Immunological Interactions Between Gammadelta T Cells and West Nile Virus in the Infected Host

ABSTRACT

Between mosquitoes and birds, West Nile virus (WNV) is a neurotropic flavivirus, an arthropod‐borne pathogen involved in an enzootic cycle. Additionally, it can infect both people and horses, leading to severe illness. Since 1999, WNV has spread across North and South America, including Mexico and the Caribbean. It is endemic in several regions of Europe, Africa, the Middle East, and Asia. WNV affects the central nervous system (CNS), causing severe disease in a small percentage of infected individuals, especially in immunocompromised or elderly hosts. This re‐emerging pathogen was identified during an outbreak in July 2025 in the Lazio region of Italy, rekindling interest in an area where the virus had not circulated for some time and raising several questions about the WNV vector and its spread. Gammadelta T (γδ T) lymphocytes are innate cells that can respond rapidly and non‐specifically to viral infections and other pathogens, thereby linking innate and adaptive immunity. Several studies in mice and humans suggest they play distinct roles in controlling WNV infection by communicating with other immune cells, underscoring their antiviral role, which is essential for containing viral dissemination in the host. This review will discuss recent studies on the role of γδ T cells in viral pathogenesis and in protective immunity during WNV infection.

1. Introduction

West Nile virus (WNV) is a neurotropic flavivirus with a single‐stranded RNA genome and has posed public health challenges in North America for over a decade. Initially identified in Uganda in 1937, WNV was first reported in Europe in the late 20th century [1]. Subsequently, it became the leading cause of epidemic viral encephalitis across Europe [2]. The virus follows an enzootic transmission cycle, utilising mosquitoes as vectors and birds as hosts that amplify its presence. While mammals can contract the virus through mosquito bites, they are considered dead‐end hosts; humans and horses serve as incidental hosts (Figure 1).

FIGURE 1.

WNV vector cycle. WNV is typically transmitted to humans by infected mosquitoes. These mosquitoes become infected by feeding on infected birds. The mosquitoes then spread the virus, and birds help it propagate and increase in number. The virus can also be transmitted between humans through blood transfusions and organ transplants. Additionally, it can spread from mother to child during pregnancy and breastfeeding, or through laboratory exposure.

WNV has reached Europe via migratory birds from sub‐Saharan Africa, North Africa, and the Middle East, with mosquitoes serving as vectors during the warmer months [3] In Europe, the primary vectors of WNV transmission include several species, such as Culex pipiens and Culex modestus. However, Aedes albopictus and Aedes (Ochlerotatus) detritus also contribute to its spread [4]. Most human infections occur between July and September when mosquito activity peaks. These vectors facilitate transmission among both birds and mammals and act as bridge vectors, enhancing epizootic transmission to humans and equids [5]. Birds expel high concentrations of the virus through oral and cloacal secretions, with evidence indicating direct viral transmission among certain bird species, such as geese [6, 7].

Since 2018, significant outbreaks of WNV have been recorded across Europe, Tunisia, Israel, and the United States [8, 9]. Recently, Italy's Lazio region experienced an epidemic with over 101 cases reported in July 2025 alone [10]. Specifically, from July 18 to August 2025, there were 171 confirmed locally acquired cases of WNV infection, including 110 instances of WNV fever, 57 cases of neuroinvasive syndrome, four asymptomatic blood donors, and nine deaths [11], raising new questions about managing this infection in previously unaffected areas of Italy. These findings highlight the need for further research to implement future public health strategies, improve the sensitivity of existing surveillance systems, and emphasise the importance of differential diagnosis for febrile and/or neuroinvasive cases. Furthermore, it is crucial for immunocompromised host populations because of the potential for WNV transmission through organ transplantation and the increased risk of neuroinvasive disease in these patients [12, 13]. WNV transmission during solid organ transplantation (SOT) was identified in several clusters in the US and Italy from 2002 to 2023 [14].

Research on WNV infection has utilised various animal models, including hamsters, mice, monkeys, and horses [15, 16, 17, 18]. Investigations involving these models—as well as cell cultures or samples from infected patients—have yielded critical insights into how host immunity reacts to WNV.

γδ T cells, which function as innate lymphocytes linking innate and adaptive immune responses in humans and mouse models, recognise non‐peptidic antigens called Phosphoantigens (PhAgs) [19]. They respond quickly and non‐specifically to viral infections without MHC restriction [19]. Their roles in immune responses against viruses have been detailed across multiple disease models; notably, γδ T cells exhibit both protective and pathogenic effects in humans and mice alike [20]. However, the mechanisms of γδ T cell activation during WNV infection remain unclear.

