Al amplifier of the underlying peptide-MHCTCR recognition event which avoids false

Al amplifier of the underlying peptide-MHCTCR recognition event which avoids false

Al amplifier of the underlying peptide-MHCTCR recognition event which avoids false positives due to nonspecific binding. The LCI imaging platform is fundamentally compatible with a segmented culture system that will allow for isolation of rare cells that may be lost in the current open perfusion cell culture system. LCI may therefore provide a viable alternative for the identification and isolation of rare effector T cells killing autologous tumor cells or HLA-matched cancer cell lines. T cells against cancer-associated antigens are generally anticipated to bear lower affinity TCRs if they are raised against a selfantigen and presumably escaped thymic selection and tolerance induction [29]. The affinity between the TCR and peptide-MHC is considered to play a crucial role in the outcome of T cell stimulation [30]. The classic method to assess TCR-peptide-MHC affinity entails the measurement of on and off-rates using surface plasmon resonance. The surface bound peptide-MHC-TCR interaction does not accurately mimic the multiple receptormediated Title Loaded From File Title Loaded From File interactions that occur during recognition of a target cell by a CTL. Evidence suggests that these measurements provide limited information regarding lymphocyte effector function [30,31]. In a transfection system, TCRs engineered with higher affinity for cognate peptide-MHC ligands compared to their wild type counterpart exhibited increased CTL activity [31]. An affinity model suggests that activation of T cells is related to the number of receptors engaged. Higher affinity interactions require less TCRpeptide-MHC engagements to activate a T cell into a cytotoxic state [32]. It is conceivable that higher affinity TCR-peptideMHC interactions drive a more rapid response than their lower affinity counterparts, and the LCI approach may also potentially discriminate between these interactions.Supporting InformationFigure S1 Averaged, normalized mass versus time plots for control target cell growth conditions showing robust growth on the LCI stage, and specificity of T cell mediated cytotoxicity. (A) Unaffected M202 cells (n = 632) during treatment with F5 TCR transduced, CD8+ T cells. (B) M202 cells (n = 117) prior to treatment with F5 TCR transduced, CD8+ T cells. (C) M202 cells (n = 2058) treated with F5 TCR negative, CD8+ T cells. (D) Antigen-irrelevant, PC-3 prostate cancer cells (n = 1006) treated with F5 TCR transduced, CD8+ T cells. Blue line shows mean normalized mass versus time (normalized relative to mass at first timepoint). Light blue region shows the mean +/2 SD. (TIF) Figure S2 Averaged, normalized mass versus time for unresponsive T cells showing steady growth on the LCI stage. (A) Unresponsive F5 TCR transduced CD8+ T cells (n = 101) plated with M202 target cells. (B) Untransduced CD8+ T cells (n = 146) plated with M202 target cells. (C) F5 TCR transduced CD8+ T cells (n = 950) plated with antigen-irrelevant, PC-3 prostate cancer target cells. (TIF) Figure S3 Intensity images of cells on the interferometer stage after 18 h of imaging showing typical target cell conditons. Left column shows the full image frame, the right column shows a subset of the full image frame. (A)?D) MMass Changes During CTL Target Cell Killingtarget cells plated with F5 TCR transduced, CD8+ T cells showing nearly complete death of target cells. For comparison, (A) and (B) show the same field of view as in Fig. 2 A . (C), (D) show a single living cell. E, F. M202 target cells plated with untransduced CD8+ T cells.Al amplifier of the underlying peptide-MHCTCR recognition event which avoids false positives due to nonspecific binding. The LCI imaging platform is fundamentally compatible with a segmented culture system that will allow for isolation of rare cells that may be lost in the current open perfusion cell culture system. LCI may therefore provide a viable alternative for the identification and isolation of rare effector T cells killing autologous tumor cells or HLA-matched cancer cell lines. T cells against cancer-associated antigens are generally anticipated to bear lower affinity TCRs if they are raised against a selfantigen and presumably escaped thymic selection and tolerance induction [29]. The affinity between the TCR and peptide-MHC is considered to play a crucial role in the outcome of T cell stimulation [30]. The classic method to assess TCR-peptide-MHC affinity entails the measurement of on and off-rates using surface plasmon resonance. The surface bound peptide-MHC-TCR interaction does not accurately mimic the multiple receptormediated interactions that occur during recognition of a target cell by a CTL. Evidence suggests that these measurements provide limited information regarding lymphocyte effector function [30,31]. In a transfection system, TCRs engineered with higher affinity for cognate peptide-MHC ligands compared to their wild type counterpart exhibited increased CTL activity [31]. An affinity model suggests that activation of T cells is related to the number of receptors engaged. Higher affinity interactions require less TCRpeptide-MHC engagements to activate a T cell into a cytotoxic state [32]. It is conceivable that higher affinity TCR-peptideMHC interactions drive a more rapid response than their lower affinity counterparts, and the LCI approach may also potentially discriminate between these interactions.Supporting InformationFigure S1 Averaged, normalized mass versus time plots for control target cell growth conditions showing robust growth on the LCI stage, and specificity of T cell mediated cytotoxicity. (A) Unaffected M202 cells (n = 632) during treatment with F5 TCR transduced, CD8+ T cells. (B) M202 cells (n = 117) prior to treatment with F5 TCR transduced, CD8+ T cells. (C) M202 cells (n = 2058) treated with F5 TCR negative, CD8+ T cells. (D) Antigen-irrelevant, PC-3 prostate cancer cells (n = 1006) treated with F5 TCR transduced, CD8+ T cells. Blue line shows mean normalized mass versus time (normalized relative to mass at first timepoint). Light blue region shows the mean +/2 SD. (TIF) Figure S2 Averaged, normalized mass versus time for unresponsive T cells showing steady growth on the LCI stage. (A) Unresponsive F5 TCR transduced CD8+ T cells (n = 101) plated with M202 target cells. (B) Untransduced CD8+ T cells (n = 146) plated with M202 target cells. (C) F5 TCR transduced CD8+ T cells (n = 950) plated with antigen-irrelevant, PC-3 prostate cancer target cells. (TIF) Figure S3 Intensity images of cells on the interferometer stage after 18 h of imaging showing typical target cell conditons. Left column shows the full image frame, the right column shows a subset of the full image frame. (A)?D) MMass Changes During CTL Target Cell Killingtarget cells plated with F5 TCR transduced, CD8+ T cells showing nearly complete death of target cells. For comparison, (A) and (B) show the same field of view as in Fig. 2 A . (C), (D) show a single living cell. E, F. M202 target cells plated with untransduced CD8+ T cells.

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