Cytokine requirements for the differentiation and expansion of IL-17A- and IL-22-producing human Vgamma2Vdelta2 T cells

Abstract

Human gammadelta T cells expressing the Vgamma2Vdelta2 TCR play important roles in immune responses to microbial pathogens by monitoring prenyl pyrophosphate isoprenoid metabolites. Most adult Vgamma2Vdelta2 cells are memory cytotoxic cells that produce IFN-gamma. Recently, murine gammadelta T cells were found to be major sources of IL-17A in antimicrobial and autoimmune responses. To determine if primate gammadelta T cells play similar roles, we characterized IL-17A and IL-22 production by Vgamma2Vdelta2 cells. IL-17A-producing memory Vgamma2Vdelta2 cells exist at low but significant frequencies in adult humans (1:2762 T cells) and at even higher frequencies in adult rhesus macaques. Higher levels of Vgamma2Vdelta2 cells produce IL-22 (1:1864 T cells), although few produce both IL-17A and IL-22. Unlike adult humans, in whom many IL-17A+ Vgamma2Vdelta2 cells also produce IFN-gamma (Tgammadelta1/17), the majority of adult macaques IL-17A+ Vdelta2 cells (Tgammadelta17) do not produce IFN-gamma. To define the cytokine requirements for Tgammadelta17 cells, we stimulated human neonatal Vgamma2Vdelta2 cells with the bacterial Ag, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate, and various cytokines and mAbs in vitro. We find that IL-6, IL-1beta, and TGF-beta are required to generate Tgammadelta17 cells in neonates, whereas Tgammadelta1/17 cells additionally required IL-23. In adults, memory Tgammadelta1/17 and Tgammadelta17 cells required IL-23, IL-1beta, and TGF-beta, but not IL-6. IL-22-producing cells showed similar requirements. Both neonatal and adult IL-17A+ Vgamma2Vdelta2 cells expressed elevated levels of retinoid-related orphan receptor gammat. Our data suggest that, like Th17 alphabeta T cells, Vgamma2Vdelta2 T cells can be polarized into Tgammadelta17 and Tgammadelta1/17 populations with distinct cytokine requirements for their initial polarization and later maintenance.

FIGURE 1. Frequency of Vγ2Vδ2 T cells producing IL-17A and IL-22 in adult human and rhesus macaque donors

A. PBMC from 10 normal donors were stimulated with PMA and ionomycin and intracellular cytokine staining for IL-17A and IL-22 was performed. Viable T cells were gated using live/dead blue and anti-CD3 after which the different T cell subsets discriminated using anti-Vδ2 (to identify Vγ2Vδ2 T cells) and anti-pan γδ (to identify total γδ T cells). αβT cells were defined as CD3⁺,γδ⁻. B. Numbers of IL-17A⁺ Vγ2Vδ2, IL-22⁺ Vγ2Vδ2 and IL-17A⁺, IL-22⁺ Vγ2Vδ2 T cells per milliliter of blood were calculated (Table 1). C. Representative IL-17A staining for Vγ2Vδ2 T cells (abbreviated Vδ2 T cells), total γδ T cells, and total αβ T cells. D. Representative IL-22 staining for Vγ2Vδ2 T cells (abbreviated Vδ2 T cells), total γδ T cells, and total αβ T cells. E. Representative IL-17A staining and average frequency of IL-17A⁺ Vδ2 T cells among eight rhesus macaques. Each point refers to one donor and bars depict means.

FIGURE 2. Cytokine profile and memory phenotype of IL-17A⁺ Vγ2Vδ2 T cells from adult human and monkey donors

A. IFN-γ production by IL-17A-producing Vδ2 T cells. PBMC were stimulated with PMA and ionomycin and stained intracellularly for IL-17A, IFN-γ, and IL-22. Shown is a representative human (top) and monkey (bottom) donor. B. Representative surface staining for memory markers, CD27 and CD28 on total Vγ2Vδ2 T cells or IL-17A⁺ gated Vγ2Vδ2 T cells. C. Frequency of total human Vγ2Vδ2 T cells or IL-17A⁺ gated Vγ2Vδ2 T cells belonging to T early + naïve (CD27⁺, CD28⁺), T intermediate (CD27⁺, CD28⁻) or T late (CD27⁻, CD28⁻) memory subsets. Each point represents one donor and bars depict means.