This review aims to gather recent findings on these unique immune cells, focussing on both WNV pathogenesis and protective immunity.

2. WNV Disease: Clinical Features and Sequelae

The WNV has become a worldwide concern, leading to outbreaks and cases of viral encephalitis. This virus impacts the central nervous system (CNS) and can result in serious illness for a small fraction of those infected, particularly among immunocompromised individuals or the elderly. In humans, the incubation period typically ranges from 2 to 6 days; however, it can extend up to 21 days in those with weakened immune systems [11]. Viraemia levels in humans are generally low, occurring one to 3 days post‐infection and may persist for as long as 11 days [21]. Still, this viraemia is not regarded as infectious to mosquitoes. Factors that may elevate the risk of WNV include hypertension, advanced age, brain tumours, haematological disorders, diabetes and kidney issues, alcohol abuse, and genetic issues. Although human‐to‐human transmission is rare, it can occur through substances of human origin (SoHO), such as blood transfusions or organ transplants from an infected individual [21, 22, 23]. Reports suggest that individuals with WNV could have a mortality rate reaching up to 10% [22]. In the United States, instances of vertical transmission from mother to child via placental transfer and breastfeeding have been documented [23, 24, 25]. Additionally, infections related to occupational exposure—such as during mosquito collection or autopsy procedures—have been reported [26, 27, 28]. Approximately 80% of human infections with WNV are asymptomatic; about 20% manifest as febrile illness (West Nile fever), while less than 1% progress to neuroinvasive disease (WNND), including meningitis and encephalitis [29]. On rare occasions, WNV infection may lead to Guillain–Barré syndrome or other demyelinating neuropathies [11]. The overall mortality rate for patients with WNND is around 10%, increasing to 20% for those aged 70 and older and ranging between 30% and 40% for patients with haematologic cancers [30], SOT [12], or those undergoing treatment with B‐cell–depleting monoclonal antibodies [31]. In patients hospitalised for WNV disease, over half experience long‐term consequences such as fatigue, weakness, myalgia, memory impairment, and depression. Currently, no specific therapeutic agents are available to treat WNV infection, and no approved human vaccines are available to prevent WNV infection and/or disease.

3. γδ T Cells: Innate Lymphocytes in Infectious Diseases

γδ T cells play an essential role in infectious diseases in both human beings and mice. γδ T cells represent a small fraction of unconventional CD3+ T cells in lymphoid tissues and blood, as well as at epithelial and mucosal surfaces [32]. Unlike conventional αβ T cells, which recognise peptide antigens presented by MHC molecules on antigen‐presenting cells (APCs) [32], γδ T cells have a unique TCR rearrangement that allows them to recognise a broad variety of peptide or non‐peptide antigens, including microbial products, stress molecules, and self‐antigens, both via pattern recognition receptors (PRRs) and via the TCR [19]. γδ T cells also have on their surface the natural killer receptors (NKRs), such as the NKG2D, and their ligands include MHC class I‐related chain A or B (MICA or MICB) and the UL‐16 binding proteins (ULBP1–6) in humans and the H60, MULT‐1, and RAE‐1 in mice [19]. Upon encountering different antigens, γδ T cells rapidly produce pro‐inflammatory cytokines [Interferon gamma (IFN‐γ), Tumour Necrosis Factor alpha (TNF‐α), and Interleukin‐17 (IL‐17)] to kill pathogens or infected cells [33]. These cytokines are also essential for recruiting and activating other immune cells, such as neutrophils, dendritic cells, macrophages, and NK cells, to kill infected or tumour cells and promote tissue repair [19]. Moreover, activated γδ T cells can directly eliminate infected cells by releasing cytotoxic molecules, such as perforin and granzymes, thereby inducing apoptosis [19]. γδ T cells can also produce modulatory cytokines or, through direct cell‐to‐cell contact, communicate with other immune cells (CD4 and CD8 T cells, B cells, and NK cells) [34]. These interactions are essential for enhancing innate and adaptive immune responses against pathogens, facilitating rapid pathogen clearance [34, 35].

γδ T cells exert a potent antiviral activity against several types of viral diseases (e.g., WNV, CMV, HIV, Enterovirus) [34, 35, 36, 37, 38], bacterial (e.g., Mycobacterium tuberculosis ‐MTB) [39], and parasitic pathogens such as Plasmodium spp [40] and Listeria Monocytogenes [41] through cytolytic and noncytolytic mechanisms [42].

Human γδ T cells can be divided into five main populations (δ1, δ2, δ3, δ4, and δ5) [43], based on the T cell receptor δ (TCRδ) chain expression [44, 45] and distributed in an organ‐specific manner [43, 46]. The last three subpopulations had limited literature, whereas Vδ2 T cells were the most studied in humans.

