Differential Cytokine Utilization and Tissue Tropism Results in Distinct Repopulation Kinetics of Naïve vs. Memory T Cells in Mice

Naïve and memory T cells co-exist in the peripheral T cell pool, but the cellular mechanisms that maintain the balance and homeostasis of these two populations remain mostly unclear. To address this question, here, we assessed homeostatic proliferation and repopulation kinetics of adoptively transferred naïve and memory T cells in lymphopenic host mice. We identified distinct kinetics of proliferation and tissue-distribution between naïve and memory donor T cells, which resulted in the occupancy of the peripheral T cell pool by mostly naïve-origin T cells in short term (<1 week), but, in a dramatic reversal, by mostly memory-origin T cells in long term (>4 weeks). To explain this finding, we assessed utilization of the homeostatic cytokines IL-7 and IL-15 by naïve and memory T cells. We found different efficiencies of IL-7 signaling between naïve and memory T cells, where memory T cells expressed larger amounts of IL-7Rα but were significantly less potent in activation of STAT5 that is downstream of IL-7 signaling. Nonetheless, memory T cells were superior in long-term repopulation of the peripheral T cell pool, presumably, because they preferentially migrated into non-lymphoid tissues upon adoptive transfer and additionally utilized tissue IL-15 for rapid expansion. Consequently, co-utilization of IL-7 and IL-15 provides memory T cells a long-term survival advantage. We consider this mechanism important, as it permits the memory T cell population to be maintained in face of constant influx of naïve T cells to the peripheral T cell pool and under competing conditions for survival cytokines

Suppression of Dendritic Cell-Derived IL-12 by Endogenous Glucocorticoids Is Protective in LPS-Induced Sepsis

<div><p>Sepsis, an exaggerated systemic inflammatory response, remains a major medical challenge. Both hyperinflammation and immunosuppression are implicated as causes of morbidity and mortality. Dendritic cell (DC) loss has been observed in septic patients and in experimental sepsis models, but the role of DCs in sepsis, and the mechanisms and significance of DC loss, are poorly understood. Here, we report that mice with selective deletion of the glucocorticoid receptor (GR) in DCs (GR^CD11c-cre) were highly susceptible to LPS-induced septic shock, evidenced by elevated inflammatory cytokine production, hypothermia, and mortality. Neutralizing anti-IL-12 antibodies prevented hypothermia and death, demonstrating that endogenous GC-mediated suppression of IL-12 is protective. In LPS-challenged GR^CD11c-cre mice, CD8⁺ DCs were identified as the major source of prolonged IL-12 production, which correlated with elevations of NK cell-derived IFN-γ. In addition, the loss of GR in CD11c⁺ cells rescued LPS-induced loss of CD8⁺ DCs but not other DC subsets. Unlike wild-type animals, exposure of GR^CD11c-cre mice to low-dose LPS did not induce CD8⁺ DC loss or tolerance to subsequent challenge with high dose, but neutralization of IL-12 restored the ability of low-dose LPS to tolerize. Therefore, endogenous glucocorticoids blunt LPS-induced inflammation and promote tolerance by suppressing DC IL-12 production.</p></div

Loss of endotoxin tolerance in GR^CD11c-cre mice.

<p>(A) Tolerizing dose of LPS-induced loss of CD8⁺ DCs in WT but not GR^CD11c-cre mice. Mice were treated with 0.5 μg/g body weight of LPS for 24 hr and rechallenged with 3 μg/g body weight of LPS for 3 hr before splenic DCs were analyzed by flow cytometry. The experiment was repeated three times, with three mice in each group per experiment. (B) High-dose LPS induced IL-12 production by DCs in GR^CD11c-cre mice despite prior exposure to low-dose LPS. Ten to twelve wk female GR^CD11c-cre mice (<i>n</i> = 4) and control mice (<i>n</i> = 5) were treated with 0.5 μg/g body weight of LPS for 24 hr and rechallenged with 10 μg/g body weight of LPS for 6 hr. Percentage of IL-12⁺ DCs in the spleen of a representative mouse is shown (left panel). Average numbers of total IL-12⁺ DCs were calculated based on the total number of splenocytes in each mouse (right panel). (C) Body temperature of mice rechallenged with 10 μg/g body weight of LPS 24 hr after tolerization. Results were pooled from two independent experiments, <i>n</i> = 8 for WT and <i>n</i> = 5 for GR^CD11c-cre. (D) Serum levels of TNF-α, IL-1β, IFN-α, and IFN-γ from mice in (B) were measured 6 hr after rechallenge. (E) Body temperature of mice rechallenged with 10 μg/g body weight LPS 24 hr after tolerization and 1 hr after injection with 250 μg anti-IL-12 or isotype control. Results were pooled from two independent experiments with one to two mice per condition, respectively, <i>n</i> = 3 per condition. The asterisk indicates the time by which all GR^CD11c-cre mice had died. All WT and the GR^CD11c-cre animals treated with anti-IL-12 survived. The data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002269#pbio.1002269.s001" target="_blank">S1 Data</a>.</p

Elevated proinflammatory cytokines in GR^CD11c-cre mice.

