Terminal osteoblast differentiation, mediated by runx2 and p27KIP1, is disrupted in osteosarcoma

The molecular basis for the inverse relationship between differentiation and tumorigenesis is unknown. The function of runx2, a master regulator of osteoblast differentiation belonging to the runt family of tumor suppressor genes, is consistently disrupted in osteosarcoma cell lines. Ectopic expression of runx2 induces p27KIP1, thereby inhibiting the activity of S-phase cyclin complexes and leading to the dephosphorylation of the retinoblastoma tumor suppressor protein (pRb) and a G1 cell cycle arrest. Runx2 physically interacts with the hypophosphorylated form of pRb, a known coactivator of runx2, thereby completing a feed-forward loop in which progressive cell cycle exit promotes increased expression of the osteoblast phenotype. Loss of p27KIP1 perturbs transient and terminal cell cycle exit in osteoblasts. Consistent with the incompatibility of malignant transformation and permanent cell cycle exit, loss of p27KIP1 expression correlates with dedifferentiation in high-grade human osteosarcomas. Physiologic coupling of osteoblast differentiation to cell cycle withdrawal is mediated through runx2 and p27KIP1, and these processes are disrupted in osteosarcoma

Bacillus anthracis TIR Domain-Containing Protein Localises to Cellular Microtubule Structures and Induces Autophagy

Toll-like receptors (TLRs) recognise invading pathogens and mediate downstream immune signalling via Toll/IL-1 receptor (TIR) domains. TIR domain proteins (Tdps) have been identified in multiple pathogenic bacteria and have recently been implicated as negative regulators of host innate immune activation. A Tdp has been identified in Bacillus anthracis, the causative agent of anthrax. Here we present the first study of this protein, designated BaTdp. Recombinantly expressed and purified BaTdp TIR domain interacted with several human TIR domains, including that of the key TLR adaptor MyD88, although BaTdp expression in cultured HEK293 cells had no effect on TLR4- or TLR2- mediated immune activation. During expression in mammalian cells, BaTdp localised to microtubular networks and caused an increase in lipidated cytosolic microtubule-associated protein 1A/1B-light chain 3 (LC3), indicative of autophagosome formation. In vivo intra-nasal infection experiments in mice showed that a BaTdp knockout strain colonised host tissue faster with higher bacterial load within 4 days post-infection compared to the wild type B. anthracis. Taken together, these findings indicate that BaTdp does not play an immune suppressive role, but rather, its absence increases virulence. BaTdp present in wild type B. anthracis plausibly interact with the infected host cell, which undergoes autophagy in self-defence

Interleukin-4 Regulates Eomesodermin in CD8⁺ T Cell Development and Differentiation

<div><p>Interleukin (IL)-4 is a cytokine classically associated with CD4⁺ T helper type 2 differentiation, but has been recently shown to also be required for the development of CD8⁺ innate-like lymphocytes. CD8⁺ innate-like lymphocytes are non-conventional lymphocytes that exhibit characteristics typically associated with memory CD8⁺ T cells, including expression of the T-box transcription factor Eomesodermin (Eomes). Here we investigate the signaling pathways required for IL-4 induction of Eomes and CD8⁺ innate-like lymphocyte markers in murine CD8SP thymocytes and peripheral CD8⁺ T cells. We demonstrate that IL-4 is sufficient to drive Eomes expression and the CD8⁺ innate-like lymphocyte phenotype through cooperation between STAT6- and Akt-dependent pathways. Furthermore, we show that while IL-4 has little effect on the induction of Eomes in the setting of robust T cell receptor (TCR) activation, this cytokine promotes Eomes in the setting of attenuated TCR stimulation in mature CD8⁺ T cells suggesting that cytokine signaling pathways may direct cell fate when TCR signals are limiting.</p></div

IL-4 upregulates Eomes in memory CD8⁺ T cells in an Akt-dependent manner.

