If desired, this direct effect could be avoided by using antigen-specific cellular stimuli, such as antigen-specific T-lymphocytes stimulated with antigen pulsed presenting cells. CD3/28 antibody as the T-lymphocyte stimulus. We propose model-specific validation of microbead-based MDSC assays, or use of an alternative stimulation approach such as plate bound CD3/28 antibodies. evaluation of MDSCs is usually complicated by their poor survival in culture and tendency to differentiate into mature myeloid cells when cultured in the presence of growth factors such as GM-CSF [7]. A variety of assays have been used to measure the immunosuppressive capacity of MDSCs. Mixed leukocyte assays evaluating the impact of MDSCs on T-lymphocytes stimulated with anti-CD3/anti-CD28 coated microbeads have become popular due to their relative simplicity and the potency of the CD3/28-mediated T-cell stimulation. In these assays, reduced T-cell proliferation or IFN production in the presence of MDSCs has been interpreted as an accurate indication of MDSC suppressive function. However, concerns in both our lab and others have begun to arise as to the physiologic accuracy and potential for artifact in this polystyrene microbead-based assay [8]. Here, using splenic MDSCs isolated from mice bearing syngeneic, carcinogen-induced oral cavity carcinomas produced subcutaneously in wild-type mice, we demonstrate artefactual suppression of CD3/28 microbead stimulated T-lymphocyte proliferation by MDSCs due to sequestration of beads away from T-lymphocytes in a mixed leukocyte assay. This effect could not be reversed with inhibitors of known MDSC immunosuppressive mechanisms, and was likely due in part to early phagocytic activity and death of sorted peripheral MDSCs. Reversible and dose-dependent inhibition of T-lymphocyte proliferation by MDSCs was achieved with elimination of polystyrene beads from the assay. We propose model-specific validation of microbead-based MDSC assays, or use of an alternative stimulation approach such as plate bound CD3/28 antibodies. 2. Materials and Methods 2.1 Murine tumor model The murine oral cancer (MOC) model is a carcinogen-induced model of oral cavity malignancy that is transplantable into fully immunocompetent C57BL/6 (B6) mice [9]. MOC1 cells were provided by Dr. R. Uppaluri (Washington University School of Medicine). MOC cells were cultured as previously described [10]. All animal experiments were approved by the NIDCD Animal Care and Use Committee (ASP #1364-14). To generate syngeneic tumor-bearing mice, 4106 MOC1 cells were injected subcutaneously in matrigel into the flank of WT C57BL/6 (B6) mice. Tumors were engrafted and allowed to reach at least 500 mm3 before MDSC isolation. 2.2 Cell sorting Splenic single cell suspensions were generated from WT B6 or MOC1 tumor-bearing mice through mechanical dissociation and RBC lysis (Biolegend). To isolate responder T-cells, WT B6 splenocytes were stained and sorted on an autoMACS magnetic sorter (Miltenyi Biotec) using the pan T-cell unfavorable selection kit from Miltenyi (#130-095-130) per the manufacturers instructions. For MDSC isolation, splenic single cell suspensions were stained with the anti-Ly6G microbead kit from Miltenyi (#130-092-332) per the manufacturers instructions and isolated on an autoMACS magnetic sorter. 2.3. Flow cytometry Cell surface staining was performed using fluorophore conjugated anti-mouse CD4 clone GK1.5, CD8 clone 53-6.7, Gr1 clone RB6-8c5, and CD11b clone M1/70 antibodies from Biolegend. Dead cells were excluded via 7AAD negativity. Data was acquired on a FACSCanto using FACSDiva software (BD Biosciences) and analyzed on FlowJo software vX10.07r2. 2.4 T-Cell proliferation assay WT B6 T-cells were labeled with 5 M carboxyfluorescein succinimidyl ester (CFSE, Sigma Aldrich) as previously described [11]. 8104 CSFE-labelled T-lymphocytes were stimulated with a 1:1 ratio of anti-CD3/anti-CD28 coated dynabeads (ThermoFisher) in round-bottom 96-well plates in the presence of MDSCs as indicated for 3-4 days. For plate-bound CD3/28 stimulation, 5 g/mL each of anti-CD3 (clone 145-2C11, eBioscience) and anti-CD28 (clone 37.51, eBioscience) was diluted in PBS and coated onto flat-bottom 96-well CEP-18770 (Delanzomib) plates (Corning) overnight at 4C. CFSE labeled Plxnc1 T-cells were co-cultured with the indicated ratios of MDSCs for four hours, then added to the prepared CD3/28 coated plate (wells were washed with PBS 2 to CEP-18770 (Delanzomib) removed unbound antibody prior to adding cells). Where indicated, MDSCs and T-lymphocytes were exposed to 300 M of nor-NOHA (arginase inhibitor) or CEP-18770 (Delanzomib) L-NMMA (iNOS inhibitor) for 4 hours before T-lymphocyte stimulation with either CD3/28 microbeads or plate bound antibody. After 3 days in culture, T-cell CFSE peak distribution was quantified by flow cytometry. T-cells and MDSCs were cultured in complete media (RPMI 1640 supplemented with 10% FCS, 1.5% HEPES, 1% glutamine, 1% nonessential amino acids, 1% sodium pyruvate, 1% Pen/Strep, 0.1% gentamycin, 50 M beta-mercaptoethanol). T-lymphocyte proliferation was quantified as the average number of divisions for all those cells in the culture (division index) using FlowJo software. Percept inhibition of proliferation was calculated using the following: ([Proliftest -?ProlifUnstim]/[Prolifmax -?Prolifunstim]).