A Novel Bacterial Artificial Chromosome-Transgenic Podoplanin–Cre Mouse Targets Lymphoid Organ Stromal Cells in vivo

Stromal cells provide the structural foundation of secondary lymphoid organs (SLOs), and regulate leukocyte access and cell migration within the different compartments of spleen and lymph nodes (LNs). Furthermore, several stromal cell subsets have been implied in shaping of T cell responses through direct presentation of antigen. Despite significant gain of knowledge on the biology of different SLO-resident stromal cell subsets, their molecular and functional characterization has remained incomplete. To address this need, we have generated a bacterial artificial chromosome-transgenic mouse model that utilizes the podoplanin (pdpn) promoter to express the Cre-recombinase exclusively in stromal cells of SLOs. The characterization of the Pdpn–Cre mouse revealed transgene expression in subsets of fibroblastic reticular cells and lymphatic endothelial cells in LNs. Furthermore, the transgene facilitated the identification of a novel splenic perivascular stromal cell subpopulation that forms web-like structures around central arterioles. Assessment of the in vivo antigen expression in the genetically tagged stromal cells in Pdpn–Cre mice revealed activation of both MHC I and II-restricted TCR transgenic T cells. Taken together, stromal pdpn–Cre expression is well-suited to characterize the phenotype and to dissect the function of lymphoid organ stromal cells

An Innate Checkpoint Determines Immune Dysregulation and Immunopathology during Pulmonary Murine Coronavirus Infection.

Hallmarks of life-threatening, coronavirus-induced disease include dysregulated antiviral immunity and immunopathological tissue injury. Nevertheless, the sampling of symptomatic patients overlooks the initial inflammatory sequela culminating in severe coronavirus-induced disease, leaving a fundamental gap in our understanding of the early mechanisms regulating anticoronavirus immunity and preservation of tissue integrity. In this study, we delineate the innate regulators controlling pulmonary infection using a natural mouse coronavirus. Within hours of infection, the cellular landscape of the lung was transcriptionally remodeled altering host metabolism, protein synthesis, and macrophage maturation. Genetic perturbation revealed that these transcriptional programs were type I IFN dependent and critically controlled both host cell survival and viral spread. Unrestricted viral replication overshooting protective IFN responses culminated in increased IL-1β and alarmin production and triggered compensatory neutrophilia, interstitial inflammation, and vascular injury. Thus, type I IFNs critically regulate early viral burden, which serves as an innate checkpoint determining the trajectory of coronavirus dissemination and immunopathology

HDAC1 Controls CD8⁺ T Cell Homeostasis and Antiviral Response

<div><p>Reversible lysine acetylation plays an important role in the regulation of T cell responses. HDAC1 has been shown to control peripheral T helper cells, however the role of HDAC1 in CD8⁺ T cell function remains elusive. By using conditional gene targeting approaches, we show that <i>LckCre</i>-mediated deletion of HDAC1 led to reduced numbers of thymocytes as well as peripheral T cells, and to an increased fraction of CD8⁺CD4^– cells within the CD3/TCRβ^lo population, indicating that HDAC1 is essential for the efficient progression of immature CD8⁺CD4^– cells to the DP stage. Moreover, CD44^hi effector CD8⁺ T cells were enhanced in mice with a T cell-specific deletion of HDAC1 under homeostatic conditions and HDAC1-deficient CD44^hi CD8⁺ T cells produced more IFNγ upon <i>ex vivo</i> PMA/ionomycin stimulation in comparison to wild-type cells. Naïve (CD44^l°CD62L⁺) HDAC1-null CD8⁺ T cells displayed a normal proliferative response, produced similar amounts of IL-2 and TNFα, slightly enhanced amounts of IFNγ, and their <i>in vivo</i> cytotoxicity was normal in the absence of HDAC1. However, T cell-specific loss of HDAC1 led to a reduced anti-viral CD8⁺ T cell response upon LCMV infection and impaired expansion of virus-specific CD8⁺ T cells. Taken together, our data indicate that HDAC1 is required for the efficient generation of thymocytes and peripheral T cells, for proper CD8⁺ T cell homeostasis and for an efficient <i>in vivo</i> expansion and activation of CD8⁺ T cells in response to LCMV infection.</p></div

Alternative NF-κB signaling regulates mTEC differentiation from podoplanin-expressing presursors in the cortico-medullary junction

The thymic epithelium forms specialized niches to enable thymocyte differentiation. While the common epithelial progenitor of medullary and cortical thymic epithelial cells (mTECs and cTECs) is well defined, early stages of mTEC lineage specification have remained elusive. Here, we utilized in vivo targeting of mTECs to resolve their differentiation pathways and to determine whether mTEC progenitors participate in thymocyte education. We found that mTECs descend from a lineage committed, podoplanin (PDPN)-expressing progenitor located at the cortico-medullary junction. PDPN(+) junctional TECs (jTECs) represent a distinct TEC population that builds the thymic medulla, but only partially supports negative selection and thymocyte differentiation. Moreover, conditional gene targeting revealed that abrogation of alternative NF-κB pathway signaling in the jTEC stage completely blocked mTEC development. Taken together, this study identifies jTECs as lineage-committed mTEC progenitors and shows that NF-κB-dependent progression of jTECs to mTECs is critical to secure central tolerance

Adoptively transferred HDAC1-null transgenic P14 CD8⁺ T cells display impaired expansion and activation.

