Fas-Mediated Apoptosis Regulates the Composition of Peripheral αβ T Cell Repertoire by Constitutively Purging Out Double Negative T Cells

BACKGROUND: The Fas pathway is a major regulator of T cell homeostasis, however, the T cell population that is controlled by the Fas pathway in vivo is poorly defined. Although CD4 and CD8 single positive (SP) T cells are the two major T cell subsets in the periphery of wild type mice, the repertoire of mice bearing loss-of-function mutation in either Fas (lpr mice) or Fas ligand (gld mice) is predominated by CD4(-)CD8(-) double negative alphabeta T cells that also express B220 and generally referred to as B220+DN T cells. Despite extensive analysis, the basis of B220+DN T cell lymphoproliferation remains poorly understood. In this study we re-examined the issue of why T cell lymphoproliferation caused by gld mutation is predominated by B220+DN T cells. METHODOLOGY AND PRINCIPAL FINDINGS: We combined the following approaches to study this question: Gene transcript profiling, BrdU labeling, and apoptosis assays. Our results show that B220+DN T cells are proliferating and dying at exceptionally high rates than SP T cells in the steady state. The high proliferation rate is restricted to B220+DN T cells found in the gut epithelium whereas the high apoptosis rate occurred both in the gut epithelium and periphery. However, only in the periphery, apoptosis of B220+DN T cell is Fas-dependent. When the Fas pathway is genetically impaired, apoptosis of peripheral B220+DN T cells was reduced to a baseline level similar to that of SP T cells. Under these conditions of normalized apoptosis, B220+DN T cells progressively accumulate in the periphery, eventually resulting in B220+DN T cell lymphoproliferation. CONCLUSIONS/SIGNIFICANCE: The Fas pathway plays a critical role in regulating the tissue distribution of DN T cells through targeting and elimination of DN T cells from the periphery in the steady state. The results provide new insight into pathogenesis of DN T cell lymphoproliferation

Seroprevalence of Anti-SARS-CoV-2 Antibodies in Benadir Region, Somalia

Only little is known about the true extent of COVID-19 in Somalia. The study aims to assess the seroprevalence of the COVID-19 pandemics in the Benadir region using SARS-CoV-2 antibodies and estimate the number of inhabitants infected with SARS-CoV-2. Population-based cross-sectional survey was conducted to measure the seroprevalence of antibodies against SARS-CoV-2 in the Benadir region (Mogadishu city). In the study, we enrolled 2500 Mogadishu city residents aged ≥18 years who did not receive the SARS-CoV-2 vaccine. The overall seroprevalence of IgG/IgM anti-SARS-CoV-2 antibodies was 44.8%. The seropositivity in females (56.6%) was higher than in males (46.2%). The trend in seropositivity increased with age; however, the variation was only significant in the age group 38–57 with an odds ratio and p-value of 4.11 (1.475–11.47), p = 0.007. Families with >5 members (47.2%) were more likely to test positive than those with <5 members (37%). Participants who reported COVID-19 symptoms during the pandemics or who had contact with COVID-19 patients had significantly increased IgG prevalence. Participants with larger families, individuals working in the public sector, and students showed significant seropositive results. Therefore, precautionary measures should be heightened for individuals working in the public sector

Phenotypic similarities of peripheral and intestinal DN T cells.

<p>(A) Single cell suspensions from spleens of gld mice or intestine (small and large intestines were combined) of gld or wt mice were stained for APC-TCRβ, PerCP-CD4, and PerCP-CD8α, and FITC-CD2, CD5 or B220. TCR⁺ cells were gated and expression of indicated molecules by DN and SP subsets were determined. Spleens of wt mice are not included in the analysis because of the paucity of DN T cells. (B) Similar distribution of Vβ expression by DN T cells. Peripheral T cells and IEL were isolated from 12-week-old C3H-gld/gld mice and surface-stained for TCR, CD4, CD8, and Vβ6, Vβ8, Vβ13 or Vβ14. After gating on TCR⁺ cells, the frequencies of different Vβs among DN, CD4 or CD8 T cells were determined. Data show mean±SD from one of two independent experiments. (C) Downregulation of sdc1 by TCR activation. Top panel: Analysis of TCR (left dot plot); and CD4 and CD8α expression (right dot plot) by freshly isolated DN cells prior to culture. The isolated cells were stained with TCR, CD4 and CD8 specific antibodies and their purity assessed by flow cytometry (>95% of isolated cells expressed TCR (left dot plot) and lacked CD4 and CD8 expression (middle dot plot). The histogram shows CFSE intensity in DN T cells before (day 0) and after (day 4) TCR activation. Bottom panel: Kinetics of sdc1 and CD62L downregulation by DN T cells in response to CD3/CD28 stimulation. Data from one of two independent experiments are shown.</p

Sdc1⁺ B220+ DN T cells accumulate in an age-dependent manner in the gut epithelium of wt mice with intact Fas pathway.

