Flow cytometric evaluation of the γδ T cell receptor (TCR) repertoire in control vs. early and late stage tumor-bearing mice (distribution on left panels, x ± SD, right panels, * = p<0.05).

<p>There were no significant changes in the Vγ1, Vγ4, or Vγ7 subsets from controls either post-tumor injection day 11±1 or at end stage. Additionally, there were no changes in the proportions of Vδ1, Vδ4, and Vδ6.3 at day 10. A significant decrease of the %Vδ6.3 population at end stage was offset by a parallel increase in %Vδ4 cells. TCR subsets Vγ3 and Vδ3 were also measured, however, relative proportions were negligible <i>(data not shown)</i>.</p

Cytotoxicity and growth inhibition assessment of activated γδ T cells against GL261 tumors.

<p><i>(a)</i> Activated γδ T cells were incubated with GL261 cells either in suspension (closed circles) or attached to plastic (closed squares) show robust <i>in vitro</i> cytotoxicity against the non-adherent GL261 cells in a 4h standard assay (x ± SD, * = p<0.05). The insert is a high-power confocal micrograph of adherent GL261 cells cytoplasm stained with wheat germ agglutinin (green) showing microfilopodia spreading from the attached cells as well as tumor from an athymic nude mouse was harvested 10 days following GL261 cells, chopped and disaggregated in media, fixed/post-fixed in in glutaraldehyde and osmium tetroxide, dehydrated in ethanol, and embedded in epoxy resin. Filopodia (clear arrows) and microspikes (filled arrows) are seen on the cell surface in these transmission electron micrographs from tumor sections. <i>(b)</i> Injection of 5 x 10⁵ GL261 cells followed 15m later by 1.5 x 10⁶ activated γδ T cells (dashed line) did not improve median survival when compared to saline-injected mice (solid line). <i>(c)</i> Evidence of local tumor inhibition was noted in histologic examination. Representative histologic specimens show a much smaller tumor at the injection site (arrow) in the γδ T cells injected mouse (B) than from a control mouse (A) at 25 days.</p

Circulating γδ T cell enumeration and function in tumor-bearing mice.

<p><i>(a)</i> The circulating T cell count (distribution on left panels, x ± SD, right panels, * = p<0.05) was increased in tumor-bearing mice at 10 days post-GL261 injection independent of the total T cell count. Terminal γδ T cell counts in tumor-bearing mice also fell significantly lower than controls and day 10 glioma-bearing mice. <i>(b)</i> Approximately 12% of γδ T cells constitutively produced IFN-γ with no additional increase following PMA/Ionomycin stimulation. <i>(c)</i> Annexin V expression was upregulated on γδ T cells from glioma-bearing mice, also independently from the total T cell population indicating simultaneous γδ T cell proliferation and apoptosis likely due to activation-induced cell death (AICD). <i>(d)</i> The γδ T cell count and Annexin V expression were measured in unmanipulated (e.g. no intracranial injection) mice and 10 days following IC injection of the methylcellulose vehicle in which GL261 cells were suspended to determine if IC injection-related injury produced the same increase in γδ T cell count and Annexin V expression as tumor injection. There was no difference in either parameter between sham-injected mice and mice that received intracranial methylcellulose vehicle alone.</p