FIGURE 3. IL-1β, TGF-β, and IL-6 induce maximal polarization of IL-17A⁺ neonatal CD4⁻ Vγ2Vδ2 T cells upon antigen stimulation with HMBPP

Umbilical cord blood mononuclear cells were expanded in the presence or absence of HMBPP, IL-23, IL-1β, TGF-β, IL-6, neutralizing anti-IL-6, or neutralizing anti-IL-23 for 13 days (n = 8 individuals for IL-17A data and n = 4 for IL-22 data). IL-2 was added on day 3. On the final day, cells were restimulated with PMA and ionomycin and intracellular staining for IL-17A, IL-22, and IFN-γ performed. Expanded cord blood CD4⁻ Vγ2Vδ2 T cells (defined as Vδ2⁺, CD3⁺, CD4⁻) were divided into IFN-γ⁻, IL-17A⁺ Vγ2Vδ2 T cells (termed Tγδ17), IFN-γ⁺, IL-17A⁺ Vγ2Vδ2 T cells (termed Tγδ1/17), IFN-γ⁺, IL-17A⁻ Vγ2Vδ2 T cells (termed Tγδ1), and IFN-γ^+/−, IL-22⁺ Vγ2Vδ2 (termed Tγδ22). A. (Top) Median number of total IL-17A⁺ CD4⁻ Vγ2Vδ2 T cells (combined Tγδ1/17 and Tγδ17) or Tγδ22 Vγ2Vδ2 T cells among total CD4⁻ Vγ2Vδ2 T cells for each cytokine condition. (Middle) Median number of Tγδ17 or Tγδ1/17 CD4⁻ Vγ2Vδ2 T cells among total CD4⁻ Vγ2Vδ2 T cells for each condition. (Bottom) Median percent of maximum Tγδ17 or Tγδ1/17 CD4⁻ Vγ2Vδ2 T cells expanded for each condition. B. Representative cytokine staining on viable CD4⁻ Vγ2Vδ2 T cells expanded in the presence of HMBPP, IL-1β, TGF-β, IL-6, and anti-IL-23, either unstimulated (left) or restimulated with PMA and ionomycin (right). Bars depict medians and error bars depict median absolute error. *p < 0.05, Kruskal-Wallis comparison with condition 2.

FIGURE 4. IL-23 is required for expansion of adult IL-17⁺ Vγ2Vδ2 T cells

IL-17A-producing Vγ2Vδ2 T cells, in serum-supplemented media, were measured in PBMC after expansion with HMBPP, IL-1β, IL-6, neutralizing anti-IL-4, and neutralizing anti-IFN-γ in the presence or absence of IL-23. IL-2 was added on day 3. On day 12, cells were re-stimulated with PMA and ionomycin, after which the supernatants and cells were harvested for analysis. Expanded Vγ2Vδ2 T cells were defined as Vδ2⁺, CD3⁺. Representative of 2 donors. A. Cytokine profile of expanded Vγ2Vδ2 T cells. Intracellular staining for IL-17A, IL-22, and IFN-γ, (or isotype control) in the presence or absence of IL-23 is shown. B. Percent and total number of IL-17⁺ Vγ2Vδ2 T cells in the presence or absence of exogenous IL-23 (top two panels). Total expanded Vγ2Vδ2 T cells on day 12 (third panel). Total IL-17A protein released into culture as determined by ELISA (bottom panel).

