Results: 4

1.
Fig. 2

Fig. 2. Ihh, Ptch1 and Smo are localised on intracellular vesicles that polarise towards the immunological synapse. From: Hedgehog signaling controls T-cell killing at the immunological synapse.

OT-I CTL transduced with Ptch1-YFP and labelled with antibodies to YFP (green), CD8 (red) and Ihh (white). (A, B) show OT-1 CTL alone and (C) conjugated with EL4 target cells. (D) Endogenous Smo (green) and CD8 (white) in OT-I CTL. Single x-y confocal sections are shown. Nuclei are stained with Hoechst (blue). (A:n>75;B,C:n>35;D:n>85. 2-4 independent experiments each). (E) Individual frames of a movie (Suppl. Movie 1) showing OT-I CTL nucleofected with Smo-EGFP and PACT-RFP (centrosome marker, red), forming a synapse with EL4 target cells (blue). Time after initial contact is shown in minutes (n=33). Scale bars: 5μm.

Maike de la Roche, et al. Science. 2013 December 6;342(6163):1247-1250.
2.
Fig. 1

Fig. 1. TCR activation triggers Hh signaling and expression of Hh components in CD8 T cells. From: Hedgehog signaling controls T-cell killing at the immunological synapse.

(A) Graphs showing mRNA levels of Gli1 in naïve CD8 T cells (left) and CTL (right) at times shown after TCR cross-linking with plate-bound anti-CD3 antibody relative to CD3ε as a reference gene. Similar results were obtained using TBP as a reference gene; n=3 (naïve) or 2 (CTL); data are mean +/− SD. Cells plated without anti-CD3 showed no Gli1 induction over 12h. (B) Immunoblot analysis of protein expression of Ptch, Gli1, Ihh and actin at 0, 24 and 48h after TCR stimulation in naïve CD8 T cells; n=3. Molecular weights are shown in kDa. Similar results were also obtained from CD8 T cells derived from C57BL/6 and BALB/c mice (not shown). (C and D) Naïve CD8 T cells were purified from spleens of WT or Lckoff mice and stimulated for 12h with plate-bound anti-CD3 antibody. (C) Graphs showing mRNA levels of Lck (left) and Gli1 (right) in Lckoff CD8 T cells relative to WT control; n=2, data are mean +/− SD. (D) Immunoblot analysis of Ptch, Gli1, Lck and actin in Lckoff and WT control CD8 T cells after 12h of TCR stimulation; n=3. Molecular weights are shown in kDa.

Maike de la Roche, et al. Science. 2013 December 6;342(6163):1247-1250.
3.
Fig. 4

Fig. 4. Hh signaling in CTL controls Rac1 expression and actin reorganisation at the immunological synapse. From: Hedgehog signaling controls T-cell killing at the immunological synapse.

(A-C) Mx1-Cre Smocond/+(A) and Mx1-Cre Smocond/cond CTL (B) were conjugated to P815 target cells for 15min, fixed and stained using antibodies against CD8, γ-tubulin, and actin. Single x-y confocal sections and en face (y-z) constructions through the synapse are shown, demonstrating that the actin ring does not form properly in Mx1-Cre Smocond/cond CTL. Nuclei stained with Hoechst (blue). Scale bars: 5μm. (C) Quantitation of actin clearance at the immunological synapse, depicting the percentage of CTL in which actin remains distributed throughout the synapse (not cleared), show an intermediate phenotype or is cleared to form an actin ring (Smocond/+n=47; Mx1-Cre Smocond/condn=62). Immunoblot analyses of protein expression of Rac1, actin and calnexin (D) at 0, 24 and 48h after TCR stimulation in naïve CD8 T cells (n=3) and (E) Mx1-Cre Smocond/+ and Mx1-Cre Smocond/cond CTL (n=2). Molecular weights are shown in kDa. (F) Graph showing mRNA levels of Rac1 in Mx1-Cre Smocond/cond CTL relative to Mx1-Cre Smocond/+ control CTL n=3, data are mean +/− SD.

Maike de la Roche, et al. Science. 2013 December 6;342(6163):1247-1250.
4.
Fig. 3

Fig. 3. Hh signaling is required for CTL killing and centrosome polarisation to the immunological synapse. From: Hedgehog signaling controls T-cell killing at the immunological synapse.

(A) qPCR analysis of Smo mRNA expression in Mx1-Cre Smocond/cond and control Mx1-Cre Smocond/+ CTL relative to CD3ε as a reference gene (left); n=4, data are mean +/− SD. qPCR analysis showing increase in Gli1 mRNA levels 2h after TCR activation with plate-bound anti-CD3 antibody for Mx1-Cre Smocond/+ and Mx1-Cre Smocond/cond CTL relative to unstimulated (right); n=3, data are mean +/− SD. (B) Representative staining of endogenous Smo (green) expression in Mx1-Cre Smocond/+ and Mx1-Cre Smocond/cond CTL. Nuclei stained with Hoechst (blue); n>100. Scale bars: 5μm. (C) Mx1-Cre Smocond/+ and Mx1-Cre Smocond/cond CTL were stimulated with plate-bound anti-CD3 antibody for times indicated and blotted for protein expression of pERK and ERK; n=2. Molecular weights are shown in kDa. (D) Percentage lysis of P815 target cells by Mx1-Cre Smocond/+ and Mx1-Cre Smocond/cond CTL at effector:target (E:T) ratios shown (n=6). (E) Centrosome position relative to clustered Lck was classified as <1μm (docked), 1-3μm (proximal) or >3μm (distal) as percentage of conjugates of Mx1-Cre Smocond/+ (n=129) and Mx1-Cre Smocond/cond (n=121) with P815 targets as depicted in Figure S4 (n=3), data are mean +/− SD. (F) Immunoblots of cell lysates from OT-I CTL treated with 5μM vismodegib for 36h, probed with antibodies against Gli1, Ptch, ERK, Lck, granzyme A, perforin and actin; n=2. (G) OT-I CTL treated with 10μM cyclopamine for 24h, stimulated with plate-bound anti-CD3 antibody for times indicated and blotted for protein expression of pERK and ERK; n=2. Molecular weights are shown in kDa. (H) Percentage lysis of EL4 target cells by OT-I CTL treated with vismodegib at concentrations stated; n=5. x-axis shows varying CTL effector to target (E:T) ratios. (I) OT-I CTL treated with vismodegib (5μM) were labelled with antibodies against Lck, γ-tubulin, and CD8. Quantitation of centrosome polarisation after treatment is shown (n>60).

Maike de la Roche, et al. Science. 2013 December 6;342(6163):1247-1250.

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