Shared reactivity of V{delta}2(neg) {gamma}{delta} T cells against cytomegalovirus-infected cells and tumor intestinal epithelial cells

Abstract

Long-lasting expansion of Vdelta2(neg) gammadelta T cells is a hallmark of cytomegalovirus (CMV) infection in kidney transplant recipients. The ligands of these cells and their role remain elusive. To better understand their immune function, we generated gammadelta T cell clones from several transplanted patients. Numerous patient Vdelta1(+), Vdelta3(+), and Vdelta5(+) gammadelta T cell clones expressing diverse Vgamma chains, but not control Vgamma9Vdelta2(+) T clones, displayed strong reactivity against CMV-infected cells, as shown by their production of tumor necrosis factor-alpha. Vdelta2(neg) gammadelta T lymphocytes could also kill CMV-infected targets and limit CMV propagation in vitro. Their anti-CMV reactivity was specific for this virus among herpesviridae and required T cell receptor engagement, but did not involve major histocompatibility complex class I molecules or NKG2D. Vdelta2(neg) gammadelta T lymphocytes expressed receptors essential for intestinal homing and were strongly activated by intestinal tumor, but not normal, epithelial cell lines. High frequencies of CMV- and tumor-specific Vdelta2(neg) gammadelta T lymphocytes were found among patients' gammadelta T cells. In conclusion, Vdelta2(neg) gammadelta T cells may play a role in protecting against CMV and tumors, probably through mucosal surveillance of cellular stress, and represent a population that is largely functionally distinct from Vgamma9Vdelta2(+) T cells.

Figure 1.

Vδ2^neg γδ T cells produce TNFα when cocultured with CMV-infected MRC5 cells. (A) MRC5 cells were infected for 1–4 d with CMV or left uninfected, and cocultured with two representative Vδ2^neg γδ T clones (5–4 and 4–29) or medium alone for TNFα release quantification by ELISA. Results are presented as mean ±SD of culture triplicates. (B) Vδ2^neg γδ T cells were cultured in medium alone, with PMA and ionomycin, or with uninfected or CMV-infected MRC5 cells. Cells were labeled with anti-Cδ mAb and anti-TNFα mAb and analyzed by flow cytometry. Only γδ T cells are shown in the histograms and the percentages of TNFα- expressing cells are indicated. (C) Anti-CMV reactive 5-4 and 4–29 clones have been cultured for 4 h in medium alone, with PMA and ionomycin, or on MRC5 cells uninfected or infected with TB40/E, AD169, or Towne strains of CMV, or with clinical isolates of VZV or HSV-1. Results are mean ± SD of TNFα secreted in supernatant of culture triplicates. All results are representative of at least three independent experiments.

Figure 2.

Vδ2^neg γδ T cells can lyse CMV-infected cells and limit CMV replication in vitro. (A) FSF uninfected (dashed lines) or infected with CMV (solid lines) were labeled with ⁵¹Cr and incubated with four different anti-CMV γδ T cell clones. Results are the mean of specific lysis of culture triplicates. SD was always <15% of the mean value (not depicted). (B) As in A, but blocking mAbs directed against TNFα (20 μg/ml) or Fas (1 μg/ml) or 20 μg/ml control mAb were added at the start of cell contact. When indicated, γδ T cell clone was pretreated with SrCl or NaCl at 20 mM before contact with FSF. Viability of T cells after this treatment was >90%. Effector/target ratio was 10/1. As positive control for mAb efficacy, WEHI or Jurkat cells were labeled with ⁵¹Cr and incubated with 50 pg/ml TNFα or Fas-L⁺ 1A12 cells (10/1 effector/target ratio), respectively. Same results were obtained with the 5–4 clone. (C) Uninfected or infected FSF were labeled as in A and mixed with the same number of either cold uninfected FSF or cold infected FSF, and incubated with the 4–29 anti-CMV γδ T cell clone at a 10/1 effector/target ratio. (D) FSF were incubated for 2 h with CMV, washed, and incubated in medium alone, with IFNγ (100, 10, and 1 U/ml), or with the anti-CMV 4-29 or unrelated T clones (Vδ1 or a Vγ9Vδ2 T cells). Anti-IFNγ blocking mAb or an unrelated control mAb (20 and 2 μg/ml) were added to 4-29 T cell cocultures. After 4 d, infectious CMV was titered in culture supernatants by plaque assay on MRC5 cells. Results are mean ± SD of culture triplicates and are representative of three independent experiments.

Figure 3.