In mice, γδ T cells predominantly comprise Vγ1, Vγ2, Vγ4, Vγ5, Vγ6, and Vγ7 subsets [43], which showed both direct and indirect functions in immune defence against various pathogens [47, 48].

In this part of the manuscript, we describe the principal human and murine γδ T cell subpopulations, highlighting their profiles and functions in the context of WNV infection.

4. Human γδ T Cells

Vδ2 T cells are the predominant subset of γδ T cells in the peripheral blood of adult humans, comprising approximately 90% of total circulating γδ T cells. During TCR rearrangement, the Vδ2 chain almost exclusively pairs with the Vγ9 chain, forming the widely distributed Vγ9Vδ2 T cell population [45]. As immune cells that do not rely on MHC, γδ T cells have recently been shown to use a non‐restricted recognition mechanism linked to Vγ9. Phosphoantigens (PhAgs), which gather in infected cells, do not directly bind to Vγ9Vδ2 T cells but act as ‘molecular glues,’ promoting intracellular interactions between butyrophilin subfamily three members A1 (BTN3A1 and BTN2A1) on the target cells. This interaction enables the extracellular domain of BTN2A1 to bind the Vγ9 TCR chain, thereby activating γδ T cells [19]. As a result, PhAgs or BTN‐monoclonal Abs can be used to specifically stimulate Vγ9Vδ2 T cells. Besides recognising through the Vγ9Vδ2 TCR‐dependent pattern, Vδ2 T cells can identify virus‐infected cells by the up‐regulation of NKG2D ligands (such as MICA, MICB, and ULBP) on the infected cells via NKG2D, inducing target cell death through the Fas‐Fas ligand (Fas‐FasL) and TNF‐related apoptosis‐inducing ligand‐death receptor 5 (TRAIL‐DR5) pathways [49]. Moreover, Vδ2 T cells may also express the Fcγ receptor III (CD16), enhancing their antiviral activity through antibody‐dependent cellular cytotoxicity [50]. Vδ1 T cells account for 0.1%–0.5% of γδ T cells in the peripheral blood of adult humans and represent the ‘adaptive’ subset of γδ T cells. Despite their low frequency in blood, they are widely distributed in peripheral tissues and organs, including the intestinal epithelium, skin, liver, and spleen, where they contribute to tissue homoeostasis [43]. Vδ1 T cells do not associate with a specific Vγ chain, nor do they pair with Vγ9, and therefore cannot be activated by PhAgs, unlike Vδ2 T cells [51]. Instead, Vδ1 T cells recognise several MHC‐like proteins, including members of the CD1 family and the MHC class I‐related molecule (MR1), which present lipid antigens or small‐molecule metabolites [52]. Interestingly, Vδ1 T cells can also recognise target cells through their TCR, by interacting with the MHC II complex HLA‐DR, or via the NKG2D pathway, similarly to Vδ2 T cells. Other molecules, such as Natural Killer Receptors (NKRs) and activating receptors (DNAM‐1), further enhance their cytotoxic activity. These diverse recognition mechanisms broaden their immune effector functions against pathogens [49, 53].

Vδ3 T cells are generally absent from the peripheral blood of healthy people but become more prevalent in individuals with reactivated human cytomegalovirus (HCMV) infections [51]. These cells are predominantly found in the liver, intestines, and lamina propria [54]. Notably, Vδ3 T cells exhibit functional similarities and antigen recognition patterns to those of Vδ1 T cells [55]. It has been observed that Vδ3 T cells recognise CD1d and respond by killing CD1d⁺ target cells, releasing Th1, Th2, and Th17 cytokines, and promoting the maturation of DC into APCs [55]. However, research on Vδ3, Vδ4, and Vδ5 T cells remains limited, primarily due to the lack of specific monoclonal antibodies that would allow for efficient immunophenotyping and cell sorting.

