Proliferation and cytokine secretion from wild-type and Sfpi1^lck−/− γδ T cells.

<p>(A) Wild-type and <i>Sfpi1</i>^lck−/− mice were injected with BrdU and after 24 hours thymocytes were stained with antibodies to γδ TCR and BrdU. Results are average ± SEM. (B–C) Splenocytes were stained with CFSE and stimulated with anti-CD3 and anti-CD28, and analyzed for the CFSE staining on γδ T cells after 72 hours. CFSE staining profiles are shown (B). The mean fluorescence intensity of CFSE and the proliferation index of the populations are presented as the mean ± SEM of three mice. (D) γδ T cells from spleen of wild-type and <i>Sfpi1</i>^lck−/− mice were stained with PE-Annexin V and percentages of annexin V positive cells are indicated. (E) RNA was isolated from sorted γδ T cells from spleen and thymus of wild-type and <i>Sfpi1</i>^lck−/− mice before analysis of mRNA using qPCR. Results are the average ± SEM of 5–6 mice. (F-G) γδ T cells were purified from spleen by flow cytometry and stimulated with anti-CD3 for 3 days before IFN-γ and IL-17 concentrations in supernatants were quantified using ELISA. Data are representative of two independent experiments and represent mean ± SEM of replicate samples. (H) γδ T cells in spleen from wild-type and <i>Sfpi1</i>^lck−/− mice were stained using antibody specific for CCR6. Numbers are γδ T cells in each subpopulation and are averages ± SEM of three mice. *, Significantly different from the WT, p<0.05 determined by Student's t test.</p

Increased γδ T cells in <i>Sfpi1</i>^lck−/− mice.

<p>(A) γδ TCR+ thymocytes and γδTCR+ splenocytes were sorted from wild-type and <i>Sfpi1</i>^lck−/− mice and genomic DNA was analyzed for the presence of the wild-type (WT; +), floxed (fl), or deleted (Δ) allele. (B) Flow cytometric analysis of γδ T cells in spleen, thymus and intestine (intra-epithelial lymphocytes) from wild-type and <i>Sfpi1</i>^lck−/− mice using antibodies specific for CD3 and pan-γδ TCR. Numbers in dot plots represent the mean ± SEM of 10–12 mice. The absolute number of γδ T cells was calculated by multiplying the total cell number recovered from each organ by the percentage of γδ T cells. Results are an average of 10–12 mice. (C) Flow cytometric analysis of γδ T cells in spleen from wild-type and <i>Sfpi1</i>^lck−/− mice using antibodies specific for pan-γδ TCR, CD62L and CD44. Numbers are γδ T cells in each subpopulation and are averages ± SEM of three mice. *Significantly different from WT, p<0.05 determined by Student's t test.</p

T cell development is normal in <i>Sfpi1</i>^lck−/− mice.

<p>(A) Flow cytometric analysis of cells in spleen and thymus from <i>Sfpi1</i>^lck−/− mice and wild-type control mice. The CD3+ αβTCR+ and CD3+ NK1.1+ T cell percentages are presented as mean ± SEM of six mice. Percentages were not significantly different between WT and <i>Sfpi1</i>^lck−/− mice (p>0.05). (B) Thymocytes from wild-type control mice were sorted into double-negative subpopulations and RNA was analyzed for <i>Sfpi1</i> expression. (C) The indicated T cell populations were sorted and RNA was analyzed for <i>Sfpi1</i> expression by qPCR. Results are averages ± SEM of values from three mice. ND, not detected.</p

data_sheet_1_PU.1 Is Required for the Developmental Progression of Multipotent Progenitors to Common Lymphoid Progenitors.PDF

<p>The transcription factor PU.1 is required for the development of mature myeloid and lymphoid cells. Due to this essential role and the importance of PU.1 in regulating several signature markers of lymphoid progenitors, its precise function in early lymphopoiesis has been difficult to define. Here, we demonstrate that PU.1 was required for efficient generation of lymphoid-primed multipotent progenitors (LMPPs) from hematopoietic stem cells and was essential for the subsequent formation of common lymphoid progenitors (CLPs). By contrast, further differentiation into the B-cell lineage was independent of PU.1. Examination of the transcriptional changes in conditional progenitors revealed that PU.1 activates lymphoid genes in LMPPs, while repressing genes normally expressed in neutrophils. These data identify PU.1 as a critical regulator of lymphoid priming and the transition between LMPPs and CLPs.</p

The Closely Related CD103⁺ Dendritic Cells (DCs) and Lymphoid-Resident CD8⁺ DCs Differ in Their Inflammatory Functions

