T-cell suppression is induced by a nonapoptotic pathway. T cells cultured for 2 days in the presence of IL-15 and TNF-α–activated neutrophils were harvested for flow cytometry or separated into small and large T cells by FACS for proteomic comparison, electron microscopy imaging, adenosine triphosphate (ATP) level determination, and MitoTracker staining. (A) Representative FACS plots of annexin V and Hoechst binding in cell gate for large T cells or FSC^low T cells; the red squares indicate the FSC^low T cells induced by MDSC activity of activated neutrophils (n = 3). (B) Flow cytometric analysis of cytoplasmic presence of cleaved caspase-3 in T cells after indicated culture conditions of 2 days (n = 4). (C) Principal component analysis of mass spectrometry label-free quantification (LFQ) intensities of small (S) and large (L) T cells. (D) Volcano plot representation of a 2-sided non-paired Student t test (small vs large T cells) with an false discovery rate of 0.05 and an S0 value of 2. Proteins more abundant in small T cells compared with large T cells are red and proteins less abundant are blue (see supplemental Table 1 for protein identification). (E) Heat plot representation of LFQ values of proteins annotated for mitochondrial localization (Human MitoCarta 2.0; 1158 entries) affected in this analysis. (F) ATP levels were measured in T cells cultured for 2 days either unstimulated (purple bars), stimulated by IL-15 and TNF-α (blue bars), or in the presence of neutrophils activated by IL-15 and TNF-α. The latter T cells were separated into large (green bars) and small (red bars) T cells (n = 3). (G) Live cell imaging of mitochondria stained with MitoTracker Green and Hoechst in small and large T cells. Shown are representative images of 3 experiments (magnification ×40). (H) Representative electron microscopy photos of small and large T cells (n = 4). Scale bar for top left image is 1 μm; scale bars for other images are 2 μm. *P < .05, ***P < .001. Stim, stimulated; Unstim, unstimulated.