Phenotypic and functional alterations of Vgamma2Vdelta2 T cell subsets in patients with active nasopharyngeal carcinoma

Abstract

Introduction: Human Vgamma2Vdelta2 T cells play important role in immunity to infection and cancer by monitoring self and foreign isoprenoid metabolites with their gammadelta T cell antigen receptors. Like CD4 and CD8 alphabeta T cells, adult peripheral Vgamma2Vdelta2 T cells represent a pool of heterogeneous cells with distinct functional capabilities.

Purpose: The aim of this study was to characterize the phenotypes and functions of various Vgamma2Vdelta2 T cell subsets in patients with nasopharyngeal carcinoma (NPC). We sought to develop a better understanding of the role of these cells during the course of disease and to facilitate the development of immunotherapeutic strategies against NPC.

Results: Although similar total percentages of peripheral blood Vgamma2Vdelta2 T cells were found in both NPC patients and normal donors, Vgamma2Vdelta2 T cells from NPC patients showed decreased cytotoxicity against tumor cells whereas Vgamma2Vdelta2 T cells from normal donors showed potent cytotoxicity. To investigate further, we compared the phenotypic characteristics of Vgamma2Vdelta2 T cells from 96 patients with NPC and 54 healthy controls. The fraction of late effector memory Vgamma2Vdelta2 T cells (T(EM RA)) was significantly increased in NPC patients with corresponding decreases in the fraction of early memory Vgamma2Vdelta2 T cells (T(CM)) compared with those in healthy controls. Moreover, T(EM RA) and T(CM) Vgamma2Vdelta2 cells from NPC patients produced significantly less IFN-gamma and TNF-alpha, potentially contributing to their impaired cytotoxicity. Radiotherapy or concurrent chemo-radiotherapy further increased the T(EM RA) Vgamma2Vdelta2 T cell population but did not correct the impaired production of IFN-gamma and TNF-alpha observed for T(EM RA) Vgamma2Vdelta2 T cells.

Conclusion: We have identified distinct alterations in the Vgamma2Vdelta2 T cell subsets of patients with NPC. Moreover, the overall cellular effector function of gammadelta T cells is compromised in these patients. Our data suggest that the contribution of Vgamma2Vdelta2 T cells to control NPC may depend on the activation state and differentiation of these cells.

Fig. 1

Vγ2Vδ2 T cells from NPC patients demonstrate decreased tumor cytolytic activity but are present in normal numbers. a Purity of MACS-purified Vγ2Vδ2 T cells. Vγ2Vδ2 T cells were isolated from 6 NPC patients and 5 NOR controls as described in “Materials and methods” and analyzed by flow cytometry. b Effector Vδ2 T cells from NPC patients exhibit reduced tumor cytolytic activity. Purified Vγ2Vδ2 T cells were incubated with K562 or HK-1 tumor cells for 4 hr at an E:T of 10:1. Note that Vδ2 T cells from healthy controls (NOR) showed higher cytolytic activity against K562 and HK-1 compared with Vδ2 T cells from NPC patients. c No significant difference in the frequency of Vγ2Vδ2 T cells in NPC patients and healthy controls (NOR). The percentage of peripheral blood Vγ2Vδ2 T cells among CD3 positive T cells was measured by flow cytometry as described in the “Materials and methods”. d No significant differences observed in the percentage of Vδ2 T cells in males and females in the NPC and NOR cohorts. Vγ2Vδ2 T cells were assessed as in c

Fig. 2

The proportion of T_{EM RA} Vγ2Vδ2 T cells is increased in patients with active NPC compared with healthy controls. a Vγ2Vδ2 T cells can be divided into four distinct populations based on CD27 and CD28 surface expression. Representative staining is shown. b Vγ2Vδ2 T cell subset distributions differ between NPC patients and healthy controls. An example of the γδ T cell subset distribution in an NPC patient and a NOR is shown (left panel). T_CM (CD27⁺CD28⁺) Vγ2Vδ2 T cells were significantly reduced and T_{EM RA} (CD27⁻CD28⁻) cells significantly increased in NPC patients compared with NOR (right panel). c The increase in T_{EM RA} cells, and decrease in T_CM cells in NPC patients was not related to the age of the patients compared with NOR. No relationship exists between age and T_CM and T_{EM RA} cells among NPC patients (closed symbols) and healthy controls (open symbols)