5. Murine γδ T Cells

According to variations in the TCR γ chain, mouse γδ T cells are classified into six distinct subpopulations: Vγ1, Vγ2, Vγ4, Vγ5, Vγ6, and Vγ7 T cells. Notably, γδ T cells in the lungs of naïve mice are primarily composed of the Vγ1+ and Vγ4+ subsets [56, 57]. Research across various disease models has shown that specific TCR‐expressing Vγ‐chain T cells perform specialised functions [58]. For instance, while Vγ1+ T cells enhance airway responsiveness, Vγ4+ T cells have been shown to mitigate it [59]. Despite growing evidence of γδ T cell functions in multiple mouse models, such as Klebsiella pneumoniae [60] and cryptococcal pneumonia [61], the role of different γδ T cell subsets in lung inflammation remains underexplored. Recent findings highlight that Vγ4+ T cells are the main producers of IL‐17 in both infectious and non‐infectious conditions. Their ability to produce IFN‐γ is significantly reduced during the later stages of Plasmodium berghei blood‐stage infection [62]. In the infectious models of Listeria Monocytogenes [41, 63], Escherichia coli [64], Bacillus subtilis [65], and Vγ4+ T cells quickly secreted large amounts of IL‐17, combined with IL‐23 produced by DC during infection. Splenic Vγ1+ γδ T cells preferentially expand and express CD40 L; additionally, both Vγ1+ and Vγ4+ γδ T cells secrete pro‐inflammatory factors during infections. While CD40 L expression on Vγ1+ γδ T cells remains stable throughout infection, their IFN‐γ positivity decreases in late‐phase infections due to dysfunction within the γδ T‐cell population [62]. Notably, mouse γδ T‐cells lack a Vγ9 chain and therefore cannot be activated by PhAgs [66]. Furthermore, there are significant differences in tissue distribution between murine and human γδ T cells [43, 67]. For example, Vγ5+ dendritic epidermal T cells are the most abundant subpopulation in the mouse epidermis, whereas the human epidermis contains both αβ and γδ T cells, with Vδ1 T cells present in both epidermal and dermal layers [67, 68]. Despite differences in their composition and distribution, murine γδ T cells exhibit functional similarities to their human counterparts. For instance, CD1d serves as a ligand for Vδ1 T cells in both species [68]. Furthermore, both human and murine epidermal γδ T cells contribute to wound healing by producing keratinocyte growth factor (KGF) and insulin‐like growth factor 1 (IGF‐1) [69]. As such, studies using mouse models can provide valuable insights into the biology and function of human γδ T cells.

6. γδ T Cells' Protective Control Role During WNV Infection

γδ T cells exhibit potent antiviral activity through both direct and indirect mechanisms. Directly, they release granzyme B and perforin to kill infected cells or through death receptor pathways (Fas/FasL and TRAIL) in in vitro experimental model [19, 34, 70]. γδ T cells are also able to recognise virally infected cells via stress‐induced molecules (e.g., NKG2D) [49, 50] or metabolic pathogen‐associated markers like isopentenyl pyrophosphate (IPP) [19].

Indirectly, PhAg‐activated Vδ2 T cells produced a wide amount of soluble antiviral/immunoregulatory factors [35] able to interfere with viral replication in vitro [36, 37, 71, 72, 73, 74]; in fact, they produce pro‐inflammatory cytokines, such as IFN‐γ and TNF‐α, which contribute to the elimination of infected cells [35, 38, 70, 71, 72, 73, 74, 75]. In addition, the release also β‐chemokines (e.g., RANTES, MIP‐1α, MIP‐1β) for blocking viral entry, for example against viruses like HIV, by occupying CCR5 co‐receptors [76]. γδ T cells protective antiviral activity was observed also against Flavivirus [70, 71, 72, 73, 74, 75], and specifically against WNV [77, 78, 79] (Figure 2).

FIGURE 2.

γδ T cell cytolytic and non‐cytolytic antiviral activity. In humans and mice, γδ T cells can mount robust antiviral responses through both direct and indirect mechanisms. Specifically, they can release perforin and granzymes to kill WNV‐infected cells directly or, after encountering infected cells via γδ receptors, ‘sense’ their modifications and produce potent soluble antiviral factors (IFN‐γ and TNF‐α), eliminating the infected cells.

In murine models, it was observed that γδ T cell‐deficient mice exhibited elevated WNV viraemia, and more severe encephalitis, and were significantly more susceptible to WNV infection than wild‐type controls [80]. One study showed that γδ T cells can expand rapidly as early as 2 days after WNV infection and are a major source of IFN‐γ, along with NK cells and CD8 T cells [81]. Another group demonstrated that mice depleted of Vγ1+ T cells displayed a phenotype similar to that observed in TCR−/− mice in response to WNV infection, highlighting that Vγ1+ T cells are a significant γδ subset producing pro‐inflammatory factors [82]. One study demonstrated that irradiated mice reconstituted with IFN‐γ‐deficient γδ T cells had significantly higher WNV viral loads in the blood and brain during the infection than those reconstituted with IFN‐γ‐producing γδ T cells [79].