<p>A) <i>Right</i>, Flow cytometric analysis of Eomes expression in WT peripheral CD8⁺ T cells. <i>Left</i>, Percentage of Eomes⁺ cells among total live CD8⁺ T cells after culture for 20 h in the indicated conditions (n = 6, 3 independent experiments). B) <i>Top</i>, Flow cytometric analysis of Eomes expression in live TCRβ⁺ CD8⁺ T cells from WT naïve (CD44⁻CD62L⁺) and memory (CD44⁺CD62L⁺) CD8⁺ splenocytes cultured in indicated conditions for 20 h. <i>Bottom</i>, Percentage of Eomes⁺ cells among total live CD8⁺ T cells (n = 8 for naïve CD8⁺ cells, 3 independent experiments; n = 4 for memory CD8⁺ T cells, 2 independent experiments). C) <i>Top</i>, Flow cytometric analysis of intracellular Eomes expression in WT memory CD8⁺ T cells treated for 20 h in indicated conditions. <i>Bottom</i>, Percentage of Eomes⁺ cells among live memory CD8⁺ T cells (n = 4, 2 independent experiments). Live lymphocyte gate was determined by forward and side scatter gating. Numbers in flow plots represent the percent of the gated population. Graphs show the average percentage of the indicated population and standard error of mean. Statistical significance calculated using Student’s t-test (A, B) or one-way ANOVA with Tukey’s multiple comparison post-test (C).</p

IL-4 sensitizes naïve CD8⁺ T cells to TCR stimulation and promotes Eomes expression during low TCR activation.

<p>A) <i>Left</i>, Flow cytometric analysis of intracellular IFNγ expression in naïve CD8⁺ T cells activated for 3d with anti-CD3/CD28 with varying concentrations of IL-4, rested for 2d and then re-stimulated with PMA and ionomycin. <i>Right</i>, Percentage of IFNγ⁺ T cells among total live cells after stimulation in indicated conditions. B) <i>Left</i>, Flow cytometric detection of intracellular Eomes expression in naïve CD8⁺ T cells activated for 3d with anti-CD3/CD28 with varying concentrations of IL-4 then rested for 2d. <i>Right</i>, Percentage of Eomes⁺ cells among total live cells after stimulation in indicated conditions. Data are pooled from five independent experiments. Graphs show the average percentage of the indicated population and standard error of mean.</p

mTORC1 is partially required for IL-4 regulation of Eomes in CD8SP thymocytes.

<p><i>Top</i>, Flow cytometric analysis of intracellular Eomes expression in WT TCRβ⁺ CD8P thymocytes after culture in the indicated conditions for 20 h. <i>Bottom left</i>, Percentage of Eomes⁺ cells among total CD8SP thymocytes (n = 8, 3 independent experiments). <i>Bottom right</i>, Flow cytometric analysis of intracellular phopho-S6 expression in WT CD8SP thymocytes after culture in indicated conditions for 20 h, representative of n = 5 per group, 2 independent experiments. Numbers in flow plots represent the percent of the gated population. Graphs show the average percentage of the indicated population and standard error of mean. Statistical significance calculated one-way ANOVA with Tukey’s multiple comparison post-test.</p

STAT6 is required for IL-4 regulation of Eomes in CD8SP thymocytes.

<p>A) Flow cytometric analysis of Eomes expression in WT and STAT6^−/− TCRβ⁺ CD8SP thymocytes after culture with or without IL-4 for 20 h. <i>Right top</i>, percentage of Eomes⁺ thymocytes among total CD8SP cells. <i>Right lower</i>, quantification of fold induction of Eomes in IL-4-treated CD8SP thymocytes of indicated genotypes. All data are representative of n = 3–4/genotype from 2 independent experiments. B) <i>Left</i>, relative Eomes expression in cDNA isolated from sorted CD8SP thymocytes in WT and STAT6^−/− thymocytes cultured in the absence or presence of IL-4 for 20 h, relative to WT CD8SP thymocyte population treated in media alone. <i>Right</i>, quantification of fold induction of Eomes expression in cDNA isolated from IL-4-treated CD8SP thymocytes of indicated genotypes, normalized to matched samples treated with media alone. Data are representative of n = 5/genotype, 2 independent experiments. C) Flow cytometric analysis of IL4Ra on CD8SP cells from WT thymocytes cultured as above. <i>Right</i>, percentage of IL4Ra⁺ cells among total CD8SP thymocytes in indicated conditions (n = 5/genotype, 2 independent experiments). D) Flow cytometric analysis of surface CD44 expression on CD8SP thymocytes treated under indicated conditions as above. <i>Right</i>, percentage of CD44⁺ cells among total CD8SP thymocytes (n = 5/genotype, 2 independent experiments). Numbers in flow plots (A, C, D) represent the percent of the gated population. Graphs show the average percentage (A, C, D) or fold induction (A, B) of the indicated population and standard error of mean. Statistical significance calculated using Student’s t-test.</p

