A Role for Bcl-2 in Notch1-Dependent Transcription in Thymic Lymphoma Cells

Notch1 is a transcription factor important for T-cell development. Notch1 is active in double negative (DN) thymocytes, while being depressed in double positive (DP) thymocytes. Synchronously, the expression of Bcl-2 becomes downregulated during the transition from DN to DP thymocytes. We previously observed that overexpression of an intracellular active Notch1 (ICN) in Bcl-2-positive 2B4 T cells leads to the transcription of Notch1-regulated genes. However, these genes were not induced in Bcl-2-negative DP PD1.6 thymic lymphoma cells overexpressing ICN. Here we show that, when Bcl-2 is simultaneously introduced into these cells, Notch-regulated genes are transcribed. Only in the presence of both Bcl-2 and ICN, PD1.6 thymic lymphoma cells become resistant to glucocorticoid (GC)-induced apoptosis. Our data suggest that Bcl-2 plays a role in modulating Notch1 function in T cells

Human T Cell Crosstalk Is Induced by Tumor Membrane Transfer

<div><p>Trogocytosis is a contact-dependent unidirectional transfer of membrane fragments between immune effector cells and their targets, initially detected in T cells following interaction with professional antigen presenting cells (APC). Previously, we have demonstrated that trogocytosis also takes place between melanoma-specific cytotoxic T lymphocytes (CTLs) and their cognate tumors. In the present study, we took this finding a step further, focusing on the ability of melanoma membrane-imprinted CD8⁺ T cells to act as APCs (CD8⁺T-APCs). We demonstrate that, following trogocytosis, CD8⁺T-APCs directly present a variety of melanoma derived peptides to fraternal T cells with the same TCR specificity or to T cells with different TCRs. The resulting T cell-T cell immune synapse leads to (1) Activation of effector CTLs, as determined by proliferation, cytokine secretion and degranulation; (2) Fratricide (killing) of CD8⁺T-APCs by the activated CTLs. Thus, trogocytosis enables cross-reactivity among CD8⁺ T cells with interchanging roles of effectors and APCs. This dual function of tumor-reactive CTLs may hint at their ability to amplify or restrict reactivity against the tumor and participate in modulation of the anti-cancer immune response.</p></div

The effect of CD8⁺T-APCs on effector CTLs is mediated by tumor-derived pMHC.

<p><b>(A)</b> Ova-expressing EG7 and parental EL4 target cell lines (<i>Left column</i>) and target-entrained CD8⁺T-APC (generated following co-culture of OT-I CD8⁺ T cells with designated targets, <i>right column</i>) were labeled with Ova_257–264/H-2Kb-specific mAb (<i>black histogram</i>). <i>Grey histogram</i>, background staining with isotype control antibody. <b>(B)</b> Proliferation of OT-I CD8⁺ T cells stimulated with CD8⁺T-APCs. CFSE-labeled OT-I T cells were left untreated (no target) or co-cultured with the following T-APCs: OT-I CD8⁺ pre-incubated with EL4 (EL4) or OT-I CD8⁺ pre-incubated with EG7 cells (EG7). ConA stimulation was used as positive control (right). <i>Scale bars</i>, proliferating lymphocytes that divided at least twice. <i>Numbers</i>, percentage of dividing CD8⁺ T cells. Data are representative of two independent experiments.</p

Assessment of CTL fratricide activity based on detection of cleaved caspase-3.

<p>MART-1- or MUC-1- reactive CTLs were co-cultured with DDAO-SE-tagged CD8⁺T-APCs and non T-APCs, generated by co-incubation of 2E2 cells with 624<i>mel</i> and M171 melanoma cells, respectively. T-APC damage was examined based on detection of intracellular cleaved caspase-3 in the DDAO-SE⁺CD8⁺ population. Numbers in upper right quadrants indicate the percentage of cleaved caspase 3-positive CD8⁺T-APC cells. Data are representative of three independent experiments.</p

CD8⁺ T-APCs induce degranulation of effector CTLs with different antigen specificity.

<p>(<b>A, B</b>) CD8⁺ clones with different antigen specificity were used as CD8⁺T-APCs and effector CTLs. Biotinylated effector CTLs were co-cultured with CD8⁺T-APCs, stained with anti-CD107A mAb and streptavidin-allophycocyanin and analyzed by flow cytometry. (<b>A</b>) The gp100_154–162-specific clone 1G2 was used as CD8⁺T-APC for the MART-1_26–35-specific CTL clones (2E2 and 2D11). <b>(B)</b> The MART-1_26–35-specific clones 2E2 and 2D11 were used as CD8⁺T-APC for the gp100_154–162-specific clone (1G2). Numbers in upper right quadrants indicate the percentage of CD107A⁺streptavidin⁺ lymphocytes, gated on the CD8⁺ population (effector CTLs). Data are representative of three independent experiments. (<b>C</b>) Graphic presentation of intra- and inter-clonal T cell cross talk.</p

CD8⁺T-APCs activate anti-tumor CD8 T cells of the same antigen specificity.

<p><b>(A-C)</b> Cytokine production by effector CTLs in response to activation by T-APCs. <b>(A)</b> DiIC₁₈-labeled CD8⁺T-APCs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118244#sec002" target="_blank">materials and methods</a>) were incubated for 6 hours with surface-biotinylated effector CTLs. Cytokine-producing effector CTLs were defined based on intracellular IFN-γ or TNF-α staining of CD8⁺streptavidin⁺ lymphocytes. Numbers in upper right quadrants indicate the percentage of IFN-γ ⁺ (upper panel) or TNF-α ⁺ (lower panel) effector CTLs. Labels indicate cells used as targets for CTLs: CTLs co-cultured with 624<i>mel</i> melanoma cells are designated T-APC; CTLs co-cultured with irrelevant M171 melanoma cells are designated non T-APC. <b>(B)</b> Confocal images of cytokine-producing effector CTLs. Calcein AM labeled CD8⁺T-APCs (<i>green</i>, upper panel) or non T-APCs (<i>green</i>, lower panel) were co-cultured for 6 hours with streptavidin-allophycocyanin-stained effector CTLs (<i>red</i>). Intracellular TNF-α production (<i>blue</i>) by effector CTLs is shown. Scale bars are 10 μm (upper panel) and 20 μm (lower panel). <b>(C)</b> Time period that CD8⁺T-APCs activate effector CTLs. Effector CTLs were co-cultured with CD8⁺T-APCs either immediately or 6, 24 and 48 hours after CD8⁺T-APC purification. Data are mean ± SE (n = 3 replicates/group) percentage of IFN-γ ⁺ effector CTLs, gated on CD8⁺ T cells. <b>(D)</b> CD8⁺T-APCs trigger degranulation of effector CTLs. CD8⁺T-APCs were generated as described above (1A) and co-cultured with effector CTLs. Cytolytic activity of T cells was measured by detection of surface CD107A on CD8⁺streptavidin⁺ effector CTLs. Number in upper right quadrants indicates the percentage of CD107A⁺ streptavidin⁺ effector CTLs. Data are representative of at least three independent experiments.</p