Effects of Hepatitis B Surface Antigen on Virus-specific and Global T Cells in Patients With Chronic HBV infection

10.1053/j.gastro.2020.04.019Gastroenterolog

Hepatitis B virus-specific T cells associate with viral control upon nucleos(t)ide-analogue therapy discontinuation.

BACKGROUND: The clinical management of chronic hepatitis B virus (HBV) patients is based exclusively on virological parameters that cannot independently determine in which patients nucleos(t)ide-analogue (NUC) therapy can be safely discontinued. NUCs efficiently suppress viral replication, but do not eliminate HBV. Thus, therapy discontinuation can be associated with virological and biochemical relapse and, consequently, therapy in the majority is life-long. METHODS: Since antiviral immunity is pivotal for HBV control, we investigated potential biomarkers for the safe discontinuation of NUCs within immune profiles of chronic HBV patients by utilizing traditional immunological assays (ELISPOT, flow cytometry) in conjunction with analyses of global non-antigen-specific immune populations (NanoString and CyTOF). Two distinct cohorts of 19 and 27 chronic HBV patients, respectively, were analyzed longitudinally prior to and after discontinuation of 2 different NUC therapy strategies. RESULTS: Absence of hepatic flares following discontinuation of NUC treatment correlated with the presence, during NUC viral suppression, of HBV core and polymerase-specific T cells that were contained within the ex vivo PD-1+ population. CONCLUSIONS: This study identifies the presence of functional HBV-specific T cells as a candidate immunological biomarker for safe therapy discontinuation in chronic HBV patients. Furthermore, the persistent and functional antiviral activity of PD-1+ HBV-specific T cells highlights the potential beneficial role of the expression of T cell exhaustion markers during human chronic viral infection. FUNDING: This work was funded by a Singapore Translational Research Investigator Award (NMRC/STaR/013/2012), the Eradication of HBV TCR Program (NMRC/TCR/014-NUHS/2015), the Singapore Immunology Network, the Wellcome Trust (107389/Z/15/Z), and a Barts and The London Charity (723/1795) grant.This work was funded by a Singapore Translational Research Investigator Award (NMRC/STaR/013/2012), the Eradication of HBV TCR Program (NMRC/TCR/014-NUHS/2015), the Singapore Immunology Network, the Wellcome Trust (107389/Z/15/Z), and a Barts and The London Charity (723/1795) grant

Hormone-sensing cells require Wip1 for paracrine stimulation in normal and premalignant mammary epithelium

10.1186/bcr3381Breast Cancer Research15

Expression analysis of rare cellular subsets: Direct RT-PCR on limited cell numbers obtained by FACS or soft agar assays

10.2144/000114019BioTechniques544208-211BTNQ

Transcriptional Repressor Tbx3 Is Required for the Hormone-Sensing Cell Lineage in Mammary Epithelium

<div><p>The transcriptional repressor Tbx3 is involved in lineage specification in several tissues during embryonic development. Germ-line mutations in the Tbx3 gene give rise to Ulnar-Mammary Syndrome (comprising reduced breast development) and Tbx3 is required for mammary epithelial cell identity in the embryo. Notably Tbx3 has been implicated in breast cancer, which develops in adult mammary epithelium, but the role of Tbx3 in distinct cell types of the adult mammary gland has not yet been characterized. Using a fluorescent reporter knock-in mouse, we show that in adult virgin mice Tbx3 is highly expressed in luminal cells that express hormone receptors, and not in luminal cells of the alveolar lineage (cells primed for milk production). Flow cytometry identified Tbx3 expression already in progenitor cells of the hormone-sensing lineage and co-immunofluorescence confirmed a strict correlation between estrogen receptor (ER) and Tbx3 expression in situ. Using in vivo reconstitution assays we demonstrate that Tbx3 is functionally relevant for this lineage because knockdown of Tbx3 in primary mammary epithelial cells prevented the formation of ER+ cells, but not luminal ER- or basal cells. Interestingly, genes that are repressed by Tbx3 in other cell types, such as E-cadherin, are not repressed in hormone-sensing cells, highlighting that transcriptional targets of Tbx3 are cell type specific. In summary, we provide the first analysis of Tbx3 expression in the adult mammary gland at a single cell level and show that Tbx3 is important for the generation of hormone-sensing cells.</p></div

Tbx3 is required for the generation of hormone-sensing cells.

<p>(A) Primary MECs from wildtype or KI mice were transduced with a non-silencing lentiviral vector (control) or with two independent short hairpins against Tbx3 (sh-1 and sh-2). Cells were injected into mammary fat pads devoid of endogenous epithelium and outgrowths were analyzed 8–10 weeks later for the identity of lentivirally transduced cells (recognized by tGFP expression). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110191#pone.0110191.s004" target="_blank">File S4B</a> for a schematic experimental design. Each bar represents one fat pad and 46 to 569 tGFP+ luminal cells were counted per fat pad. There is a significant bias against the formation of HS cells by cells with Tbx3 knockdown (Chi square of shRNA versus control transplant <0.01). (B) Paraffin section of a cleared mammary fat pad transplanted with MECs that were exposed to the non-silencing control vector. Transduced cells are identified with an antibody staining against tGFP (green), luminal cells are identified by cytokeratin 8 (blue) and HS cells are identified by the estrogen receptor (ER, red). White arrow indicates transduced cells contributing to the hormone-sensing lineage. (C) Paraffin section of a mammary fat pad transplanted with MECs exposed to the first short hairpin against Tbx3. White arrow head indicated transduced cells in the luminal alveolar (ER-negative) lineage. The background of immunohistochemistry is higher in transplanted samples (arguably due to fibrosis). Where ER staining was ambiguous due to high background, we used progesterone receptor (PR, red) staining as an alternative marker for HS cells. (D) Paraffin section of a mammary fat pad transplanted with MECs exposed to the second short hairpin against Tbx3. White arrow head indicates transduced cells in the luminal alveolar (ER-negative) lineage. White scale bar is 20 µm and yellow scale bar is 10 µm.</p

Tbx3 marks the hormone sensing lineage, including ER+ progenitor cells.