IL-4 promotes Eomes expression and CD8⁺ ILL development.

<p>A) Flow cytometric analysis of intracellular Eomes expression in CD8SP cells from WT thymocytes cultured in the absence or presence of IL-4 (20 ng/ml) for 20 h. Plots are gated on live, TCRβ⁺ CD8SP lymphocytes. <i>Right</i>, Percentage of Eomes⁺ cells among total CD8SP thymocytes in indicated conditions is shown (n = 7, 3 independent experiments). B) Representative flow cytometric analysis of IL4Ra on CD8SP cells from WT thymocytes cultured as above. <i>Right</i>, percentage of IL4Ra⁺ cells among total CD8SP thymocytes in indicated conditions (n = 5, 2 independent experiments). C) Flow cytometric analysis of surface CD44 and CD122 expression on CD8SP thymocytes treated under indicated conditions as above. <i>Right</i>, percentage of CD44⁺CD122⁺ cells among total CD8SP thymocytes (n = 8, 3 independent experiments). D) Flow cytometric analysis of intracellular Eomes expression in CD8SP thymocytes from d8 WT FTOC treated under indicated conditions. <i>Right</i>, percentage of Eomes⁺ cells among total CD8SP thymocytes in indicated conditions (n = 9, 2 independent experiments). E) Flow cytometric analysis of intracellular expression of phopsho-STAT6 (pSTAT6) and phospho-Akt T308 (pAkt) in Eomes⁺ CD8⁺ ILLs versus Eomes⁻ CD8SP thymocytes directly <i>ex vivo</i> from SLP-76 Y145F mice (n = 2–5). F) Flow cytometric analysis of intracellular expression of pSTAT6 and pAkt in TCRβ⁺ CD8SP thymocytes cultured with or without IL-4 <i>in vitro</i> for 20 h. <i>Right</i>, mean fluorescence intensity (MFI) of pSTAT6 and pAkt T308 under indicated conditions (n = 5, 2 independent experiments). Flow plots are gated on live, TCRβ⁺ CD8SP lymphocytes. Numbers in flow plots (A–D) represent the percent of the gated population. Graphs (A, B, C and D) show the average percentage of the indicated population and standard error of mean. Statistical significance calculated using Student’s t-test.</p

Two cases of challenging cutaneous lymphoid infiltrates presenting in the context of COVID-19 vaccination: A reactive lymphomatoid papulosis-like eruption and a bona fide lymphoma

COVID-19 infection and vaccination may be associated with a wide variety of cutaneous and immune manifestations. Here, we describe two patients who presented with monoclonal cutaneous T-cell infiltrates that showed cytologic and immunophenotypic features concerning for lymphoma shortly following COVID-19 vaccination. In one case, the eruption completely resolved. The second patient showed initial resolution, but her disease recurred and progressed following a breakthrough SARS-CoV-2 infection. These cases suggest that immune stimulation following exposure to SARS-Cov-2 protein(s) in vaccine or infection may facilitate the development of a lymphoma or lymphoproliferative disorder in susceptible individuals. Moreover, they show that separating these cases from pseudolymphomatous reactive conditions is often challenging and requires close clinical correlation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/175916/1/cup14371_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/175916/2/cup14371.pd