Azathioprine therapy selectively ablates human Vδ2+ T cells in Crohn’s disease
Neil E. McCarthy, Charlotte R. Hedin, Theodore J. Sanders, Protima Amon, Inva Hoti, Ibrahim Ayada, Vidya Baji, Edward M. Giles, Martha Wildemann, Zora Bashir, Kevin Whelan, Ian Sanderson, James O. Lindsay, Andrew J. Stagg
Neil E. McCarthy, Charlotte R. Hedin, Theodore J. Sanders, Protima Amon, Inva Hoti, Ibrahim Ayada, Vidya Baji, Edward M. Giles, Martha Wildemann, Zora Bashir, Kevin Whelan, Ian Sanderson, James O. Lindsay, Andrew J. Stagg
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Research Article Immunology

Azathioprine therapy selectively ablates human Vδ2+ T cells in Crohn’s disease

Abstract

Tumor-derived and bacterial phosphoantigens are recognized by unconventional lymphocytes that express a Vγ9Vδ2 T cell receptor (Vδ2 T cells) and mediate host protection against microbial infections and malignancies. Vδ2 T cells are absent in rodents but readily populate the human intestine, where their function is largely unknown. Here, we assessed Vδ2 T cell phenotype and function by flow cytometry in blood and intestinal tissue from Crohn’s disease patients (CD patients) and healthy controls. Blood from CD patients included an increased percentage of gut-tropic integrin β7–expressing Vδ2 T cells, while “Th1-committed” CD27-expressing Vδ2 T cells were selectively depleted. A corresponding population of CD27+ Vδ2 T cells was present in mucosal biopsies from CD patients and produced elevated levels of TNFα compared with controls. In colonic mucosa from CD patients, Vδ2 T cell production of TNFα was reduced by pharmacological blockade of retinoic acid receptor-α (RARα) signaling, indicating that dietary vitamin metabolites can influence Vδ2 T cell function in inflamed intestine. Vδ2 T cells were ablated in blood and tissue from CD patients receiving azathioprine (AZA) therapy, and posttreatment Vδ2 T cell recovery correlated with time since drug withdrawal and inversely correlated with patient age. These results indicate that human Vδ2 T cells exert proinflammatory effects in CD that are modified by dietary vitamin metabolites and ablated by AZA therapy, which may help resolve intestinal inflammation but could increase malignancy risk by impairing systemic tumor surveillance.

Authors

Neil E. McCarthy, Charlotte R. Hedin, Theodore J. Sanders, Protima Amon, Inva Hoti, Ibrahim Ayada, Vidya Baji, Edward M. Giles, Martha Wildemann, Zora Bashir, Kevin Whelan, Ian Sanderson, James O. Lindsay, Andrew J. Stagg

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Figure 4

Activated human Vδ2 T cells are highly sensitive to AZA exposure.

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Activated human Vδ2 T cells are highly sensitive to AZA exposure. Periph...
Peripheral blood mononuclear cells were labeled with CellTrace Violet (CTV) dye, seeded into 96-well round-bottom plates in complete medium (4 × 105 cells/well) and stimulated with 1 nM HDMAPP phosphoantigen (1-hydroxy-2-methyl-2-buten-4-yl 4-diphosphate, tebu-bio, Peterborough, UK) together with anti-CD2/3/28 beads to activate conventional T cells (Miltenyi Biotec) in the presence of 0-50 μM AZA (Sigma-Aldrich) for 5 days at 37°C, 5% CO2. Both Vδ2 T cells and conventional αβ T cells expanded markedly over the 5-day culture, as assessed by flow-cytometry. Addition of low-dose AZA to these cultures significantly impaired the proliferation of Vδ2 T cells while exerting little effect on αβ T cell expansion. This difference in population growth was evident even at clinically relevant concentrations of AZA (5 μM) (33). Impairment of αβ T cell proliferation comparable to that observed for Vδ2 T cells was achieved only at high doses of AZA. Two-way repeated-measures ANOVA was used to test the influence of cell type and drug dose on proliferated cell number (*P = 0.022, **P = 0.005; compared with Vδ2 T cells subjected to the same concentration of AZA. There was a statistically significant interaction between cell type and drug dose; P = 0.018). Shown are grouped data from n = 6 independent experiments and a representative example of cell proliferation analysis by flow-cytometry. Cell frequencies were normalized to maximum proliferated cell number to control for intra/interindividual variability in expansion of the different cell types. US, Unstimulated cells in the absence of AZA.