<p>(A) Combined density/contour plot of mammary epithelial cells (from a pool of 5 Tbx3^+/Venus mice) separated into basal (red) and luminal (blue) cells based on CD24 and alpha6-integrin (CD49f) expression. (B) Colony forming potential of 1000 sorted cells from each population, representative of two independent experiments. (C) Histogram of luminal mammary epithelial cells Venus^High (luminal) and Venus^Low (luminal) cells sorted for colony forming assay (from a pool of 5 Tbx3^+/Venus mice per experiment). (D) Colony forming potential of 1000 sorted cells from each population, representative of two independent experiments. (E) FACS profile of hormone-sensing (CD49b^high and CD49b^low) and alveolar progenitor cells that were used for colony assays. Cells are color-coded based on Venus expression (green = Venus^High, grey = Venus^Low). (F) Fold change in Tbx3, progesterone receptor (PR) and estrogen receptor (ER) mRNA expression of sorted CD49b^high and CD49b^low hormone-sensing cells and alveolar progenitor cells, relative to CD49b^low hormone-sensing cells (dark purple bar). Fold change in Elf5 and cKit mRNA expression is shown relative to luminal alveolar cells (orange bar). (G) Colony forming potential of 1000 sorted luminal cells: CD49b^low and CD49b^high hormone-sensing cells and alveolar progenitor cells. (H) Quantification of colony forming assays with HS cells (Sca1^highCD49b^low), HS progenitor cells (Sca1^highCd49b^high) and alveolar progenitor cells (Sca1^lowCD49b^high). Bars represent the mean of three independent pools of 5–6 adult virgin Tbx3^+/Venus animals ± SD. HS progenitor cells form more colonies than HS cells (p = 0.02, paired t-test) and there is no significant difference in colony forming potential between HS progenitor and alveolar progenitor cells (p = 0.21, paired t-test).</p

Tbx3 marks hormone sensing cells.

<p>(A) Luminal cells from wildtype mammary glands are separated into hormone-sensing (HS, Sca1^hiCD49b^lo, purple) and alveolar (Sca1^l°CD49b^hi, orange) subsets based on Sca1 and alpha2-integrin (CD49b) expression. (B) There is no significant (n.s.) difference in the proportion of hormone-sensing (HS, purple) and alveolar cells (orange) between Tbx3^+/+ (wildtype, WT) and Tbx3^+/Venus (Knock-in, KI), paired t-test p = 0.53 for HS and p = 0.60 for Alv. (C) Tbx3 mRNA levels in sorted populations as indicated. (D) Tbx3^+/Venus luminal cells were first gated for Low or High Venus expression (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110191#pone-0110191-g001" target="_blank">Figure 1D</a>), and then plotted based on Sca1 and CD49b expression. (E) Proportion of hormone-sensing (HS, purple) and alveolar cells (orange) that are Venus^Low (grey) or Venus^High (green), measured by FACS in 3 independent Tbx3^+/Venus animals. (F) Fold change in mRNA expression in luminal Venus^Low (left panel) or Venus^High (right panel) cells, relative to total luminal population. Data are presented as mean ± SD of three adult virgin Tbx3^+/Venus animals.</p

Fluorescent reporter reveals distinct Tbx3 expression in mammary epithelial cell subsets.

<p>(A) Epithelial cells isolated from mammary glands of wildtype (Tbx3^+/+) or knock-in (Tbx3^+/Venus) mice show three peaks with different levels of Venus expression (Low, Medium and High). (B) Mammary epithelial cells (MECs) from 3 independent Tbx3^+/Venus animals were sorted according to Venus signal intensity. qPCR on 500 directly lysed cells shows that both Venus and Tbx3 mRNA correlates tightly with Venus fluorescence intensity. (C) MECs were labeled with fluorescent antibodies against CD24 and α6-integrin (CD49f) to distinguish the luminal (blue) and basal (red) cell populations. (D) When plotted separately, luminal cells from Tbx3^+/Venus mammary glands show two main populations; Venus^Low (cells with low Tbx3 expression) and Venus^High (cells with high Tbx3 expression). Basal cells express intermediate level of Venus (and Tbx3). (E) Quantification of the percentage of Venus-Low, -Medium and -High cells in the luminal and in the basal population of Tbx3^+/Venus epithelium. Data are presented as mean ± SD of three individual adult virgin Tbx3^+/Venus animals. (F) Populations gated based on Tbx3 expression (Venus-Low, -Medium and -High) plotted on a CD24/CD49f contour plot.</p

Tbx3 expression in epithelial cells in intact mammary glands.

<p>(A–C) Confocal immunofluorescence on paraffin sections of mammary glands from wildtype adult virgin mice. (A) Ductal structure probed with antibodies for Tbx3 (green), the estrogen receptor (ER, red) and the luminal cell marker cytokeratin-8 (CK8, blue). Nuclei are stained with DAPI (grey). (B) Duct probed for Tbx3 (green), the basal marker smooth muscle actin (SMA, red) and CK8 (blue). (C) Duct probed for Tbx3 (green), E-Cadherin (E-cad, red) and CK8 (blue). Images are representative of staining performed on paraffin sections of 3 independent animals. Scale bar is 20 µm or 10 µm for the inset.</p