Fig. 2

Serial fractionation of Tu167 conditioned growth medium (CGM) identifies that the >200 kDa fraction induces CD3⁺ T cells to have a suppressor phenotype: CD27/CD28 loss, suppression of responder T cell proliferation, and their reduction in the antitumor cytokine IFN-γ. After 70% confluence of the head and neck carcinoma tumor cell line Tu167, its CGM was spun down for 15 min at 3000 x g to remove cell debris. a. Tu167 CGM was first added to the ultrafiltration centrifuge tube that had the largest MWCO filter. Flow-through permeate was collected and added to the next smallest MWCO filter. This process was repeated until after the smallest MWCO filter. Retentate from each filter and permeate from smallest MWCO filter were then incubated with freshly purified T cells for 6 h. T cells were then washed and cultured for 7 days before staining for CD27/CD28 loss by flow cytometry. Dot plots are gated on CD3⁺. b. Bar graphs are the combination of five independent experiments and error bars represent ± SDEV. c to d. T cells were incubated under the above conditions and then mixed at a 1:2 ratio with autologous responder T cells for 3 days under anti-CD3/CD28 stimulation. Cells were then permeabilized and labeled with BV785, PE-Cy7, APC-R700-conjugated antibodies towards CD3, Ki67, and IFN-γ, respectively. Bar graphs represent the percent CD3⁺Ki67⁺ (c), and CD3⁺ IFN-γ⁺ (F) cells. Data represents the mean +/− SD of 3. To compare means, a one-way analysis of variance was used with a Bonferroni multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 for the indicated groups vs. unfractionated conditioned media