<p>(A–C, E, and F) Kinetics of LPS-induced cytokine levels in WT and GR^CD11c-cre mice. The sera used in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002269#pbio.1002269.g001" target="_blank">Fig 1C</a> were also used to determine cytokine concentrations at each time point. Data shown are from four to eight animals per strain per time point pooled from multiple independent experiments. (D) Semiquantitative RT-PCR analysis of IFN-α4 transcription in DC subsets from WT and GR^CD11c-cre mice 1 hr after LPS challenge. β-actin was used as internal control. Results shown are one representative of two independent experiments with pooled splenocytes from two WT and two GR^CD11c-cre mice. (G) Splenocytes were prepared from mice 12 hr after LPS injection, and cultured in vitro for 4 hr with Brefeldin A. Intracellular IFN-γ was measured for gated NK and T cells by flow cytometry. One mouse from each genotype is shown out of two independent experiments, each using three mice per group. (H) Mean fluorescence intensity (MFI) and percentage of IFN-γ⁺ cells in WT and GR^CD11c-cre mice 12 hr post LPS challenge (<i>n</i> = 3). (I) GR^CD11c-cre or WT mice, each represented by an individual symbol, were injected with the indicated doses of neutralizing anti-IL-12 antibody 1 hr before challenge with a lethal dose of LPS (10 μg/g body weight). Surface body temperature at 30 hr is plotted. Results shown are pooled from two independent experiments, each with three to four mice per group. The data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002269#pbio.1002269.s001" target="_blank">S1 Data</a>.</p

GC suppresses in vivo IL-12 production by CD8⁺ DCs.

<p>(A) Six hr after LPS challenge, WT and GR^CD11c-cre splenocytes were analyzed for intracellular IL-12 levels. The right panels are histograms of CD8 staining of CD11c⁺ IL-12⁺ cells. (B) Intracellular IL-12 levels in CD8⁻ and CD8⁺ DCs between WT (shaded) and GR^CD11c-cre (solid line). One representative pair of a group of three is shown. (C) GC suppression of IL-12 production by CD8⁺ DCs ex vivo. Splenocytes were prepared from mice 3 hr after LPS injection and cultured with Brefeldin A in the presence or absence of corticosterone for 4 hr. Intracellular IL-12 levels on gated CD8⁺ DCs from one experiment are shown. The data are representative of three independent pairs of mice.</p

LPS-induced loss of CD8⁺ DCs is GR-dependent.

<p>(A–C) Splenic DCs from WT and GR^CD11c-cre B10.A mice were analyzed by flow cytometry at indicated times after injection with LPS (3 μg/g body weight). Only B220⁻TCRβ⁻ cells are shown. Results shown are from one representative experiment of three independent ones, each with three to five mice per group (D) Percentage of CD8⁺ DCs in WT and GR^CD11c-cre mice at time 0 (steady state). Results shown are from three pooled independent experiments, with 3–4 mice per group. (E) Total number of CD8⁺ and CD8⁻ DCs at time 0, 6 hr, and 24 hr post LPS injection. Results shown are from one representative experiment of three independent ones, each with three to five mice per group. (F) Sensitivity of DC subsets to GC-induced death in vitro. Total splenocytes from WT and GR^CD11c-cre mice were incubated with 100 nM corticosterone in vitro for 6 hr, and the percentage of dead cells in indicated populations are shown. The data shown are representative of four pairs of mice in two independent experiments. The data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002269#pbio.1002269.s001" target="_blank">S1 Data</a>.</p

GR^CD11c-cre mice are more susceptible to LPS-induced septic shock than WT.

<p>(A) and (B) Immunoblot for GR in sorted DCs and other immune subsets from WT and GR^CD11c-cre mice. β-Actin was blotted as loading control. Some lanes were reordered for clarity, which is indicated by vertical white lines. (C) Plasma corticosterone concentrations after LPS challenge. Age- and sex-matched WT and GR^CD11c-cre mice were injected with LPS (3 μg/g body weight), and blood was drawn at indicated time points. Data shown are from four to eight animals per strain per time point pooled from multiple independent experiments. (D) GR^CD11c-cre mice (<i>n</i> = 4–6 per strain for each dose of LPS) were injected with the indicated amount of LPS, surface body temperature was recorded at the indicated times. * Four out of five GR^CD11c-cre mice died by 24 hr after LPS challenge. The data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002269#pbio.1002269.s001" target="_blank">S1 Data</a>.</p

Histone H3 Lysine 27 demethylases Jmjd3 and Utx are required for T-cell differentiation

International audienceAlthough histone H3 lysine 27 trimethylation (H3K27Me3) is associated with gene silencing, whether H3K27Me3 demethylation affects transcription and cell differentiation in vivo has remained elusive. To investigate this, we conditionally inactivated the two H3K27Me3 demethylases, Jmjd3 and Utx, in non-dividing intrathymic CD4+ T-cell precursors. Here we show that both enzymes redundantly promote H3K27Me3 removal at, and expression of, a specific subset of genes involved in terminal thymocyte differentiation, especially S1pr1, encoding a sphingosine-phosphate receptor required for thymocyte egress. Thymocyte expression of S1pr1 was not rescued in Jmjd3- and Utx-deficient male mice, which carry the catalytically inactive Utx homolog Uty, supporting the conclusion that it requires H3K27Me3 demethylase activity. These findings demonstrate that Jmjd3 and Utx are required for T-cell development, and point to a requirement for their H3K27Me3 demethylase activity in cell differentiation