Vγ9Vδ2 T (Vδ2) cells survey cholesterol biosynthesis by sensing metabolic intermediates in an innate-like, MHC-unrestricted fashion [1], [2]. The cholesterol metabolites with stimulatory activity, overall defined as phosphoantigens (PAg) due to their pyrophosphate moiety, include high potency microbial molecules, in particular E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), and low-potency compounds produced by host cells, such as isopentenyl pyrophosphate (IPP) [3], [4], [5], [6]. Phosphoantigens are sensed via an inside out mechanism not yet completely elucidated. They bind to an intracellular domain of the membrane molecule Butyrophilin 3A1 (BTN3A1), expressed by a broad range of target/accessory cells [7], [8], [9], [10], [11]. PAg binding causes changes in BTN3A1 conformation and membrane mobility/distribution, ultimately resulting in Vδ2 cell activation [7], [12], [13], [14]. While several details about this process are still unknown, it appears that other butyrophilins interacting with BTN3A1 are also needed for Vδ2 cell activation [15], [16].

Vδ2 cells display potent cytotoxic function against diverse infected or malignant cells and employ various strategies to kill their targets [17], [18]. Killing of target cells often entails the release of granules containing cytotoxic mediators such as perforin, granzymes and granulysin [19], [20], [21], [22], [23]. Potential target cells can be rendered (more) susceptible to Vδ2-mediated killing by inhibition of an enzyme in the mevalonate pathway, the farnesyl pyrophosphate (FPP) synthase, as this results in intracellular accumulation of IPP [24], [25], [26], [27], [28], [29]. FPP is effectively inhibited by treatment with aminobisphosphonates (ABPs), drugs routinely employed for cancer chemotherapy and activators of Vδ2 cells [26], [30], [31]. ABPs such as Zoledronic acid (ZOL) are more effective than PAgs for stimulation of neonatal Vδ2 cells [32], [33], [34], thus serve as useful tools to study differentiation and function of cord blood Vδ2 cells in vitro.

In neonates born to healthy women, cord blood Vδ2 cells are mostly naïve and display different functional features compared to their adult or infant counterpart [32], [33], [35], [36], [37], [38], [39], [40], [41]. In particular, they have little cytotoxic potential, but quickly acquire it after birth [37], [38], [39], in a process likely driven by gut microbial colonization and exposure to environmental microorganisms. The molecular pathway(s) involved in the differentiation of naïve cells into cytotoxic effectors has not been identified, and it is unclear whether the same mechanism(s) regulates cytotoxic potential before and after birth. We therefore assessed the involvement of PD-1, an inhibitory receptor expressed prominently at birth compared to later in life, in the modulation of Vδ2 cell cytotoxic potential at birth.

We previously observed that neonatal Vδ2 cells express the inhibitory receptor PD-1 much longer than their adult counterparts after in vitro activation. The engagement of this molecule dampens cord blood Vδ2 cell cytokine production and degranulation, suggesting that it serves as a key regulatory factor for Vδ2 cell function in early life [42]. We also noted that acquisition of adult-like Vδ2 cell cytotoxic potential between birth and 12 months of life coincided with decreasing PD-1 and increasing NKG2A expression [38], consistent with the results of another report, which timed the acquisition of cytotoxic potential to the first ten weeks of life [39]. In the current study, we thus investigate if the expression of PD-1 (or lack thereof) is related to the differentiation of cytotoxic effectors in early life. In our in vitro culture system, PD-1 expression, especially in the absence of CD56, displayed a strong negative association with cytotoxic potential, measured as the proportion of perforin+ Vδ2 cells. Transcriptomic profiles of sorted Vδ2 subsets revealed that PD-1+CD56− cells (with the lowest cytotoxic potential based on perforin production) expressed significantly lower levels of several genes related to cytotoxicity and NK function compared to PD-1−CD56+ cells (the subset with the highest cytotoxic potential), consistent with the flow cytometry results. Overall, PD-1 expression on resting Vδ2 lymphocytes at birth serves as a marker to identify cells less likely to have acquired cytotoxic potential and it becomes a strong predictor of low cytotoxic potential in the absence of CD56 expression.