In humans, nitrogen‐containing bisphosphonates (N‐BPs), such as ZA, indirectly stimulate Vγ9Vδ2 T cells by inhibiting farnesyl diphosphate synthase, a key enzyme in the mevalonate pathway, thereby causing intracellular accumulation of IPP [83] (Figure 3) for Vδ2‐activation. ZA is currently the most potent third‐generation bisphosphonate, is safe and widely used to treat osteoporosis, hypercalcaemia, and bone metastases in patients with breast and prostate cancer [84]. Once activated with N‐BPs, Vδ2 cells are potent cytotoxic effector cells [85] against malignancies [86, 87] and HIV‐infected cells [88, 89, 90, 91].

FIGURE 3.

Mevalonate pathway. The Mevalonic Acid Cycle is critical for protein prenylation and cholesterol biosynthesis in eukaryotic organisms. Blocking this pathway by N‐BPs interferes with both prenylation and cholesterol synthesis. However, the ‘post‐IPP’ N‐BP block increases the accumulation of IPP, a potent endogenous stimulator of Vγ9Vδ2 T lymphocytes.

Our group demonstrated the strong anti‐WNV activity of the soluble factors released by Zoledronic Acid (ZA)‐activated human Vδ2 T cell lines [78]. We assessed the cytotoxicity of the Vδ2 T cell lines against WNV‐infected cells in an in vitro model of WNV replication. The ZA activation of Vδ2 T cell lines inhibited WNV replication by inducing IFN‐γ release, which is involved in the specific anti‐WNV activity. Additionally, the Vδ2 T cell lines directly killed WNV‐infected cells, possibly via perforin‐mediated cytotoxicity, in a dose‐ and time‐dependent manner, providing insight into the effector functions of human Vδ2 T cells against WNV infection.

Another key mechanism of γδ T cells' viral control is their ability to be ‘cytotoxic cells’ [92, 93, 94, 95, 96, 97]. γδ T cells perform their cytolytic activity by releasing perforin and granzymes after cell‐cell contact with the infected or tumour cells [97, 98] in humans and mice. A similar cytotoxic capacity of Vδ2 T cells was also reported against influenza‐ and SARS‐CoV‐infected cells [94, 95]. The cytotoxicity of Vδ2 T cells can be induced by several pathways, involving T cell receptor (TCR) and/or natural killer (NK) receptors, and can be mediated by the secretion of various soluble factors, such as perforin [98, 99]. Mice depleted of γδ T cells and infected with WNV exhibited lower levels of perforin in splenocytes after 6 days post‐infection, contributing to viral control [81], suggesting a possible involvement of perforin in the Vδ2‐mediated killing of WNV‐infected cells, as previously shown for CD8+ T‐cells [99]. In contrast, depleting γδ T cells from mice primarily infected with WNV before secondary infections does not appear to influence host susceptibility [100]. This suggests that γδ T cells may not play a direct role in the development of memory responses to WNV or substantially impact antibody responses during primary or secondary infections. The involvement of γδ T‐cell responses has been documented in human and primate studies of bacterial infections [101, 102]. In addition, a reduction of CD4+ and CD8+ memory T cell responses was observed in TCRδ−/− mice [103]. Furthermore, γδ T cells may also regulate the responses of CD4+ and CD8+ T cells during WNV infection, as observed in TCR−/− mice [104], suggesting a role in regulating the memory T cell response during WNV infection. Several studies have shown that γδ T cells are required for optimal priming and/or expansion of WNV‐specific CD8+ T cells, thereby influencing their cytotoxic efficiency and CNS viral clearance in TCR‐/‐ mice [103, 105, 106]. Moreover, γδ T cells upregulate perforin, granzymes, and death ligands and can directly lyse WNV‐infected cells in the spleen and other peripheral sites [106]. This function is particularly important before full CD8 T cell expansion, bridging the early innate and delayed adaptive phases [106].

B cells are critical for surviving during WNV infection. They prevent the virus from spreading throughout the body and into the CNS by producing neutralising antibodies. Individuals with impaired B‐cell function face a significantly higher risk of severe neurological complications. Also, B lymphocytes were influenced by γδ T cells during WNV infection, promoting higher humoral and neutralising antibody responses [105].

In the complex interplay between γδ T cells and other innate cells, DCs are among the most important antigen‐presenting cells (APC), as they possess the unique capacity to initiate primary T cell responses. Crosstalk between T cells and DCs plays a crucial role in promoting DC maturation and T‐cell priming during WNV infection and antigen presentation capability via cytokines (IFN‐γ, TNF‐α) and costimulatory interactions, thereby shaping the strength and quality of adaptive responses [103, 107, 108]. In WNV‐infected mice, splenic DCs exhibited impaired presenting cell activity and lower levels of CD40, CD80, and CD86 markers compared to wild‐type mice [108]. WNV‐infected γδ T cells contribute pro‐inflammatory cytokines like IFN‐γ, TNF‐α, and IL‐6, which aid DC maturation processes. Evidence suggests significant upregulation of co‐stimulatory molecules along with MHC class II expression on DCs co‐cultured with infected versus uninfected γδ T cells, indicating that factors secreted by these infected subsets are crucial for promoting both DC maturation and subsequent CD4+ priming [109].

