Distinct cytokine-driven responses of activated blood gammadelta T cells: insights into unconventional T cell pleiotropy

Abstract

Human Vgamma9/Vdelta2 T cells comprise a small population of peripheral blood T cells that in many infectious diseases respond to the microbial metabolite, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), expanding to up to 50% of CD3(+) cells. This "transitional response," occurring temporally between the rapid innate and slower adaptive response, is widely viewed as proinflammatory and/or cytolytic. However, increasing evidence that different cytokines drive widely different effector functions in alphabeta T cells provoked us to apply cDNA microarrays to explore the potential pleiotropy of HMB-PP-activated Vgamma9/Vdelta2 T cells. The data and accompanying validations show that the related cytokines, IL-2, IL-4, or IL-21, each drive proliferation and comparable CD69 up-regulation but induce distinct effector responses that differ from prototypic alphabeta T cell responses. For example, the Th1-like response to IL-2 also includes expression of IL-5 and IL-13 that conversely are not induced by IL-4. The data identify specific molecules that may mediate gammadelta T cell effects. Thus, IL-21 induces a lymphoid-homing phenotype and high, unexpected expression of the follicular B cell-attracting chemokine CXCL13/BCA-1, suggesting a novel follicular B-helper-like T cell that may play a hitherto underappreciated role in humoral immunity early in infection. Such broad plasticity emphasizes the capacity of gammadelta T cells to influence the nature of the immune response to different challenges and has implications for the ongoing clinical application of cytokines together with Vgamma9/Vdelta2 TCR agonists.

FIGURE 1

Flow cytometric analysis of activated γδ T cells. PBMC were cultured in the absence (∎) or presence () of HMB-PP, together with the cytokines indicated, and analyzed by flow cytometry. Cell proliferation was assessed by expansion of Vγ9+CD3+ cells within the CD3+ lymphocyte population (n = 12). Expression of CD69 on Vγ9+ CD3+ cells was determined after 18 h (n = 8), expression of ICAM-1 (n = 6), and LFA-1 (n = 4) after 6 days.

FIGURE 2

IL-2-dependent gene expression. Expression of IFN-γ (n = 3–6) and TNF-α (n = 6) in Vγ9⁺ CD3⁺ cells was analyzed by intracellular flow cytometry after 72 h. GM-CSF (n = 4), CCL3 (n = 3), and IL-13 (n = 4) were detected in 72 h culture supernatants, IL-5 (n = 2) after 6 days. Solid and hatched bars as in Fig. 1 legend.

FIGURE 3

IL-4-dependent gene expression. A, Expression of IL-4 and LT-α mRNAs by γδ T cells purified from PBMC after 72 h, as analyzed by real-time RT-PCR. Expression levels were normalized to values in medium controls and represent mean values and SDs from duplicate experiments. B, Expression of IL-4 and GATA-3 mRNAs by γδ T cells purified from PBMC after 72 h. Semiquantitative RT-PCR data shown are representative of three blood donors. C, Surface expression of CD27 (n = 6–7) as analyzed by flow cytometry on day 6.

FIGURE 4

IL-21-dependent gene expression. A, Surface expression of CD25 (n = 6), intracellular expression of CD152 (n = 6), and CXCL10 secretion into the culture supernatants (n = 4–9) were detected after 72 h. Surface expression of CD244 (n = 8–11), KLRG1 (n = 3), and CD62L (n = 5) was measured after 6 days (note that KLRG1 expression is depicted as mean fluorescence values as at baseline Vγ9/Vδ2 T cells were already ~80% KLRG1⁺). Solid and hatched bars as in Fig. 1 legend. B, Expression of IL-21 and IL-21R mRNAs by γδ T cells purified from PBMC after 72 h in culture was analyzed by semiquantitative RT-PCR; data shown are representative of three individual blood donors (ctrl, whole PBMC stimulated with PHA).

FIGURE 5. IL-21-dependent expression of CXCL13

A, M/A plot of IL-21-stimulated vs IL-2-stimulated Vγ9/Vδ2 T cells.The locations of the IL-2-induced genes encoding for GM-CSF and LT-α and the IL-21-induced gene encoding for CXCL13 are indicated. B and C, Expression of CXCL13 mRNA by γδ T cells purified from PBMC cultured for 72 h in medium alone or with HMB-PP and the cytokines indicated. Semiquantitative RT-PCR data shown are representative of three blood donors; real-time data are mean values and SDs from duplicate experiments. D, Detection of CXCL13 protein in the supernatant of PBMC cultured for 72 h with and without HMB-PP, in the presence of the cytokines indicated (n = 6–9). E, CXCL13 production by PBMC cultured for 72 h in medium alone or with HMB-PP plus IL-21 after depletion of γδ T cells, monocytes, or B cells (n = 3). F, CXL13 production by purified γδ T cells in the absence or presence of monocytes as feeder cells after 72 h (n = 4). G, Inhibition of the IL-21-induced γδ T cell proliferation by IFN-β. PBMC were cultured for 6 days with HMB-PP plus IL-2 or HMB-PP plus IL-21 in the absence or presence of IFN-β (n = 3). H, Inhibition of the IL-21-dependent CXCL13 expression by IFN-β. PBMC were cultured for 72 h with HMB-PP plus IL-2 or HMB-PP plus IL-21 in the absence or presence of IFN-β (n = 5).

FIGURE 6. A role for γδ T cells in regulating follicular B cell maturation

A, Immunohistochemical analysis of serial sections of an active gut follicle from a patient with Yersinia ileitis, with γδ T cells and CXCL13, respectively, stained in red. Colocalization of CXCL13-producing cells and γδ T cells is indicated by arrows. Note that a similar picture of the same section stained for γδ T cells was already published earlier (23 ), but has been included here for the sake of optimum comparison. B, Proposed interaction between γδ T cells, B cells, and T_FH cells in secondary lymphoid tissue. Expression of CCR7 by naive and central memory αβ T cells permits entry to the LNs, and subsequent colocalization with CCR7⁺ DC in the T zone that is defined by the CCR7 ligands CCL19 and CCL21. T cell priming results in the generation of TFH cells expressing CXCR5, thereby conferring responsiveness to the B zone-specific chemokine CXCL13. After relocalization of T_FH cells and CXCR5⁺ B cells to the follicles, mutual interaction through ICOS and CD40 with their respective ligands induces plasma cell maturation on one hand, and gives rise to effector αβ T cells on the other hand (see also Refs. 2 and 60 ). Upon infection with microbial pathogens, peripheral γδ T cells recognize HMB-PP at sites of inflammation and contribute to the local immune response by secretion of proinflammatory cytokines and chemokines, and lysis of infected cells. Alternatively or additionally, they acquire a LN homing phenotype by up-regulation of CCR7 and CD62L (Ref. and this study), and HMB-PP released in the periphery may reach local LN through tissue drainage and act on LN resident γδ T cells. Data presented here show that γδ T cells moving into the GCs will respond to T_FH-derived IL-21 by producing CXCL13 and CXCL10, thus contributing to the molecular definition of the B zone and aiding further recruitment of CXCR5⁺ B cells, T_FH cells, and monocytes. HMB-PP triggered γδ T cells may also directly present microbial Ags to T_FH cells at an early stage of infection (65 ).