Rational subpopulation manipulation can change TIL anti‐tumor reactivity and is accompanied by a shift in subpopulation signature. Rational subpopulation.

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Rational subpopulation manipulation can change TIL anti‐tumor reactivity and is accompanied by a shift in subpopulation signature. Rational subpopulation manipulation can change TIL anti‐tumor reactivity and is accompanied by a shift in subpopulation signature. (A) IFN‐γ levels of 12 TILs before (red bars) and after (blue bars) rational subpopulation depletion and enrichment are compared. Nine of the original nonreactive TILs show significant increase in IFN‐γ levels with up to 106‐fold increase observed for TIL #2. Incubation of TILs in the control experiments with culture media or unrelated melanoma (white bars) indicates that the increase in IFN‐γ secretion does not occur spontaneously and is tumor (HLA) specific. (B) The shift in reactivity can be explained in terms of a shift in subpopulation signature. The subpopulation fractions of 10 TILs before and after subpopulation manipulation are shown. The rows and columns correspond to different subpopulations and TILs, respectively. Two ways unsupervised clustering was performed on the rows and columns. The 10 nonreactive TILs prior to the manipulation are designated by a red color and the letter ‘P.’ Ten TILs after manipulation are designated by the letter ‘A’ with blue and yellow corresponding to reactive and nonreactive, respectively. Eight of the nonreactive TILs before manipulation became reactive, seven of which also showed a shift from a nonreactive subpopulation signature to a reactive one as indicated by the blue arrows going from right to left. The two TILs that remained nonreactive after manipulation exhibited either a minor change or a negative change in subpopulation signature as indicated by the red arrows. (C) The transformation of a nonreactive TIL to a reactive one can be described as a path between two points in the subpopulation space. In order to visualize the TILs positions in the multidimensional subpopulation space, we applied PCA, which is a method for dimensionality reduction (see Materials and methods). This enabled us a simple 2D visualization of the different TILs. The x and y‐axes are the principal components capturing 49 and 24% of the total variance in the data. The x‐axis captures a shift from CD8+ and CD28− enriched subpopulation to CD4+ and CD28+ subpopulations, whereas the y‐axis reflects a combination of additional subpopulations (see Supplementary Figure S4 for subpopulations coefficients defining each of the principal components). The figure shows a subspace region that is overpopulated with reactive TILs. The change in reactivity can be visualized as a path from a nonreactive TIL to a TIL that resides in the reactive subspace (e.g. see dotted arrow). Kfir Oved et al. Mol Syst Biol 2009;5:265 © as stated in the article, figure or figure legend