Cytokine-mediated activation of human ex vivo-expanded Vγ9Vδ2 T cells

Abstract

Vγ9Vδ2 T cells, the major subset of the human peripheral blood γδ T-cell, respond to microbial infection and stressed cells through the recognition of phosphoantigens. In contrast to the growing knowledge of antigen-mediated activation mechanisms, the antigen-independent and cytokine-mediated activation mechanisms of Vγ9Vδ2 T cells are poorly understood. Here, we show that interleukin (IL) -12 and IL-18 synergize to activate human ex vivo-expanded Vγ9Vδ2 T cells. Vγ9Vδ2 T cells treated with IL-12 and IL-18 enhanced effector functions, including the expression of IFN-γ and granzyme B, and cytotoxicity. These enhanced effector responses following IL-12 and IL-18 treatment were associated with homotypic aggregation, enhanced expression of ICAM-1 and decreased expression of the B- and T-lymphocyte attenuator (BTLA), a co-inhibitory receptor. IL-12 and IL-18 also induced the antigen-independent proliferation of Vγ9Vδ2 T cells. Increased expression of IκBζ, IL-12Rβ2 and IL-18Rα following IL-12 and IL-18 stimulation resulted in sustained activation of STAT4 and NF-κB. The enhanced production of IFN-γ and cytotoxic activity are critical for cancer immunotherapy using Vγ9Vδ2 T cells. Thus, the combined treatment of ex vivo-expanded Vγ9Vδ2 T cells with IL-12 and IL-18 may serve as a new strategy for the therapeutic activation of these cells.

In the Absence of a TCR Signal IL-2/IL-12/18-Stimulated γδ T Cells Demonstrate Potent Anti-Tumoral Function Through Direct Killing and Senescence Induction in Cancer Cells

“…TCR stimulus significantly decreased the expression of Eomes in context with IL-2/IL-12 and IL-2/IL-12/IL-18 ( Figure 3B). Transcriptome analysis suggests that the enhanced production of IFN-γ by cytokines is not mediated by T-bet or Eomes, thus potentially via IκBζ [20,32,33]. However, as protein expression may differ from mRNA expression, this point needs further investigation.…”

“…The transcription factor T-bet and Eomes is critical for IFN-γ expression in the mouse [30,31] and has a partially contributive role on human γδ T cells [20]. To investigate whether the cytokine-induced production of IFN-γ is associated with increased T-bet or Eomes expression, we examined the expression of T-bet and Eomes in cytokine-treated γδ T cells by qPCR.…”

“…While TCR or NK-receptor stimulation in the presence of IL-12 and IL-18 promotes effector functions such as cytotoxicity and IFN-γ expression, and enhances the clonal expansion of Vγ9Vδ2 T cells [16][17][18][19], a synergistic effect for IL-12 and IL-18 in a TCR-independent production of IFN-γ has been demonstrated for γδ T cells lately [20][21][22]. In an effort to dissect potentially detrimental suppressive activity induced via supraphysiologic pharmacological TCR triggering from anti-tumoral behavior, we hypothesized that this capacity of γδ T cells for TCR-independent activation, namely bypassing the TCR via cytokines, might be an option to generate physiologically activated Vγ9Vδ2 γδ T cells in reasonable numbers for anti-tumoral immunotherapy [20]. While this ability of TCR-independent activation is seen to varying degrees also in conventional CD4 + or CD8 + αβ T cells, γδ T cells have the potential of secreting cytokines more abundantly than these cells [21,23].…”

Suppressive activity of Vδ2+ γδ T cells on αβ T cells is licensed by TCR signaling and correlates with signal strength

“…To understand the role of the γδ TCR in determining functional differentiation into a regulatory phenotype, we examined peripheral Vδ2 + T cells after four different magnitudes of TCR stimulation, namely negatively freshly isolated Vδ2 + T cells (no stimulation), freshly untouched isolated Vδ2 + T cells with subsequent TCR-bypass stimulation (TCR-independent cytokine-mediated stimulation), fresh positively isolated Vδ2 + T cells (a single strong γδ TCR stimulus), and IPP-stimulated Vδ2 + T cells (strongest and continuous γδ TCR stimulation) for their suppressive activity by co-culturing them with anti-CD3/CD28 activated autologous αβ T cells. Vδ2 + T cells activated in a TCR-independent manner by inflammatory cytokines (TCR-bypass stimulation) [34][35][36] mimicked the mildest form of TCR stimulatory manipulation. In contrast, the anti-Vδ2 antibody crosslinks the TCR of Vδ2 + T cells during positive selection, resulting in one strong TCR stimulus.…”

“…Thus γδ T cells stimulated for 10 days with IPP and subsequent positive isolation via the TCR using MACS technology represented the maximum TCR stimulatory manipulation in our model system. Since IL-15 and IL-12 alone or in combination enhanced the activation of PAg-stimulated γδ T cells [24,36,38] and TGF-β/IL-15 induced Foxp3 in Vδ2 + T cells, which then suppressed the proliferation of αβ T cells [21], TCR stimulation-where indicated-was combined with either one of these cytokines or combinations thereof. Untouched Vδ2 + T cells did not exhibit any inhibitory effect on the proliferation of anti-CD3/anti-CD28 stimulated αβ T cells, while TCR-bypass stimulation with IL-12/IL-18 [39] combined with IL-15 resulted in slight effects, this constituting only 16% of the inhibition mediated by TCR-crosslink-stimulated cells (average 68% suppression, Fig.…”