NKp46-expressing human gut-resident intraepithelial Vδ1 T cell subpopulation exhibits high antitumor activity against colorectal cancer
Joanna Mikulak, … , Antonino Spinelli, Domenico Mavilio
Joanna Mikulak, … , Antonino Spinelli, Domenico Mavilio
Published November 5, 2019
Citation Information: JCI Insight. 2019;4(24):e125884. https://doi.org/10.1172/jci.insight.125884.
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Research Article Gastroenterology Immunology

NKp46-expressing human gut-resident intraepithelial Vδ1 T cell subpopulation exhibits high antitumor activity against colorectal cancer

Abstract

γδ T cells account for a large fraction of human intestinal intraepithelial lymphocytes (IELs) endowed with potent antitumor activities. However, little is known about their origin, phenotype, and clinical relevance in colorectal cancer (CRC). To determine γδ IEL gut specificity, homing, and functions, γδ T cells were purified from human healthy blood, lymph nodes, liver, skin, and intestine, either disease-free, affected by CRC, or generated from thymic precursors. The constitutive expression of NKp46 specifically identifies a subset of cytotoxic Vδ1 T cells representing the largest fraction of gut-resident IELs. The ontogeny and gut-tropism of NKp46+/Vδ1 IELs depends both on distinctive features of Vδ1 thymic precursors and gut-environmental factors. Either the constitutive presence of NKp46 on tissue-resident Vδ1 intestinal IELs or its induced expression on IL-2/IL-15–activated Vδ1 thymocytes are associated with antitumor functions. Higher frequencies of NKp46+/Vδ1 IELs in tumor-free specimens from CRC patients correlate with a lower risk of developing metastatic III/IV disease stages. Additionally, our in vitro settings reproducing CRC tumor microenvironment inhibited the expansion of NKp46+/Vδ1 cells from activated thymic precursors. These results parallel the very low frequencies of NKp46+/Vδ1 IELs able to infiltrate CRC, thus providing insights to either follow-up cancer progression or to develop adoptive cellular therapies.

Authors

Joanna Mikulak, Ferdinando Oriolo, Elena Bruni, Alessandra Roberto, Federico S. Colombo, Anna Villa, Marita Bosticardo, Ileana Bortolomai, Elena Lo Presti, Serena Meraviglia, Francesco Dieli, Stefania Vetrano, Silvio Danese, Silvia Della Bella, Michele M. Carvello, Matteo Sacchi, Giovanni Cugini, Giovanni Colombo, Marco Klinger, Paola Spaggiari, Massimo Roncalli, Immo Prinz, Sarina Ravens, Biagio di Lorenzo, Emanuela Marcenaro, Bruno Silva-Santos, Antonino Spinelli, Domenico Mavilio

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Figure 1

Identification of a subset of NKp46+ intraepithelial Vδ1 T lymphocytes highly enriched in human healthy intestine.

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Identification of a subset of NKp46+ intraepithelial Vδ1 T lymphocytes h...
(A) Representative example of flow cytometry dot plots showing the gating strategy used to identify viable CD45+/CD3+ γδ T lymphocyte both in intraepithelial (IEL) and lamina propria (LPL) compartments from specimens of human healthy colon. (B) Summary statistical graph showing within the CD45+/CD3+ lymphocytes the percentages of γδ IELs (n = 54 in white circles), γδ LPLs (n = 20 in gray circles) from human healthy colon specimens, and γδ peripheral blood mononuclear cells (PBMCs) (n = 26 in black circles) of healthy donors. (C) Summary statistical graph showing the expression percentage of CD69 and CD103 on γδ IELs (n = 20 in white circles), γδ LPLs (n = 15 in grey circles) from specimens of human healthy colon, and on γδ T cell from PBMCs (n = 20 in black circles) of healthy donors. (D) Summary statistical graph showing the expression percent of CD4, CD8, CD16, CD56, NKG2A, NKG2C, NKG2D, and killer immunoglobulin-like receptors (KIRs) on γδ IELs (n ≥ 13, white circles), γδ LPLs (n ≥ 10 in gray circles) from specimens of human healthy colon, and on γδ T cells from PBMCs of healthy donors (n ≥ 13, black circles). (E) Summary statistical graph showing the expression percent of NKp46, NKp30, and NKp44 on γδ IELs (n ≥ 25 in white circles), γδ LPLs (n ≥16 in gray circles) from specimens of human healthy colon, and on γδ T cells from PBMCs from healthy donors (n ≥25 in black circles). (F) Summary statistical analysis (upper graph) showing the expression of CD8α (white circles) and CD8β (black circles) chains within the CD8 receptor of matched CD8+ γδ IELs and CD8+/NKp46+ γδ IELs from specimens of human healthy colon (n = 15). White arrows indicate representative flow cytometry dot plots showing coexpression of CD8α and CD8β chains in CD8+ total γδ T (left) or NKp46+/γδ T IELs (right), respectively. (G) t-SNE graphs from a representative specimen of human healthy colon showing the clustering of NKp46– (C1 in blue) and NKp46+ (C2 in orange) γδ IELs within CD45+/CD3+ lymphocytes (gray; left panel) or in γδ T IELs (right panel). (H) Heatmap graph showing the degree of expression of several surface markers on matched NKp46– and NKp46+ γδ IEL clusters defined as C1 and C2 in panel G (n = 7). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.