<p>T cells were isolated from the periphery and gut epithelium of wt or gld C3H mice of different ages as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003465#s4" target="_blank">Materials and Methods</a>. Isolated cells from lymph nodes (LN), spleen, small intestine (SI), and large intestines were stained with APC-TCRβ, PerCP-CD4, PerCP-CD8α, anti-B220 and PE-sdc1 and analyzed by FACS. CD4 and CD8 T cells were included in one subset (CD4 and CD8) by simultaneously staining samples with PerCP-conjugated anti-CD4 and PerCP-conjugated anti-CD8 mAbs. This allowed us to compare directly SP (CD4⁺ and CD8⁺) and DN (CD4⁻CD8⁻) TCR⁺ subsets. Frequencies of sdc1⁺ DN T cells in the periphery and gut epithelium of wt (A) and gld (C) mice are shown. Numbers indicate the percentage of positive cells in each quadrant. Sdc1⁺ cells in the upper right quadrants in the lymph nodes and spleen of 13- and 21- to 24-week-old gld mice were CD4 T cells (not shown). Percentages of sdc1⁺ DN T cells relative to total T cells in the periphery and gut epithelium of wt (B) and gld (D) mice are shown. DN T cells isolated from the gut epithelium expressed B220 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003465#pone-0003465-g002" target="_blank">Fig. 4A</a> and data not shown). Results are expressed as mean±SD from three independent experiments with two to three mice per group. LN, Lymph nodes, SI, small intestine, Li, large intestine.</p

DN T cells have a unique transcript profile.

<p>(A) Comparison of the transcript profiles of DN T cells and autologous SP T cells (combined CD4 and CD8 T cells) isolated from inguinal and axillary lymph nodes of gld mice as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003465#s4" target="_blank">Materials and Methods</a>. The insets show purity and hallmarks properties of DN T cells including expression of TCR and B220 and lack of CD4 and CD8 on their surface. The Minus versus Average (MvA) plot shows intensity log-ratio M = log₂ (DN/SP) versus mean log-intensity A = [(log₂ (DN)+log₂ (SP)]/2. Out of 32,000 transcripts examined, expression of 160 genes was at least 3-fold higher in DN than in SP T cells. Highly expressed genes including junctional adhesion molecules, hydrolyase enzymes, and apoptotic death molecules are highlighted. Selected under-expressed genes in DN T cells relative to SP T cells are also highlighted. (B) Validation of expression of selected genes by real-time PCR. Expression level of each gene was normalized relative to expression of 18 s rRNA in the same cell subset. X-axis shows -fold change in expression of indicated genes in DN T cells relative to SP T cells. (C) Validation of specific expression of sdc1 by flow cytometry. Splenocytes were isolated from 16-week-old C3H-gld/gld mice, stained with APC-TCRβ, PerCP-CD4, FITC-CD8α, and PE-sdc1 or with APC-TCRβ, PerCP-CD4, FITC-CD8α, and PE-B220 mAbs and analyzed by FACS. Dot plot: TCR⁺ cells were gated followed by specific gating of DN (R5), CD4⁺ (R3), and CD8⁺ (R4) subsets. Histograms: Overlays show relative expression of TCR, B220, and sdc1 by gated CD4, CD8, and DN subsets.</p

SEB activation of gld CD8 or CD4 T cells in vivo does not lead to their conversion into DN T cells.

<p>C3H-gld mice (14-week-old) or their age-matched C3H-wt mice were immunized i.p. with SEB (100 µg/mouse). Frequencies of SEB-reactive Vβ8⁺ T cells among the CD8 (A), CD4 (B), and DN (C) subsets in peripheral blood were determined on the indicated days. Similar results were obtained in lymph nodes and spleens when the experiment is terminated. Frequencies of Vβ6⁺ T cells were used as negative controls. Results show mean±SD from three mice per group.</p