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.

Figure 7. Post-AZA recovery of blood Vδ2 T cells is influenced by patient age and time elapsed since drug withdrawal.

(A) Cross-sectional study of CD patients previously treated with AZA for >1 year but not receiving thiopurine therapy at the time of sampling (ex-AZA; n = 10). Evidence of blood Vδ2 T cell recovery was observed in 5 of 10 ex-AZA CD patients (recovery threshold Q1 defined as the 25th percentile of Vδ2 T cell frequency in AZA-naive CD patients), but not in any of the CD patients on concurrent AZA therapy (AZA⁺; n = 17). Fisher exact test was used to assess Vδ2 T cell recovery after AZA withdrawal. (B and C) Blood Vδ2 T cell frequency in the ex-AZA CD patients was positively correlated with time elapsed since drug withdrawal (B; n = 9), and was inversely correlated with patient age (C; n = 13). Correlations were assessed using Pearson product-moment.

Figure 6. Activation and depletion of Vδ2 T cells in AZA-treated CD.

(A–B) An analysis of CD patients with inactive disease (CDAI < 150; n = 19) indicated that the proportion of CD69⁺-activated Vδ2 T cells in the circulation was significantly enhanced compared with healthy volunteers (n = 25) (A) and also correlated with reduced cell frequency in blood (B). These data suggested that Vδ2 T cell activation and recruitment to the gut might contribute to loss of these cells from the circulation in CD. (C and D) In a cross-sectional study of AZA-treated CD patients (n = 27), ablation of circulating Vδ2 T cells was observed after >1 year continuous therapy (C) and longitudinal followup analyses confirmed a progressive loss of Vδ2 T cells with rapid onset after commencing AZA treatment (D; mean ± SEM of n = 3–5 CD patients per time point). CD patients and control groups were compared using unpaired t tests. Correlations were assessed using Spearman rank tests.

Figure 5. Selective loss of circulating Vδ2 T cells in AZA-treated CD.

(A) Flow-cytometry enabled the identification of Vδ2 T cells in addition to conventional αβ T cells in direct ex vivo analyses of peripheral blood from healthy volunteers. In contrast, blood samples from CD patients receiving AZA therapy contained only trace numbers of Vδ2 T cells, whereas αβ T cell numbers were consistent with expected frequencies in AZA-treated patients. (B and C) Loss of Vδ2 T cells in AZA-treated CD (AZA⁺; n = 17) was extensive and selective, whether assessed as a proportion of the total T cell pool (B) or by calculating the absolute number of these cells per unit volume of blood (C). CD patients not receiving AZA therapy (AZA^–; n = 20) exhibited Vδ2 T cell frequencies comparable with healthy controls (n = 36). Example dot plots in A are representative of n = 36 healthy volunteers and n = 17 AZA-treated CD patients. Bars in B and C indicate group median values. Significant differences between groups were determined using Kruskal-Wallis 1-way ANOVA on ranks.

Figure 4. Activated human Vδ2 T cells are highly sensitive to AZA exposure.

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.

Figure 3. TNFα production by mucosal Vδ2 T cells is increased in CD and impaired by blockade of RARα signaling.

(A) Vδ2 T cells in biopsy tissue from CD patients (n = 11) displayed elevated production of TNFα and IL-17A compared with mucosal Vδ2 T cells from healthy controls (n = 9). Vδ2 T cells were identified as CD3⁺Vδ2⁺ gut lymphocytes, and in some assays, fixable live/dead dye was used to confirm the viability of cells in this gate. In some experiments, IL-17A staining was omitted due to the limited number of biopsies available. (B) Enhanced TNF synthesis by Vδ2 T cells in CD intestine was not restricted to a specific cell subset, since both CD103^– and CD103⁺ populations produced comparable levels of this cytokine in the unstimulated biopsy cultures (n = 10 CD patients, n = 9 healthy controls). (C–E) Biopsy supplementation with 2nM all-trans RA over the 3d culture period exerted little effect on cytokine production by mucosal Vδ2 T cells from CD patients (n = 4), whereas inhibition of RARα signaling with the specific antagonist Ro41-5253 led to a substantial reduction in TNF synthesis. Significant differences between groups were determined by t test (A and B) or repeated-measures ANOVA (C and D). Example histograms in E are representative of n = 4 independent experiments.

Figure 2. Intestinal Vδ2 T cells in CD patients express CD27 and incorporate both CD103^– and CD103⁺ subsets.

(A) Representative examples of Vδ2 T cell subset distribution in blood from an IBS control (HC) and a patient with CD showing selective depletion of the CD45RA^–CD27⁺ subset in CD (each plot is representative of n = 8 independent experiments, and both display an equal number of cells). (B) Intestinal biopsy tissue from patients with new diagnoses of CD contained a distinct population of Vδ2 T cells that displayed a CD27⁺ phenotype analogous to that of the cells depleted from peripheral blood. (C) Mucosal Vδ2 T cells in CD patients included both CD103^– and CD103⁺ subsets in proportions comparable to those present in the healthy intestine. Shown is an example flow-cytometry analysis of colonic biopsy tissue after extensive removal of the epithelium and showing positive identification of Vδ2 T cells (thus excluding Vδ1 T cells) among the egressed leukocytes in an 18-year-old patient with active CD (B; representative of n = 11 CD patients), as well as grouped data showing Vδ2 T cell subset balance in multiple individuals (C; n = 11 CD patients, n = 9 healthy controls; comparison by t test).

Figure 1. Enhanced gut-homing potential and depletion of circulating CD27⁺ Vδ2 T cells in CD.

(A) In patients with moderately active CD about to receive de novo AZA therapy (n = 12), flow-cytometric analysis of blood lymphocytes revealed a significant increase in the proportion of β7⁺ gut-homing cells within the CD3⁺ Vδ2 T cell population when compared with healthy controls (n = 26). (B) In a separate analysis of pediatric CD patients (mean age 13 years), we observed that the overall proportion of β7⁺ Vδ2 T cells in blood was comparable with adult CD patients (NS, not shown), but the CD45RA^–CD27⁺ subset was selectively depleted compared with sex/age/ethnicity-matched IBS controls (*P < 0.001; n = 8 per group), while the subset distribution of conventional αβ T cells as defined by these markers was unaltered in CD (data not shown). (C) The CD27⁺ population of circulating Vδ2 T cells expressed significantly higher levels of β7 integrin than did any other Vδ2 T cell subset (*P < 0.05; n = 8), consistent with increased trafficking of these cells to the gut and a corresponding depletion from the blood in CD. (D) Example histograms showing integrin β7 expression levels in the different Vδ2 T cell subsets detected in blood from an IBS control (representative of n = 8). Significant differences between groups were determined by Mann-Whitney rank-sum test (A), or repeated-measures ANOVA (B and C).