Additionally, in human models, γδ T cells and WNV‐permissive DCs may exert regulatory influences on each other by inducing a human γδ T cell response to poly I:C, a TLR3 ligand, in the presence of DC‐mediated Type‐1 IFNs [110].

Regarding the innate immune response against WNV, γδ and NK cells are known to synergise to suppress WNV viraemia. They both respond rapidly and non‐specifically to WNV infection and control viral dissemination by secretion of IFN‐γ and/or cytolytic activity, emphasising timing, compartmentalisation, and subset balance in mice and humans [106]. The role of NK cells during in vivo WNV infection is complex, and their functional significance should be investigated in an organ‐specific manner, with full characterisation of the multiple activating and inhibitory receptors on their cell surface [106].

The mechanisms by which γδ T cells are activated during WNV infection and produce proinflammatory cytokines remain poorly understood. The mechanisms underlying γδ T cell activation during WNV exposure remain somewhat unclear [110]; however, it has been proposed that such infections might trigger the Toll‐like receptors (TLRs) signalling or engage other innate immune receptors involved in recognising pathogens, such as RIG‐I or MDA5 [108, 109]. In fact, TLR signalling after activation can lead to type I IFN production, which is critical for WNV control and viral inhibition through the interferon‐stimulated genes (ISG) [111, 112]. Several ISG products and their receptors, including IRF1, HPSE, RIG‐I, MDA5, and IFITM3, exhibit direct antiviral activity against WNV, underscoring the complexity and breadth of IFN‐mediated antiviral defences [113]. This IFN I/II promotes antiviral gene expression and augments the effects of type I IFN in limiting early viral replication [103, 106].

All this evidence clearly demonstrated the ability of γδ T cells to communicate with the adaptive (αβ T cells) and innate (DCs) counterparts of the immune system through direct contact and/or the release of soluble factors, therebyrestraining WNV viraemia and optimising downstream innate and adaptive immunity. Moreover, all these studies may highlight the concept of γδ T cells as a potential immunotherapeutic target for infectious diseases as well as knowledge for HIV and taking advantage of the studies on cancer treatment [114, 115, 116].

In Table 1, we reported the main γδ T cell subsets associated with WNV infection.

TABLE 1.

γδ specific cell subsets associated to WNV infection.

γδ T cells	Role	Functionality	WNV infection phase	Ref
Murine
Vγ1+	Protective	IFN‐γ production	Antiviral activity in vivo	[47, 80, 82, 92, 117, 118, 119, 120, 121, 122, 123]
Vγ4+	Pathogenetic	TGF‐β, IL‐10, and TNF‐α production	Dissemination, BBB damage, and immune response suppression	[92, 117, 121, 122, 124, 125, 126, 127, 128, 129]
Human
Vγ9Vδ2	Protective	IFN‐γ and SF production; perforin release	Antiviral activity in vitro	[79, 82, 93, 94, 95, 96, 97, 98, 99, 100, 101, 107, 110]

Abbreviations: BBB, Blood‐Brain Barrier; SF, Soluble Factors.