Fig. 3

Perforin is highly expressed in T_EM and T_{EM RA} Vγ2Vδ2 T cells. a Perforin expression in Vγ2Vδ2 T cell subsets. PBMC from NOR were surface stained with anti-Vδ2 TCR, CD27, and CD28 prior to fixation and permeabilization with BD Cytofix/CytoPerm. Intracellular perforin was detected using anti-perforin mAb. A mouse isotype control was included to determine background staining. (Left panel) Representative perforin staining. Perforin expression was higher in T_{EM RA} cells (orange shaded) compared with T_CM cells (violet shaded). Isotype controls for T_{EM RA} (orange unshaded) and T_CM (violet unshaded) are shown. (Right panel) Increased frequency of perforin expressing cells in the T_EM and T_{EM RA} Vγ2Vδ2 T cell subsets in NOR. b Comparison of perforin expression among Vγ2Vδ2 T cell subsets in NPC patients and healthy controls (NOR). Note that with the exception of T_CM, there were no statistically significant differences in perforin expression between NOR and NPC patients

Fig. 4

IFN-γ and TNF-α production is impaired in Vγ2Vδ2 T cells from patients with active NPC. a IFN-γ and TNF-α production in response to HMBPP stimulation is reduced in total Vγ2Vδ2 T cells from NPC patients. PBMC from NPC patients (n = 29) and NOR (n = 28) were stimulated with or without HMBPP (0.316 μM) for 4 h followed by intracellular cytokine staining for IFN-γ and TNF-α as described in the “Materials and methods”. Representative staining is shown in the left panel. Note that the frequency of IFN-γ or TNF-α-producing Vγ2Vδ2 T cells in NPC was decreased compared with Vγ2Vδ2 T cells from NOR. b IFN-γ and TNF-α production in response to HMBPP stimulation by T_CM and T_{EM RA} cells is reduced in NPC patients compared with NOR. Stimulation was performed as described in a. c No correlation between cytokine-producing γδ T cells and age in NPC patients or NOR. Linear regression analyses were done to assess the frequencies of cytokine-producing γδ T cells in NPC patients (filled circles) and NOR (empty circles) with their ages. d IFN-γ and TNF-α production in response to mitogen stimulation is similar between NPC patients and healthy controls (NOR). PBMC from NPC patients and NOR were stimulated with PMA and ionomycin followed by intracellular cytokine staining for IFN-γ and TNF-α for Vδ2 and CD8 T cells to evaluate antigen-independent IFN-γ and TNF-α production. The frequencies of IFN-γ- (left panel) and TNF-α- (right panel) positive cells are shown. Note that there were no significant differences in cytokine production by Vδ2 and CD8 T cells obtained from NPC patients or NOR

Fig. 5

Radiotherapy or concurrent chemo-radiotherapy further increases T_{EM RA} and decreases T_CM Vγ2Vδ2 T cells but fails to correct cytokine production. a The frequency of T_CM and T_{EM RA} Vγ2Vδ2 cells was compared between NOR, NPC patients prior to receiving treatment (PRE), and post-therapy patients (PT). For PT patients, the percentages of T_{EM RA} Vγ2Vδ2 T cells were significantly higher than for NOR and PRE subjects. Increased T_{EM RA} in PT was accompanied by a decline in early memory T_CM Vγ2Vδ2 T cells. b Treatment failed to correct the defective IFN-γ and TNF-α production detected for total Vδ2 T cells. c IFN-γ and TNF-α production by Vγ2Vδ2 T cell subsets remained defective, although IFN-γ production by T_CM cells from PT patients showed a marginal increase compared with NPC patients before therapy