Lineage specification in innate lymphocytes

International audienceInnate lymphoid cells (ILCs) are immune cells that lack specific antigen receptors but possess similar effector functions as T cells. Concordantly, ILCs express many transcription factors known to be important for T cell effector function. ILCs develop from lymphoid progenitors in fetal liver and adult bone marrow. However, the identification of ILC progenitor (ILCP) and other precursors in peripheral tissues raises the question of whether ILC development might occur at extramedullary sites. We discuss central and local generation in maintaining ILC abundance at peripheral sites

Blocked O-GlcnAc cycling disrupts mouse hematopoeitic stem cell maintenance and early t cell development

International audienceSmall numbers of hematopoietic stem cells (HSCs) balance self-renewal and differentiation to produce the diversity and abundance of cell types that make up the blood system. How nutrients are recruited to support this massive differentiation and proliferation process remains largely unknown. The unique metabolism of adult HSCs, which rely on glycolysis and glutaminolysis, suggests a potential role for the post-translational modification O-GlcNAc as a critical nutrient signal in these cells. Glutamine, glucose, and other metabolites drive the hexosamine biosynthetic pathway (HBP) ultimately leading to the O-GlcNAc modification of critical intracellular targets. Here, we used a conditional targeted genetic deletion of the enzyme that removes O-GlcNAc, O-GlcNAcase (OGA), to determine the consequences of blocked O-GlcNAc cycling on HSCs. Oga deletion in mouse HSCs resulted in greatly diminished progenitor pools, impaired stem cell self-renewal and nearly complete loss of competitive repopulation capacity. Further, early T cell specification was particularly sensitive to Oga deletion. Loss of Oga resulted in a doubling of apoptotic cells within the bone marrow and transcriptional deregulation of key genes involved in adult stem cell maintenance and lineage specification. These findings suggest that OGlcNAc cycling plays a critical role in supporting HSC homeostasis and early thymocyte development

Effector/memory CD4 T cells making either Th1 or Th2 cytokines commonly co-express T-bet and GATA-3.

Naïve CD4 T (NCD4T) cells post-activation undergo programming for inducible production of cytokines leading to generation of memory cells with various functions. Based on cytokine based polarization of NCD4T cells in vitro, programming for either 'Th1' (interferon-gamma [IFNg]) or 'Th2' (interleukin [IL]-4/5/13) cytokines is thought to occur via mutually exclusive expression and functioning of T-bet or GATA-3 transcription factors (TFs). However, we show that a high proportion of mouse and human memory-phenotype CD4 T (MCD4T) cells generated in vivo which expressed either Th1 or Th2 cytokines commonly co-expressed T-bet and GATA-3. While T-bet levels did not differ between IFNg-expressing and IL-4/5/13-expressing MCD4T cells, GATA-3 levels were higher in the latter. These observations were also confirmed in MCD4T cells from FVB/NJ or aged C57BL/6 or IFNg-deficient mice. While MCD4T cells from these strains showed greater Th2 commitment than those from young C57BL/6 mice, pattern of co-expression of TF was similar. Effector T cells generated in vivo following immunization also showed TF co-expression in Th1 or Th2 cytokine producing cells. We speculated that the difference in TF expression pattern of MCD4T cells generated in vivo and those generated in cytokine polarized cultures in vitro could be due to relative absence of polarizing conditions during activation in vivo. We tested this by NCD4T cell activation in non-polarizing conditions in vitro. Anti-CD3 and anti-CD28-mediated priming of polyclonal NCD4T cells in vitro without polarizing milieu generated cells that expressed either IFNg or IL-4/5/13 but not both, yet both IFNg- and IL-4/5/13-expressing cells showed upregulation of both TFs. We also tested monoclonal T cell populations activated in non-polarizing conditions. TCR-transgenic NCD4T cells primed in vitro by cognate peptide in non-polarizing conditions which expressed either IFNg or IL-4/5/13 also showed a high proportion of cells co-expressing TFs, and their cytokine commitment varied depending on genetic background or priming conditions, without altering pattern of TF co-expression. Thus, the model of mutually antagonistic differentiation programs driven by mutually exclusively expressed T-bet or GATA-3 does not completely explain natural CD4 T cell priming outcomes

Cells activated in non-polarizing conditions in vitro from other mouse strains and humans show co-expression of T-bet and GATA-3.

<p>(A) Dose response curve from one experiment to show secreted cytokine levels in primed and recalled cells from B6, B6aged, BALB.b and FVB. Data representative of 3 independent experiments. (B) Representative two-color plots of T-bet and GATA-3 expression in naive and primed cells as overlays. Data representative of 5 experiments. (C) Dose response curve from one experiment to show secreted cytokine levels in primed and recalled cells from B6 and IFNg-/- mice. Data representative of 3 independent experiments. (D) A representative two-color plot of T-bet and GATA-3 expression in naive and primed cells from B6 and IFNg-/- mice as overlays. Data representative of 3 experiments. (E) A representative plot to show intracellular cytokine staining in P+I treated 72 h primed human NCD4T cells. (F) Two color plots of in vitro primed human CD4 cells to show T-bet and GATA-3 expression in indicated populations as overlays. (G) Data from independent donors compiled to show relative T-bet and GATA-3 MFI increases over naive cells in IFNg+ and IL-4+ human CD4 T cells.</p

Naive CD4 T cells activated in non-polarizing conditions in vitro show pattern similar to memory cells generated in vivo.

