Regulation of GATA-3 Expression during CD4 Lineage Differentiation

GATA-3 is necessary for the development of MHC class II-restricted CD4 T cells, and its expression is increased during positive selection of these cells. TCR signals drive this upregulation, but the signaling pathways that control this process are not well understood. Using genetic and pharmacological approaches, we show that GATA-3 upregulation during thymocyte-positive selection is the result of additive inputs from the Ras/MAPK and calcineurin pathways. This upregulation requires the presence of the transcription factor c-Myb. Furthermore, we show that TH-POK can also upregulate GATA-3 in double-positive thymocytes, suggesting the existence of a positive feedback loop that contributes to lock in the initial commitment to the CD4 lineage during differentiation

A review of cleaning and disinfection guidelines and recommendations following an outbreak of classical scrapie

[EN] Classical scrapie is a prion disease of small ruminants, the infectious agent of which has been shown to be extremely persistent in the environment. Cleaning and disinfection (C&D) after a scrapie outbreak is currently recommended by many governments' veterinary advisors and implemented in most farms affected. Yet, the effectiveness of these procedures remains unclear. The aim of this study was to review existing literature and guidelines regarding farm C&D protocols following classical scrapie outbreaks and assess their effectiveness and the challenges that translation of policy and legislative requirements present at a practical level. A review of the literature was conducted to identify the on-farm C&D protocols used following outbreaks of scrapie, assess those materials with high risk for persistence of the scrapie agent on farms, and review the existing evidence of the effectiveness of recommended C&D protocols. An expert workshop was also organised in Great Britain (GB) to assess: the decision-making process used when implementing C&D protocols on GB farms, the experts' perceptions on the effectiveness of these protocols and changes needed, and their views on potential recommendations for policy and research. Outputs of the literature review revealed that the current recommended protocol for C&D [1 h treatment with sodium hypochlorite containing 20,000 ppm free chlorine or 2 M sodium hydroxide (NaOH)] is based on labo-ratory experiments. Only four field farm experiments have been conducted, indicating a lack of data on effec-tiveness of C&D protocols on farms by the re-occurrence of scrapie infection post re-stocking. Recommendations related to the control of outdoor environment, which are difficult and expensive to implement, vary between countries. The expert workshop concluded that there are no practical, cost-effective C&D alternatives to be considered at this time, with control therefore based on C&D only in combination with additional time restrictions on re-stocking and replacement with non-susceptible livestock or more genetically resistant types, where available. Participants agreed that C&D should still be completed on scrapie affected farms, as it is considered to be "good disease practice" and likely to reduce the levels of the prion protein. Participants felt that any additional protocols developed should not be "too prescriptive" (should not be written down in specific policies) because of significant variation in farm types, farm equipment and installations. Under this scenario, control of classical scrapie on farms should be designed with a level of C&D in combination with re-stocking temporal ban and replacement with livestock of limited susceptibility.The APHA would like to thank all the expert contributions at the workshop. This study was funded by Defra (UK) under Project SE1960 and the Government of education and science of the Generalitat Valenciana (Spain)Alarcon, P.; Marco-Jiménez, F.; Horigan, V.; Ortiz-Pelaez, A.; Rajanayagam, B.; Dryden, A.; Simmons, H.... (2021). A review of cleaning and disinfection guidelines and recommendations following an outbreak of classical scrapie. Preventive Veterinary Medicine. 193:1-9. https://doi.org/10.1016/j.prevetmed.2021.1053881919

The Ras/MAPK Pathway Is Required for Generation of iNKT Cells

iNKT cells derive from CD4+CD8+ DP thymocytes, and are selected by thymocyte-thymocyte interactions through signals from their invariant Vα14-Jα18 TCR and from the costimulatory molecules SLAMF1 and SLAMF6. Genetic studies have demonstrated the contribution of different signaling pathways to this process. Surprisingly, current models imply that the Ras/MAPK pathway, one of the critical mediators of conventional αβ T cell positive selection, is not necessary for iNKT cell development. Using mice defective at different levels of this pathway our results refute this paradigm, and demonstrate that Ras, and its downstream effectors Egr-1 and Egr-2 are required for positive selection of iNKT cells. Interestingly our results also show that there are differences in the contributions of several of these molecules to the development of iNKT and conventional αβ T cells

Egr1^-/-; Egr2^f/f<i>lck</i>cre mice have a complete block in iNKT development.

