Tumor-infiltrating lymphocytes are dynamically desensitized to antigen but are maintained by homeostatic cytokine
Bijan Boldajipour, Amanda Nelson, Matthew F. Krummel
Bijan Boldajipour, Amanda Nelson, Matthew F. Krummel
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Research Article Immunology Oncology

Tumor-infiltrating lymphocytes are dynamically desensitized to antigen but are maintained by homeostatic cytokine

Abstract

T cells that enter tumors are largely tolerized, but how that process is choreographed and how the ensuing “dysfunctional” tumor-infiltrating lymphocytes (TILs) are maintained are poorly understood and are difficult to assess in spontaneous disease. We exploited an autochthonous model of breast cancer for high-resolution imaging of the early and later stages of tumor residence to understand the relationships between cellular behaviors and cellular phenotypes. “Dysfunctional” differentiation began within the first days of tumor residence with an initial phase in which T cells arrest, largely on tumor-associated macrophages. Within 10 days, cellular motility increased and resembled a random walk, suggesting a relative absence of TCR signaling. We then studied the concurrent and apparently contradictory phenomenon that many of these cells express molecular markers of activation and were visualized undergoing active cell division. We found that whereas proliferation did not require ongoing TCR/ZAP70 signaling, instead this is driven in part by intratumoral IL-15 cytokine. Thus, TILs undergo sequential reprogramming by the tumor microenvironment and are actively retained, even while being antigen insensitive. We conclude that this program effectively fills the niche with ineffective yet cytokine-dependent TILs, and we propose that these might compete with new clones, when they arise.

Authors

Bijan Boldajipour, Amanda Nelson, Matthew F. Krummel

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Figure 5

Proliferation of tumor-resident T cells is independent of TCR signaling but partially dependent on IL-15 signaling.

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Proliferation of tumor-resident T cells is independent of TCR signaling ...
(A) Schematic of experiments to address TCR dependence of the proliferation of tumor-resident T cells. Genetically marked OT-I T cells expressing either a single copy of wild-type ZAP70 or the HXJ42-sensitive mutant ZAP70AS were transferred to PyMT-ChOVA mice. Fourteen days later, tumor slice cultures were prepared from tumors and incubated for 24 hours in the presence of EdU and either HXJ42 inhibitor or DMSO control. (B) Representative analysis of EdU incorporation of inhibitor-resistant OT-I/ZAP70+/– or inhibitor-sensitive OT-I/ZAP70AS T cells in the presence or absence of the inhibitor using flow cytometry. (C) The ratio of proliferation in tumor slices incubated with HXJ42 inhibitor over the proliferation in control slices was analyzed using a Mann-Whitney test. P > 0.05. Data are pooled from 3 experiments. (D) Similar to A–C, tumor slices of PyMT-ChOVA mice treated with OT-I T cells were incubated with EdU in absence or presence of an antibody that blocks the MHCI-presented ovalbumin peptide (anti-SL8). EdU incorporation was compared against the average of the control group using a Mann-Whitney test. P > 0.05. Data are pooled from 3 experiments. (E) Analysis of homeostatic cytokine expression by myeloid cells in the tumor microenvironment. Cytokine RNA expression data from ref. 7 were mined for each indicated cell type and plotted using the indicated color scale and using Gapdh expression for normalization. (F) Schematic of experiments addressing the influence of IL-15 signaling on T cell proliferation in two tumor models. KI67+ OT-I cells served as a readout of cell proliferation. (G) Statistical analysis of KI67 expression of tumor-resident OT-I T cells in B78-ChOVA and PyMT-ChOVA tumors treated with IL-15-blocking antibody or control using a Mann-Whitney test. *P < 0.05. Data are pooled from 2 experiments. (H) Statistical analysis of KI67 expression of tumor-resident OT-I T cells in B78-ChOVA tumors grown in wild-type or IL-15-deficient animals using a Mann-Whitney test. ***P < 0.001. Data are pooled from 2 experiments.