<p>(A) <i>Hdac1^f/f</i> and <i>Hdac1^f/fCd4Cre</i> P14 CD8⁺ T cells (10⁴ cells) were adoptively transferred into C57BL/6 mice. The next day, recipient mice were infected i.v. with 200 pfu LCMV Armstrong. The percentage of Vα2/Vβ8-expressing transgenic P14 CD8⁺ T cells on day 7 is shown (mean ± SEM; n = 5, analyzed in 1 experiment). (B) Mice were prepared and infected as described in A and the anti-LCMV-specific CD8⁺ T cell response was determined upon <i>in vitro</i> re-stimulation with the gp33 peptide. Diagrams show the percentage of INFγ⁺ and TNFα⁺ expressing CD45.2⁺ CD8⁺ T cells upon re-stimulation (mean ± SEM; n = 5, analyzed in 1 experiment). (C) Diagram showing the percentage of CD44^hi (left) and CD62L^lo (right) transgenic P14 CD8⁺ T cells. Data show mean ± SEM (n = 5, analyzed in 1 experiment). (D) CFSE labeled <i>Hdac1^f/f</i> and <i>Hdac1^f/fCd4Cre</i> P14 CD8⁺ T cells (10⁶ cells; CD45.2⁺) were adoptively transferred into CD45.1⁺ C57BL/6 mice. Recipient mice were infected with 200 pfu LCMV Armstrong. Histogram shows CFSE intensity in CD45.2⁺<i>Hdac1^f/f</i> and <i>Hdac1^f/fCd4Cre</i> P14 CD8⁺ T cells on day 5 after infection. The diagram at the right indicates the mean fluorescence expression (MFI) of CFSE (mean ± SD; n = 4, analyzed in 1 experiment). (E) Mice were prepared and infected as described in A (lower panel). Splenic cells were isolated on day 5 and restimulated with gp33 peptide. Contour plots to the left show intracellular IFNγ versus CD8 expression. Diagram to the right shows the summary of all experiments performed (mean ± SEM; n = 4, analyzed in 1 experiment). The numbers indicate the percentage of cells in the respective quadrants. (A–E) Statistical analysis was performed using a two-tailed non-paired Student’s t test. The P-values were defined as following: *, P<0.05; **, P<0.01; ***, P<0.001.</p

CTL effector functions in the absence of HDAC1.

<p>(A) Naïve <i>Hdac1^f/f</i> and <i>Hdac1^f/fCd4Cre</i> CD8⁺ T cells were labeled with CFSE and stimulated with anti-CD3/anti-CD28. Histograms depict CFSE intensity after 48 h of stimulation. Data are representative of 5 independent experiments. (B) Naïve cells were stimulated with anti-CD3/anti-CD28 for 48 hours. Cells were split 1∶2 on day 2, cultured for 2 additional days and stimulated with PMA/ionomycin for 4 hours. Histogram depicts intracellular IL-2 expression and the percentage of IL-2-expressing cells for all experiments is shown at the right (mean ± SEM; n = 3, performed in 3 independent experiments). (C) Naïve cells were stimulated with anti-CD3/anti-CD28. Cells were split 1∶2 on day 2, cultured for 2 additional days and re-stimulated with anti-CD3 overnight. IFNγ and TNFα levels in the supernatant were determined by ELISA (mean ± SEM; n = 3, performed in 3 independent experiments). (D) Naïve <i>Hdac1^f/f</i> and <i>Hdac1^f/fCd4Cre</i> CD8⁺ T cells were activated as described in C. The expression of <i>Gzmb</i> and <i>Prf1</i> was assessed by qRTPCR before (“resting”) and after (“react.”) overnight restimulation with anti-CD3 and normalized to <i>Hprt1</i> expression (mean ± SEM; n = 3, performed in 3 independent experiments). (E) <i>Hdac1^f/f</i> and <i>Hdac1^f/fCd4Cre</i> mice were immunized with OVA peptide (SIINFEKL) plus adjuvant. Subsequently, target cells were intravenously injected 4 days post-immunization. Target cells consisted of a 1∶1∶1 mixture of CFSE-labeled splenocytes that were either pulsed with OVA peptide (CFSE^hi), with irrelevant peptide (CFSE^med) or without peptide (CFSE^low). Eight hours after target-cell injection, the percentage of CFSE^low, CSFE^med and CSFE^hi target cells in the draining lymph node of <i>Hdac1^f/f</i> and <i>Hdac1^f/fCd4Cre</i> mice was determined. Numbers in the histogram show the percentage of cells within the indicated regions. The diagram at the right indicates the percentage of specific lysis (as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110576#s2" target="_blank">Materials and Methods</a>) and shows the summary of all <i>in vivo</i> CTL experiments performed (n = 8 for <i>Hdac1^f/f</i>; n = 9 for <i>Hdac1^f/fCd4Cre</i> mice, analyzed in 2 independent experiments). (B, C, D, E) Statistical analysis was performed using a two-tailed non-paired Student’s t test. The P-values were defined as following: *, P<0.05; **, P<0.01; ***, P<0.001.</p