<p>(A) Dose response curve for primed and recalled NCD4 T cells from B6 mice from one experiment to show secreted cytokine levels in primed and recalled cells. Data representative of >5 independent experiments. Cytokine values for naive cells were <10 pg/ml. (B) A representative profile to show T-bet and GATA-3 levels in naive and in vitro primed CD4 cells from B6 mice. (C) Data from 7 independent experiments compiled to show relative MFI values (fold increase over isotype control) for T-bet and GATA-3 (mean ± s.e.) from B6 mice. (D) Data from 5 sets of independent experiments compiled to show relative mRNA levels, normalized to NCD4T cell values, 72 h after priming (mean ± s.e.). (E) Dual color ELISpot assay to show number of spots (mean ± s.e.). Data representative of 4 independent experiments on B6 T cells. (F) Primed NCD4T cells from B6 mice show large presence of IFNg producing cells after P+I treatment. (G) Data from independent experiments on NCD4T cells from B6 mice compiled to show relative T-bet and GATA-3 MFI increases over naive cells in IFNg+ and IL-13+ cells (mean ± s.e.). (H) NCD4 T cells from BALB/c mice primed for 72 h in vitro and recalled to score single and dual cytokine producing cells. Frequency of cytokine producing cells from naive and primed T cell groups (mean ± s.e., n = 3).</p

Ova-II-specific effector/memory DO11.10 cells expressing IFNg or IL-4/5/13 co-express T-bet and GATA-3.

<p>(A) A representative plot from draining lymph node cells to show IFNg and IL-4/5/13 producing cells on recall. Lymph node cells gated as KJ1-26+CD69+CD44+ were analyzed for intracellular cytokine staining. (B) IFNg+ or IL-4/5/13+ cells from (A) showing expression of T-bet and GATA-3 as compared to naive CD4 T cells. (C) Ratios of T-bet and GATA-3 MFIs were calculated for IFNg+ or IL-4/5/13+ cells. (Mean ± s.e.).</p

Variation in the duration and dose of antigenic exposure alters Th1/Th2 balance post-priming in vitro.

<p>(A) IFNg secretion pattern after 48 or 72 h activation of DO11.10 in vitro in recall response (mean ± s.e.). Data compiled from 7 experiments. (B) IL-4 secretion pattern after 48 or 72 h activation of DO11.10 in vitro during recall (mean ± s.e.). Data compiled from 7 experiments. (C) A representative two-color plot of T-bet and GATA-3 expression in naive, 48 h primed and 72 h primed DO11.10 cells as overlays. (D) IFNg secretion pattern after low or high dose of Ova-II mediated activation of DO11.10 in vitro during recall (mean ± s.e.). Data compiled from 5 experiments. (E) IL-4 secretion pattern after low or high dose of Ova-II mediated activation of DO11.10 in vitro during recall (mean ± s.e.). Data compiled from 5 experiments. (F) A representative two-color plot of T-bet and GATA-3 expression in DO11.10 cells primed with 0.01 and 0.1 μg/ml Ova-II peptide for 72 h along with naive cells as overlays.</p

In vivo generated memory cells isolated during steady state from multiple strains show co-expression of T-bet and GATA-3.

<p>Dose response curve for IFNg (A) and IL-13 (B) production by sorted MCD4T cells stimulated for 24 h in vitro from groups as shown. One representative experiment out of three with similar trends shown. (C) Data from independent experiments to show relative T-bet and GATA-3 MFI increases over naive cells in B6, B6.aged and FVB memory cells (mean ± s.e., n = 3). The isotype control profiles showed no difference in naive and memory cell staining in each strain. (D) A two color plot to show T-bet and GATA-3 expression in NCD4T, GFP-ve MCD4T and GFP+ve MCD4T cells from 4Get mice. (E) T-bet/GATA-3 MFI ratios from GFP-ve MCD4T and GFP+ve MCD4T cells obtained from independent experiments (mean ± s.e.). (F) A representative profile of NCD4T, GFP-ve IFNg+ve and GFP+ve IL-4/5/13+ve cells from 4Get mice for T-bet and GATA-3 expression. (G) T-bet/GATA-3 MFI ratios from GFP-ve IFNg+ve MCD4T and GFP+ve IL-4/5/13+ve MCD4T cells obtained from independent experiments (mean ± s.e.). (H) Intracellular cytokine staining of MCD4T cells from B6 and IFNg-/- mice stimulated with P+I. (I) Data from independent experiments showing frequencies of IFNg+ve and IL-4/5/13+ve cells from P+I stimulated MCD4T cells from B6 and IFNg-null mice (mean ± s.e.). (J) Data from independent experiments to show relative T-bet and GATA-3 MFI increases over isotype controls in NCD4T, MCD4T and IL-4/5/13+ve MCD4T from B6 and IFNg-/- mice (mean ± s.e.).</p

Peptide-MHC mediated non-polarizing activation mimics polyclonal activation pattern.

<p>(A) Dose response curve to show secreted cytokine levels in primed and recalled DO11.10 cells (mean ± s.e.). Data compiled from 5 independent experiments. (B) A representative profile to show T-bet and GATA-3 upregulation in naive and 72 h primed DO11.10 cells. (C) Data from independent experiments compiled to show T-bet and GATA-3 MFI in naive and primed DO11.10 cells. (D) Intracellular cytokine staining of naive and primed DO11.10 cells after P+I treatment. (E) Data from independent experiments compiled to show relative T-bet/GATA-3 ratios in IFNg+ and IL-13+ DO11.10 cells (mean ± s.e.).</p