7. γδ T Cells' Role in WNV Pathogenesis

Among all γδ T cell clones in mice, circulating Vγ4+ T cells are involved in WNV pathogenesis, although the underlying mechanisms remain unclear. Data from studies on old mice showed that they were more susceptible to WNV infection than young adult mice [82, 130]. In one study, aged mice showed a higher content of V4+ cells than young adult mice [77], and, when V4+ cells were depleted in young mice, reduced mortality was observed following WNV‐induced encephalitis [82]. A hypothetical mechanism suggests that V4+ T cells contribute to WNV pathogenesis via the production of proinflammatory cytokines [82, 124]. TNF‐α is known to compromise the Blood‐Brain barrier (BBB) integrity, facilitating WNV's entry into the central nervous system (CNS) [125]. In mice depleted of Vγ4+ T cells, reduced CNS TNF‐α levels were observed, along with decreased viral load in the brain and lower mortality from WNV encephalitis [82]. Furthermore, Vγ4+ T cells also produce IL‐17, which recruits neutrophils and macrophages to sites of infection across several disease models [125, 126]. However, blocking IL‐17 in vivo did not change host susceptibility to WNV infection [124]. Interestingly, Vγ4+ T cells also negatively influence splenic Vγ1+ T cell responses during WNV infection by producing transforming growth factor‐beta (TGF‐β). This leads to increased viraemia, facilitates WNV spread into the CNS, and induces encephalitis and heightened brain inflammation [82, 124]. In WNV‐infected mice depleted of Vγ4+ T cells, a higher proliferation capability of the other γδ T cell subpopulations were observed than that of WNV‐infected controls after an ex vivo anti‐CD3 stimulation, and this difference was diminished in the presence of a TGF‐β inhibitor. Additionally, Vγ4+ T cells from WNV‐infected mice contributed thought direct and indirect pathways to the elevated IL‐10 levels, which is known to play a pathogenic role in WNV‐host immunity [127, 128]. In this scenario, Vγ1+ T cells can suppress IL‐10‐producing CD4+CD25+ T cells in the lungs of WNV‐sensitised and challenged mice [129], thereby exerting a protective role during bacterial and coxsackievirus infections [47]. Moreover, Vγ1+ T cells promote the control of WNV viraemia in aged mice treated with immunotherapy [117, 118]. A study showed that the γδ T cells of MyD88 (Myeloid differentiation factor 88) ‐deficient mice [119] and TLR‐deficient mice exhibited reduced expansion and activation capabilities compared to wild‐type mice after WNV infection, indicating a probable role for MyD88‐dependent PRR pathways in γδ T cell activation [120]. Additionally, PRRs, TLR3, and TLR7 were expressed in γδ T cells, and their innate responses were involved in both protection and pathogenesis mechanisms during WNV infection [104, 121]. TLRs are known to directly or indirectly activate γδ T cells, and TLR ligands could act as co‐stimulatory signals for TCR‐activated human γδ T cells [104, 119, 120, 121, 122]. MyD88 also showed a strong effect on WNV, inhibiting its cellular replication and modulating immune cell migration into the CNS [123]. Whether WNV directly induces T cells via their innate immune receptors, such as TLRs, or indirectly through interactions with TLR‐expressing innate immune cells needs to be investigated. We reported the γδ T cell pathogenetic/protective role across the studies in Table 2.

TABLE 2.

The γδ T cells' pathogenetic/protective role across recent studies.

Aspect	Key finding	Evidence base	Refs
Global requirement of γδ T cells for protection	γδ T cell–deficient mice exhibit higher viraemia and mortality, with decreased IFN‐γ production and impaired viral control.	γδ TCR‐/‐ mice infected with WNV; survival, viraemia, cytokines measured	[103, 105, 106]
Early IFN‐γ‐production (protection mechanism)	γδ T cells are major early IFN‐γ producer and the spleen and peripheral tissues before robust CD8+ T responses; IFN‐γ neutralisation phenocopies increased susceptibility.	Kinetics of IFN‐γ+ γδ versus other populations; IFN‐γ blockade; reconstitution experiments.	[103, 105, 106]
Promotion of CD8 T cell response	γδ T cells enhance the magnitude and functionality of WNV‐specific CD8+ T cells, improving CTL responses and viral clearance, especially in the CNS.	γδ TCR–/– versus WT: CD8+ T cell numbers, tetramer + cells, in vivo killing, brain viral loads.	[98, 103, 106]
Promotion of B‐cell humoral response/neutralisation antibodies	γδ T cells support higher neutralising antibody titres and more effective humoral immunity, correlating with improved survival.	Comparison of Ab titres, isotypes, and protection in TCR–/– versus WT; serum transfer experiments.	[98, 103]
Vγ1 T cell protective role	Vγ1+ T cells expand during infection, produce IFN‐γ, and are associated with lower viraemia and better survival; depletion of Vγ1+ cells worsens outcomes.	Subset‐specific depletion or deficiency; survival and viral load analysis.	[80, 103, 106]
Vγ4 T cell pathogenetic role	Vγ4+ cells are associated with higher viraemia and more severe encephalitis; their depletion reduces inflammation and improves outcomes.	Vγ4+specific depletion; analysis of viraemia, brain inflammation, and survival.	[80, 103, 106, 130]
IL‐17 γδ T cell‐producing	Vγ4+ cells produce IL‐17 during WNV infection and contribute to the IL‐17 milieu, but IL‐17 blockade alone does not fully reverse susceptibility.	IL 17 expression in V γ4+ cells; anti IL‐17R or IL‐17 blocking experiments	[80, 103, 106, 130]
TGF‐β production and regulation of other subsets	Vγ4+ cells produce TGF‐β, which suppresses the proliferation and the expansion of protective Vγ1+ cells; TGF‐β inhibition mitigates this suppression in vitro.	In vitro proliferation assays with/without TGF‐β inhibitor; cytokine profiling of Vγ4+ cells.	[80, 103, 106, 130]
CNS immunopathology	CNS‐infiltrating γδ T cells (enriched for Vγ4+) contribute to neuroinflammation via IL‐17 proinflammatory cytokines, and potentially cytotoxic mechanisms; Vγ4+ depletion reduces CNS inflammation.	Brain infiltrate phenotyping, cytokine expression, histopathology, and clinical scores in Vγ4‐depleted versus control mice.	[80, 103, 106, 130]
Balance between subsets	Disease outcome reflects balance between protective IFN‐γ+Vγ1+ T cells versus pathogenic Vγ4+/IL‐ 17+/TGF‐β	Comparative subset kinetics; depletion experiments; correlations with viraemia and CNS disease; altering this balance modulates susceptibility.	[70, 80, 103, 106, 130]

