalities. In mammals, Notch signaling controls key stages of lymphocyte differentiation as well as activation and several abnormalities in Notch pathway have been suggested to cause human leukemias. Cre-loxP mediated conditional gene targeting significantly contributed to our current understanding of the physiological roles of different Notch family members in hematopoietic compartment. This technique helped to overcome embryonic lethality of Notch mutants providing the opportunity to inactivate specific Notch gene in adulthood.

["Heart attack in humans"--the health belief model in comparison with the health reality of patients with myocardial infarction]. / "Czlowiek w zawale"--system prezentowanego modelu zdrowia w konfrontacji z rzeczywistoscia zdrowotna osób z zawalem miesnia sercowego.

Myocardial infarction (MI) is the direct cause of 40% of all deaths, independently of the high standard of medical treatment. The main aim of the study was to define the relationship between the declarative health model (declarations) and the real health activity of the sufferers (realization). The research was performed on 107 patients after acute MI, hospitalized in the Cardiology Department Medical University of Silesia. They were questioned in direct interviews. In addition, the modified form of Shalit's circle was applied. The results indicated the coexistence of two health models of patients. In declarations, there is a model of person with interior attribution of MI (90.65%) that has a feeling of ability to influence his/her health (e.g. through the lifestyle: 63.55%; an individual's character: 41.12%). In realization, there is a model of sick patient with exterior attribution of heart attack (e.g. political and economic crisis, innate predisposition). A similar tendency in the perception of health factors and planning of changes after MI was observed. For instance, 50.47% patients talked about "the increase caution" or "slowing down" as a new planned form of activity. Moreover, of 92% patients who have the social support only 33.64% consider that as an important factor for health. The results suggest a divergence between declarations and real health activity. The launched health model of beliefs seems to be too costly and not attractive enough. Taking into consideration the complete risk factors in MI and making the proposed health belief system more accessible seem to be essential for creating an adequate prevention program.

[Patients' opinions about the reasons for their heart attacks in the context of their health belief models--a preliminary report]. / Opinie pacjentów po zawalach serca na temat przyczyn wystapienia zawalu w kontekscie przyjetego przez nich modelu przekonan zdrowotnych--doniesienia z badan pilotazowych.

The importance of psychological factors in Myocardial Infarction (MI) is well documented. The aim of the study was to define a relationship between the perception of the reasons for MI and real health activity of the sufferers, in order to prepare an adequate psychological intervention. 30 patients after acute MI hospitalized in the Cardiology Department were included to the study. They were questioned in directed interviews. The modified form of Shalit's Circle was used to infer the model of health activity. Perceived reasons for the heart attack (first in 67% cases) appeared as a manifold area, which was divided into eight categories: stressful situations and occurrences (66.67%), an individual's character (40%), lifestyle (36.67%), connected illnesses 36.67%), genetics (20%), age (13.33%), weather (10%), "spontaneous" heart attack (6.67%). The results indicated exterior attribution of MI (e.g. political and economic crisis, innate predisposition, loss of relatives). Similar tendency in the planning of changes after heart attack was observed. For instance 43.33% patients just said they planed to restrict or give up their previous sickness-promoting behaviour, 33.33% talked laconically about "the increase of caution" or "slowing down" and only 26.67% intended to undertake new forms of activity. These opinions illustrate the general model of health beliefs (in declarations and realization), which seems to be of very limited use (or even disadvantageous) with regard to the future. Moreover, the contemporary clinical conditions are conducive to such model. Modern medical methods of treatment reduce the time spent by patients in hospital, thereby reducing the hospitalization stress. On the other hand it limits or even eliminates the most important part of treatment--the "doctor-patient" relationship. Because of the limited number of subjects a further research is needed to combine the psychological intervention aimed at health behaviour change with the high standard of cardiology treatment those patients are being given now. The psychological program should tackle emotional and social costs of heart attack and strive to change patients' health belief system.