FIGURE 5. IL-23, IL-1β, and TGF-β are sufficient for polarization of adult IL-17A⁺ Vγ2Vδ2 T cells after stimulation with HMBPP

Total PBMC, from 10 donors, were cultured in the presence or absence of HMBPP, IL-23, IL-1β, TGF-β, IL-6, neutralizing anti-IL-6, or neutralizing anti-IL-23 for seven days. IL-2 was added on day 3. On the seventh day, cells were re-stimulated with PMA and ionomycin and intracellular staining for IL-17A, IL-22, and IFN-γ was performed. Expanded PBMC Vγ2Vδ2 T cells (defined as Vδ2⁺, CD3⁺, CD4⁻) could be divided into IFN-γ⁺, IL-17A⁺ Vγ2Vδ2 T cells (termed Tγδ1/17), IFN-γ⁺, IL-17A⁻ Vγ2Vδ2 T cells (termed Tγδ1), and IFN-γ^+/−, IL-22⁺ Vγ2Vδ2 (termed Tγδ22). No IFN-γ ⁻, IL-17A⁺ Vγ2Vδ2 T cells (Tγδ17) were detected in these adult donors. A. (Top) Median number of Tγδ1/17 or Tγδ22 Vγ2Vδ2 T cells among total Vγ2Vδ2 T cells for each cytokine condition. (Bottom) Median percent of maximum Tγδ1/17 Vγ2Vδ2 T cells expanded for each cytokine condition. B. Representative cytokine staining on Vγ2Vδ2 T cells expanded in the presence of HMBPP, IL-23, IL-1β, TGF-β, and anti-IL-6, and restimulated with PMA and ionomycin. Bars depict medians and error bars depict median absolute error. * p < 0.05, Kruskal Wallis comparison with condition 2.

FIGURE 6. Expression of RORγt and T-bet by IL-17A⁺ Vγ2Vδ2 T cells

A. Representative staining for T-bet on monkey peripheral blood Vδ2 T cells, segregated into IL-17A⁺, IFN-γ⁻ Vδ2 T cells (Tγδ17) or IL-17A⁻, IFN-γ⁺ Vδ2 T cells (Tγδ1). Represents one of three monkeys examined. PBMC were isolated and stimulated with PMA and ionomycin and intracellular staining for IL-17A, IFN-γ, and T-bet performed. B. (Neonate, left) Cord blood mononuclear cells were polarized with HMBPP for 13 days in the presence of IL-1β, IL-6, TGF-β, and anti-IL-23, and (Adult, right) adult PBMC were polarized with HMBPP for 7 days in the presence of IL-1β, IL-23, TGF-β, and anti-IL-6. On the final day, cells were restimulated with PMA and ionomycin, surface stained for Vδ2 and CD3, and intracellularly stained for IL-17A, RORγt, and T-bet. Vγ2Vδ2 T cells were segregated into IL-17A⁺ and IL-17A⁻ and the MFI for each transcription factor minus the MFI of the respective isotype control is shown. Because donors had variable baseline RORγt staining, the donors are segregated into two graphs to accommodate the different magnitudes exhibited. Note that cells were not segregated based on IFN-γ production, therefore the neonatal IL-17A⁺ fraction refers to the sum of Tγδ1/17 and Tγδ17. * p < 0.05, Kruskal Wallis comparison with IL-17A⁻ group.

FIGURE 7. Steps in the differentiation and expansion of neonatal and adult Tγδ17 and Tγδ1/17 Vγ2Vδ2 T cells

A. Neonates/Infants. Naïve Vγ2Vδ2 T cells present in neonates are polarized to the Tγδ17 phenotype by antigen activation in the presence of IL-6, IL-1β, and TGF-β. These early Tγδ17 cells are characterized by elevated expression of RORγt, IL-17A production, and minimal expression of IFN-γ and T-bet. The Tγδ17 cells up-regulate IL-23R (and likely IL-12R) enabling them to maintain their Tγδ17 phenotype in the presence of IL-23, IL-1β, and TGF-β or to convert to a Tγδ1/17 phenotype in the presence of IL-23 or IL-12. B. Adults. Most adult Vγ2Vδ2 T cells are memory cells and include small but significant populations of Tγδ1/17 and Tγδ17 cells. Expansion of adult memory Tγδ1/17 and Tγδ17 cells by HMBPP requires IL-23 in addition to IL-1β and TGF-β but not IL-6. Tγδ1/17 and Tγδ17 likely have limited persistence and are either short-lived effector populations or are converted to Tγδ1 through the effects of IL-12.