Vδ2^neg γδ TCR recognition of CMV-infected cells is TCR dependent but MHC class I independent. (A) FSF were infected or not with CMV and incubated with the 4–29 anti-CMV Vδ2^neg γδ T cell clone in the presence or absence of increasing doses of anti-CD3 or anti-Cδ blocking mAbs or control mAb (ctl). Results are mean ± SD of TNFα secretion in culture duplicates and are representative of three independent experiments alternatively performed with the 4–29 or 5–4 clone. (B) MRC5 cells were infected (open triangles) or not (closed squares) with CMV and incubated with an anti-CMV 4-29 or a Vγ9Vδ2 T cell clone. T cells alone were also incubated with an agonistic anti-CD3 mAb (10 μg/ml, UCHT1, closed circles). Every 2 h, cells were harvested and stained with an anti-Cδ mAb. Results are presented as the MFI of cells and are representative of two independent experiments. (C) CMV-reactive 4-29 clone was cultured on MRC5 or TAP-deficient BRE cells either uninfected (white bars) or infected with intact (gray bars) or heat-inactivated CMV (black bars). Results are mean ± SD of TNFα secretion in culture triplicates and are representative of three independent experiments performed with 4–29 or 5–4 clones.

Figure 4.

Pattern of chemokine and homing receptor expression by Vδ2^neg and Vδ2^pos T cells. (A) PBMCs from 10 CMV-infected transplanted patients were labeled with an anti-Cδ mAb, an anti-Vδ2 mAb, and each specific mAb directed against chemokine or homing receptors, before being analyzed by flow cytometry. For one patient, the percentage of positive cells for each receptor was evaluated among both Vδ2^neg γδ T cells and Vδ2^pos γδ T cells. Results are presented as the percentage of positive cells in each patient depicted by a specific symbol. The mean expression for each receptor appears as a short line. (B) Three different CMV-specific Vδ2^neg γδ T cell clones were stained with anti–β7-integrin or anti-CCR9 mAbs (thin line) or with a control mAb (bold line), and analyzed by flow cytometry. (C) Polymerization of F-actin in response to TECK/CCL25 was tested on two Vδ2^neg γδ T clones by flow cytometry as described in Materials and methods.

Figure 5.

Anti–CMV-reactive Vδ2^neg γδ T cells are also reactive against intestinal tumor epithelial cells. (A) Vδ2^neg γδ T cell clones were cultured in medium alone, with U373 cells uninfected or infected with CMV, with PMA and ionomycin, with HT29 or Caco2 monolayers, or with Daudi or THP-1 cells. All cells were harvested, permeabilized, and labeled with anti-IFNγ and anti-TNFα mAbs. Percentages correspond to cells that expressed TNFα and/or IFNγ. Results shown are representative of experiments performed with four different clones. (B) As in Fig. 2 B with HT29 cells instead of FSF. The same results were obtained with the 5–4 clone. (C) Two Vδ2^neg γδ clones (4–29 and 5–4) or one control Vγ9Vδ2 T cell clone were incubated in medium alone (white bars), on HT29 cells (gray bars), or with PMA and ionomycin (black bars). (D) The 4–29 anti-CMV γδ T cell clone was incubated in medium alone, with an agonistic anti-CD3 mAb (10 μg/ml, UCHT1) or with HT29 cells. At indicated times, cells were harvested and stained with an anti-Cδ mAb. Results are presented as the MFI ± SD of culture triplicates and are representative of two independent experiments. (E) The 4–29 T cell clone was cultured in medium alone, with PMA and ionomycin or on normal (HIEC and FHC) or tumor (HeLa, HT29, CaCo-2) intestinal epithelial cell lines. Epithelial cells were also cultured in medium alone. After 6 h of incubation, TNFα secretion was quantified in culture supernatant.

Figure 6.

NKG2D is not involved in the activation of Vδ2^neg γδ T cells by CMV-infected fibroblasts or HT29. Vδ2^neg γδ T cell clones were incubated with ⁵¹Cr-labeled CMV-infected or uninfected FSF or HT29, in medium alone or with a control or a blocking anti-NKG2D mAb (20 μg/ml), or with the NKG2D-Fc or control ICAM2-Fc chimeric molecules (both at 20 μg/ml). Effector/target ratio was 10/1. As positive control of blocking reagent efficiency, ⁵¹Cr-labeled MICA⁺ K562 cells were incubated with NKG2D⁺ NKL cells at a 10/1 effector/target ratio. Results are expressed as mean ± SD of specific lysis in culture triplicates and are representative of three independent experiments performed with 4–29 and 5–4 T cell clones.

Figure 7.

Anti-CMV and antitumor reactivity of polyclonal γδ T cell lines isolated from CMV⁺ transplanted patients and CMV⁻ blood donors. Polyclonal γδ T cell lines were generated from five different CMV-infected transplanted patients (TP 1–5) and polyclonal Vδ2^neg γδ T cell lines were generated from three different blood donors (BD 1–3). These lines were incubated in medium alone or on monolayers of HT29 or of CMV-infected or uninfected FSF in the presence of an anti-CD107a mAb. After 6 h, T cells were stained with anti-Cδ and anti-Vδ2 mAbs and analyzed by flow cytometry. (A) Staining profiles obtained with the TP1 γδ T cell line. Indicated are percentages of CD107a⁺ cells among the Vδ2^neg (top left quadrant) or Vδ2^pos (top right quadrant) populations. (B) Percentages of CD107a⁺ cells among Vδ2^neg γδ T cells for each tested transplanted patient or blood donor.