<div><p>Migratory CD103⁺ and lymphoid-resident CD8⁺ dendritic cells (DCs) share many attributes, such as dependence on the same transcription factors, cross-presenting ability and expression of certain surface molecules, such that it has been proposed they belong to a common sub-lineage. The functional diversity of the two DC types is nevertheless incompletely understood. Here we reveal that upon skin infection with herpes simplex virus, migratory CD103⁺ DCs from draining lymph nodes were more potent at inducing Th17 cytokine production by CD4⁺ T cells than CD8⁺ DCs. This superior capacity to drive Th17 responses was also evident in CD103⁺ DCs from uninfected mice. Their differential potency to induce Th17 differentiation was reflected by higher production of IL-1β and IL-6 by CD103⁺ DCs compared with CD8⁺ DCs upon stimulation. The two types of DCs from isolated lymph nodes also differ in expression of certain pattern recognition receptors. Furthermore, elevated levels of GM-CSF, typical of those found in inflammation, substantially increased the pool size of CD103⁺ DCs in lymph nodes and skin. We argue that varied levels of GM-CSF may explain the contrasting reports regarding the positive role of GM-CSF in regulating development of CD103⁺ DCs. Together, we find that these two developmentally closely-related DC subsets display functional differences and that GM-CSF has differential effect on the two types of DCs.</p></div

CD103⁺ DCs and CD8⁺ DCs differ in expression of costimulatory molecules, inflammasomes and TLR.

<p>(<b>A</b>) Cells of pooled cutaneous LNs from Langerin-EGFP mice and Langerin-EGFP/<i>CD103−/−</i> mice were analyzed. CD103⁺ DCs were identified as CD326⁻CD205⁺langerin⁺ within migratory DCs (mDC, CD11c^intMHC II^high); CD8⁺ DCs were identified as CD205⁺ CD8⁺ within cDCs (CD11c^highMHC II^int). Histograms show the expression of CD103 and langerin-EGFP by CD8⁺ and CD103⁺ DCs. For CD103 expression, CD8⁺ DCs (grey dot line) and CD205⁺CD11b⁻ migratory DCs (equivalent of CD103⁺ DCs, black dot line) from CD103−/− mice were included. (B) CD8⁺ and CD103⁺ DCs from B6 mice were analyzed for the expression of costimulatory molecules. (C&D) CD8⁺ and CD103⁺ DCs from B6 mice were sorted. RT-qPCR was performed for the indicated transcripts with 3 reference genes as controls. One of three repeated experiments is shown.</p

CD103⁺ DCs and CD8⁺ DCs induce naïve T cells to produce different cytokines.

<p>DC subsets from peripheral LNs of B6 mice were sorted. (A&B) DCs (10⁴/well in triplicate) were cultured with CellTrace violet-labeled OT-II cells (5×10⁴/well) with or without 1 mg/mL OVA for 3 d. Cell proliferation and supernatant cytokine levels were determined. (A) CD4⁺ T cell proliferation; (B) Cytokine production. Histograms show proliferation profile of OT-II, Bar graph shows mean ± SEM (*P<0.05, **P<0.01; Two tail Student's T test). (C) DCs (10⁴/well in triplicate) were cultured with CellTrace violet-labeled OT-I cells (5×10⁴/well) with or without 0.1 mg/mL OVA for 3 d. Cell proliferation and supernatant cytokine levels were determined. Bar graph shows mean ± SEM (**P<0.01; Two tail Student's T test).</p

CD103⁺ DCs and CD8⁺ DCs have differential capacity to induce Th17 upon viral infection.

<p>(A) Proliferation of 5×10⁴ CFSE-labeled HSV-specific CD4⁺ T cells (gDT-II) after 60 h of culture together with serial dilutions of DC subsets isolated from brachial lymph nodes of mice infected withn HSV 5 d earlier. (B) Culture supernatants from (A) were measured for the indicated cytokines. (C) Proliferation and cytokine production with isolated DCs and exogenous source of antigenic peptide. Data (mean ± SEM) are one of two individual experiments. (**P<0.01; Two tailed Student's T test).</p

Elevated GM-CSF increase CD103⁺ DCs.

<p>DC-enriched LN cells were isolated from pooled LN for individual GMtg mice and wild type littermates. DC-enriched LN cells were stained for cell surface markers. (A) Gated CD11c⁺ cells were segregated into migratory DC (MHC class II^hi and CD11c^int) and lymphoid-resident DC (MHC class II^int and CD11c^hi) fractions. Numbers inside dot plots indicate percentage of gated populations. (B) CD103 expression by CD103⁺ DCs and CD8⁺ DCs is shown. (C) Mean numbers of DC subsets from cutaneous LNs of individual mice in each group are shown in bar graphs. Data are representative of >3 experiments. (D) Number of CD103⁺ DCs in ear skin. Each dot represents the data derived from an individual mouse. Horizontal line shows the mean ± SEM. Data are pooled from two experiments (*P<0.05, **P<0.01; Two tailed Student's T test).</p

CD103⁺ DCs and CD8⁺ DCs produce different cytokines and chemokines.

<p>DCs were sorted from B6 mice and cultured at 5000 DCs per well in 96-well plates with (CpG or Poly I:C) or without (Nil) stimuli for 24 h. Exogenous GM-CSF (2 ng/mL) was included in cultures. Culture supernatants were assayed for cytokines (A) and chemokines (B). Data (mean ± SEM) are one of three individual experiments (*P<0.05, **P<0.01; Two tailed Student's T test).</p