Results: 5

1.
Figure 2

Figure 2. SLP-76CKO T cells are unable to transduce TCR-mediated signals. From: Conditional deletion of SLP-76 in mature T cells abrogates peripheral immune responses1.

(A) FACS-sorted CD90.2+YFP+ T cells were left unstimulated or stimulated with anti-CD3 (500A2) for the time periods indicated followed by lysis and western blotting to detect SLP-76, pLAT and pPLCγ1. Total PLCγ1 is shown as a loading control. Representative of three individual experiments. (B) Cell suspensions from lymph node of indicated mice loaded with Indo1 were stimulated by co-crosslinking cell surface CD3 and CD4 (indicated by arrow). Intracellular calcium was measured as a ratio of FL4/FL5. Ionomycin was added to each sample during the last 30 seconds of acquisition. Representative of two experiments (C) Splenocytes stimulated for 15 minutes with 500A2 or PMA were assessed for phopsphorylation of ERK. Dot plots are gated on CD4+ cells. Numbers within the gated areas are relative percentages of YFP+ and YFP gated cells that stain positive for pERK. Representative of cells from 6 SLP-76cHET and 4 SLP-76cKO mice.

Gregory F. Wu, et al. Eur J Immunol. ;41(7):2064-2073.
2.
Figure 4

Figure 4. SLP-76CKO mice are resistant to active EAE induction. From: Conditional deletion of SLP-76 in mature T cells abrogates peripheral immune responses1.

(A) Mice were treated with tam for five consecutive days prior to immunization with MOG35-55 and assessed for clinical signs of EAE. Control mice (SLP-76WT, C57Bl/6 or C57Bl/6×129 mice) (squares; n=4) developed typical ascending paralysis starting at day 11. In contrast, SLP-76CKO mice (triangles; n=3) were asymptomatic throughout. Representative clinical scores from one of three individual experiments are shown. (B) Mononuclear cells were isolated from the CNS of mice at day 28 post-immunization with MOG35-55. Cells were stained and analyzed by flow cytometry. Left panels are gated on live, mononuclear cells and a representative contour plot is shown. Right panels are gated on CD4+ as indicated and histograms indicate levels of cell surface CD44. Representative of data from two separate experiments with a total of six mice.

Gregory F. Wu, et al. Eur J Immunol. ;41(7):2064-2073.
3.
Figure 5

Figure 5. Peripheral T cells expressing a mutant form of SLP-76 fail to transduce TCR-mediated signals despite normal development and TCR levels. From: Conditional deletion of SLP-76 in mature T cells abrogates peripheral immune responses1.

WT and SLP-76flox→Y3F mice were treated with tam for five days. (A) No differences in the expression of CD4 and CD8 (top panels) and TCRβ (bottom panels) on splenocytes analyzed by FACS was detected. Representative of four experiments (B) Cell lysates prepared from splenocytes stimulated with anti-CD3 for the indicated times were used for western blotting to detect phospho-ZAP-70 (p-ZAP-70), phospho-LAT (p-LAT), phospho-PLCγ1 (p-PLCγ1), total PLCγ, phospho-ERK (p-ERK), and total ERK. Representative of two experiments (C) Splenocytes from SLP-76WT (solid line), SLP-76flox→Y3F (dotted line), SLP-76CKO (dashed line) with anti-CD3 overnight and CD69 levels were assessed by flow cytometry on gated CD4+ T cells (top panel) and CD8+ T cells (bottom panel) Shaded areas represent staining of unstimulated cells. Representative of two experiments. (D) Thymidine incorporation was measured after overnight anti-CD3 stimulation of splenocytes from SLP-76WT (solid line), SLP-76CKO (dashed line), and SLP-76flox→Y3F (dotted line) mice. Representative of two experiments.

Gregory F. Wu, et al. Eur J Immunol. ;41(7):2064-2073.
4.
Figure 3

Figure 3. SLP-76 T is required in mature T cells for up-regulation of activation markers and for proliferation. From: Conditional deletion of SLP-76 in mature T cells abrogates peripheral immune responses1.

Splenocytes isolated from SLP-76cHET and SLP-76cKO mice were left unstimulated (shaded histograms) or were stimulated overnight with soluble anti-CD3 (open histograms). Cells were surface stained with CD4, CD8, CD69 and CD25. Expression of CD69 (A) and CD25 (B) on CD4+YFP+ and CD8+YFP+ are shown. Representative of greater than five experiments. (C) Splenocytes were labeled with CFSE and stimulated in vitro for seventy-two hours then stained with CD4 and CD8. Proliferation measured by dilution of CFSE in CD4 gated (left panel) and CD8 gated (right panel) are shown. SLP-76cKO cells are shown with a dotted line and SLP-76cHET by solid line. Representative of two experiments (D) CFSE-labeled Thy1.2+ T cells from SLP-76cHET and SLP-76cKO mice were adoptively transferred into RAG2−/−CD45SJL mice. Suspensions taken from lymph nodes seven days after transfer gated on CD45B6 donor, B220 then CD4 (left) or CD8 (right) are shown. Representative of two experiments. (E) Splenocytes from SLP-76cHet (dashed line) and SLP-76cKO (solid line) were adoptively transferred into CD45SJL and RAG2−/− CD45SJL recipients. BRDU was administered from during days 5 through 7. Lymph node suspensions gated on CD45B6 donor, YFP+ then CD4 (left) or CD8 (right) are shown. Shaded histograms represent BRDU incorporation into cells transferred into non-lymphopenic CD45SJL mice, where no LIP is expected. Representative of 2 experiments with a total of 9 recipient mice in each.

Gregory F. Wu, et al. Eur J Immunol. ;41(7):2064-2073.
5.
Figure 1

Figure 1. SLP-76 deletion in peripheral T cells. From: Conditional deletion of SLP-76 in mature T cells abrogates peripheral immune responses1.

(A) Schematic of temporal deletion approach: Mice with the SLP-76 gene floxed were crossed to mice expressing a tamoxifen-regulated Cre recombinase (CreT2). (Top) Tamoxifen is administered daily for five consecutive days by oral gavage and mice are analyzed from day ten to thirty from the start of tamoxifen. (Bottom) Prior to tamoxifen, a portion of the SLP-76 gene is floxed; a transcriptional stop cassette that blocks transcription of YFP from the Rosa26 locus is also floxed. In the presence of tamoxifen, CreT2 is active resulting in recombination between loxP sites, deletion of SLP-76 and of the stop cassette. This results in cells lacking SLP-76 but expressing YFP. (B) Following tam treatment, splenocytes from SLP-76cHET and SLP-76cKO mice were sorted based on YFP and CD4+ expression. Deletion was assessed at the genomic DNA level by PCR (top) and the RNA level by Tacman based real-time PCR (bottom). Error bars represent standard deviation of triplicate reactions for each cDNA sample (ND=non-detectable). Representative of three individual experiments. (C) Contour plot of CD4 and CD8 gated splenocytes from a SLP-76CKO mouse. Relative percentages of cells within the gated regions are shown. Representative of greater than 10 individual mice.

Gregory F. Wu, et al. Eur J Immunol. ;41(7):2064-2073.

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