<p>(<b>a</b>) Thymic profile of littermate wild-type (WT), or Egr1^-/-;Egr2^f/f<i>lck</i>cre (DKO) mice <b>(b)</b> Percentages and absolute numbers of iNKT cells in the thymus (T), spleen (S) and liver mononuclear cells (L) of WT and EgrDKO mice stained with CD4, CD8, PBS57-loaded CD1d tetramer and TCRβ. <b>(c)</b> Percentages and absolute numbers of iNKT cell subpopulations in gated thymic TCR-βhiPBS57-CD1dtet⁺ of WT and EgrDKO mice. Results representative of five independent pairs in three independent experiments. The bar graphs show the average and SEM of all the experiments. Significance as assessed using the unpaired t-test. ***<0.001, **<0.01</p

Defects in Slamf1, Slamf6 and CD1d expression in dnRas, but not Egr-1,2 double knockout mice.

<p>Slamf1, Slamf3, Slamf6, Slamf5 and CD1d expression levels in DP thymocytes mice were assessed by flow cytometry. Shown are representative histograms, and the mean and SEM of the normalized MFI of DP populations. In <b>(A)</b> WT vs. dnRas (n = 5). In <b>(B)</b> WT vs. Egr1^-/-;Egr2^f/f-<i>lck</i>-Cre (Egr-DKO) (n = 3). <b>(C)</b> SAP and Bcl_xL expression levels in DP thymocytes from WT or dnRas mice were assessed by intracellular flow cytometry. Shown are representative histograms, and the mean and SEM of the normalized MFI of DP populations (n = 5). To normalize the MFI, we averaged the MFI for the WT mice in each experiment and considered that value 1. The bar graphs show the average and SEM of all the experiments. Significance was assessed using the unpaired t-test ***<0.001, **<0.01 *<0.05<b>.</b></p

Egr1 and Egr2 contribute in a quantitatively different manner to iNKT cell development.

<p>(<b>a</b>) Contribution of WT and Egr1^-/- (Egr1KO) cells to the iNKT compartment in thymus (T), spleen (S) and liver (L) of mixed bone marrow chimeras generated by injecting Egr1^-/- (CD45.2) and F1(C57BL/6xB6-LY5.2/Cr) (CD45.1;CD45.2) bone marrow cells into lethally irradiated B6-LY5.2/Cr recipient mice (CD45.1). Mean and SEM is shown on the right (n = 5). Significance was assessed using a paired t-test. **<0.01, *<0.05. This is one out of two independent experiments. <b>(b)</b> Percentages and absolute numbers of iNKT cells in the thymus (T), spleen (S) and liver mononuclear cells (L) of WT and Egr-2^f/f-<i>lck</i>-Cre (EGR2KO) mice stained with CD4, CD8, PBS57-loaded CD1d tetramer and TCRβ. <b>(c)</b> Percentages and absolute numbers of iNKT cell subpopulations in gated TCR-β^hiPBS57-CD1dtet⁺ thymocytes from WT and EGR2KO mice. Results representative of five independent pairs in three independent experiments. The bar graphs show the average and SEM of all the experiments. Significance was assessed using an unpaired t-test. ***<0.001, **<0.01.</p

dnRas mice lack iNKT cells.

<p>(<b>a</b>) Thymic profile of WT and dnRas mice (<b>b</b>) Percentages and absolute numbers of iNKT cells in the thymus (T), spleen (S) and liver mononuclear cells (L) of normal littermate controls (WT) and dnRas mice stained with CD4, CD8, PBS57-loaded CD1d tetramer and TCRβ. (<b>c</b>) Percentages and absolute numbers of iNKT cell subpopulations in gated TCR^hiPBS57-CD1dtet⁺ thymocytes from WT and dnRas mice. Results representative of nine independent dnRas and WT pairs analyzed in five experiments<b>,</b> except for the CD44/NK1.1 histograms (n = 2). The bar graphs show the average and SEM of all the experiments. Significance as assessed using a two-tailed unpaired t-test. ***<0.001, **<0.01. (<b>d</b>) Gating strategy used to sort the different populations. (<b>e</b>) Expression of Egr-1, Egr-2 and Id3 in sorted Tet⁺ HSA^hi and Tet⁺ HSA^lo from normal littermate control (WT) and dnRas mice. Bar graphs show relative expression of dnRas compared to WT for three independent experiments. Each experiment was an independent sort of a WT and dnRas pair. Expression in each experiment was normalized to the expression levelis in Tet⁺ HSA^hi WT cells. Significance as assessed using a two-tailed unpaired t-test. **<0.01.</p