Acute Myca. treatment induces tumor cell softness, reduction in early signaling markers, and a loss of actin foci and WASP activation in tumor-CTL synapses.

(A) Schematic of the pre-treatment of B16 cells with DMSO or 1µM Mycalolide A (Myca) in B16-CTL co-culture assays. (B) AFM setup for B16 stiffness measurements. (C) comparison of DMSO (control) or Myca.-treated B16 stiffness. Each bar in the plot represents the value obtained from a single cell, the p-value of the distributions is <0 .00001, as determined using Mann-Whitney two-tailed test. (D-G) Effects of Myca treatment on events at the B16-CTL synapses. Cells were fixed after 5 min for confocal imaging (D-F) or cultured for 4 h followed by assessment of target cell killing by Sytox staining (G). Images in (D) and (F) show the maximum intensity projected en-face view of F-actin in the CTL/target interfaces. The actin foci are demarcated in white dotted circles (D, left panel), or arrows (F, left panel). The graphs show quantification of F-actin foci (D), phospho-CD3ζ and Zap70 (E), and phospho-WASP (F). Each point in the scatter plot represents the values obtained from individual cells; the bars show Mean± SEM. These experiments were repeated at least thrice with similar results. (G) In vitro killing of B16F10 target cells as a function of Effector: Target (E: T) ratio. Each data point represents values obtained from four independent samples as Mean ± SEM. This experiment was repeated twice with similar results. Scale bar in the images, 5µm.

(A-B) NPF activity of WASP is required for TCR activation at B16-CTL synapse and CTL cytotoxicity. The actin foci, associated mechanical stress, and cytotoxicity can be restored by overexpression of WASP in WASP-/- CTLs.

(A) Confocal images showing a side view of F-actin in the B16-pmel-1 cell synapses. Pmel-1 cells transfected with either GFP-WASP (WT) or GFP-WASPΔC (WASPΔC) were allowed to form synapses with B16 cells for 5min. The graphs show F-actin foci and phospho-CD3ζ intensity at B16-pmel-1 synapses. Each point in the scatter plot represents the values obtained from individual cells; the bars show Mean± SEM. This experiment was repeated thrice with similar results. p-values were obtained using Mann-Whitney two-tailed test; p***<0.0001, and **<0.005. (B) Cytotoxic activity of WT or WASPΔC expressing pmels. The data points in the graph represent Mean ± SEM from four replicates. (C) Images showing actin foci and pCasL levels in synapses formed between B16 cells and WT or WASP-/- pmel-1 CTLs, either expressing empty control vector (‘WT or WASP-/-’) or WASP construct. The graph shows the percentage of cells with discernable actin foci in the synapse. (D) Cytotoxic activity of WASP overexpressing pmels compared to the WT or WASP-/- pmels. The experiment was conducted as described in (B). Scale bar in the images, 5µm.

CTLs show an increased generation of mechanical forces and activation on stiffer substrates in a WASP-dependent manner.

(A) Experimental setup of the traction force measurements during CTL activation on gels covalently functionalized with anti-CD3 and ICAM-1. (B) Shows representative images of traction force maps in CTL-substrate interfaces, and traction force values as a function of substrate stiffness (the graph on the right) p values, ns>0.5, *=0.03 **=0.007. (C-F) The levels of pZap70, Talin, F-actin, F-actin foci, and pWASP intensities at the CTL-substrate interfaces. In graphs (D-F) the points represent mean ± SEM values. p-values were obtained using Mann-Whitney two-tailed test by comparison of values from WT and WASP-/- cells for each stiffness; p**=0.007, and *=0.03. These experiments were repeated at least twice with similar results.

WASP-dependent CD8+ T cell activation, and in-vivo anti-tumor efficacy of WASP-deficient CTLs.

Surface expression of LFA-1 (A), CD69 (B), or cytokine production (C-D) was assessed after activation of CD8+T cells with indicated reagents for 24h. (E) The activation-induced proliferation of CD8+T cells was determined using a CFSE dilution assay. All these experiments were repeated at least three times with similar results. The bar graphs represent mean ± SEM. p-value *<0.05; ***<0.005, as determined by paired two-way t-test. (F, G) C57BL/6 mice lacking WASP in their immune cells show significantly higher growth of tumors. WT or WASP-/- animals were subcutaneously injected with 0.5 million B16 cells. Tumor growth was measured using the calipers method at the indicated time points. Each data point represents data from at least six animals, and the experiment was repeated four times. (H) Adoptive transfer of WT or WASP-/- pmel-1 CTLs (left graph) or OTI CTLs in animals bearing B16 (left panels) or B16F10-ova (right panels) tumors respectively. Tumor growth was measured and plotted as described in (I-J). This experiment was repeated thrice with similar results.