8. Controversies and Unresolved Questions

In this context, it is natural to consider unresolved questions regarding the clear role of human γδ T cells in WNV infection. First, we have to consider the human γδ T cell subset (Vδ1 or Vδ2 circulating/in tissues) involved in WNV infection that mirrors the murine γδ T cell response, to better understand their role in humans using the murine experimental model. This prospective concept is still to be defined. Some questions are natural to be done: Is there a phenotypic association between the γδ T cell profile and the WNV viral load kinetics in the blood and in CSF? Is there a risk of neuroinvasion and the presence of long‐term sequelae in WNV patients? Answering these questions requires prospective, well‐phenotyped human cohorts with high‐parameter flow cytometry or single‐cell tools.

Second, the analysis of the WNV‐γδ T cell response in aged and young subjects is warranted but currently lacking. In this context, the questions should address how the ageing process could diminish the IFN‐γ‐protective γδ T cell response in WNV infection, or, indeed, how it could bypass this protective response via a more pathogenic γδ‐IL‐17/TGF‐β axis.

Third, we must consider the unresolved question of how γδ T cells recognise antigens and are activated during WNV infection. Are the γδ T cell responses generated from PhAg accumulated in the WNV‐infected cells, or do they respond to stress or metabolic molecules released or accumulated during the WNV infection? Clarifying these points may be functional for targeted therapies and for understanding the immune mechanisms involved in the pathogenesis/protection during WNV.

The fourth aspect involved the communication between γδ T cells and other innate and adaptive immune cells. In general, the literature includes studies that analyse γδ and NK cells' WNV responses separately, while investigations into the relationships among γδ T cells, MAIT, NK, NKT, and B cells are lacking. These types of studies, with a systematic analysis of all these variables, might highlight the complex network of WNV immune control and the development of the pathology.

The last aspect we have to evaluate is the types of studies conducted during the WNV epidemics. Most of these studies rarely measured the outcomes of the WNV pathology. Standardised cellular panels, with particular attention to phenotypic and functional profiles, are needed for future vaccine trials, therapeutic studies involving IFN and cytokine modulators, and observational studies of WNV cohort patients.

9. Future Directions and Conclusions

In conclusion, γδ T cells play a distinct and multifaceted role in both protective immunity and viral pathogenesis during WNV infection in mice and humans. γδ T cells, mainly Vγ1+ T cells, are involved in the immediate control of WNV dissemination in mice, partially due to their IFN‐γ‐producing activity. In contrast, Vγ4+ T cells play a pathogenic role in the mouse model by producing both pro‐inflammatory and regulatory cytokines during WNV infection, promoting WNV viraemia and/or more inflammatory responses in the brain. Moreover, γδ T cells may recognise WNV‐infected cells by producing pro‐inflammatory cytokines directly via PRRs or indirectly via other immune cells that can kill infected cells, thereby clearing the virus. The activation of γδ T cells by WNV enhances the maturation and activation of DCs, which ultimately primes T cell responses against the virus. Despite differences between human and murine γδ T cell subsets and their ligand recognition mechanisms, they exhibit significant similarities in effector functions and protective roles against pathogen infections. Data on human Vγ9Vδ2 T cells also contribute to protective immunity by enhancing cytolytic and non‐cytolytic activity against WNV‐infected target cells, thereby displaying potent antiviral activity. However, the specific role of γδ T cells in human WNV infections remains unclear. Further analyses are required to elucidate other anti‐WNV molecules among activated Vδ2‐producing soluble antiviral factors and to identify the molecular mechanisms underlying Vδ2‐mediated killing of WNV‐infected cells. Altogether, this data provides insight into the effector functions of human Vδ2 T‐cells against WNV. Additionally, targeting these cells with ZA, a commercially available drug used in humans, could offer a new prospective immunotherapeutic strategy for WNV infection. Investigating similar activities carried out by murine γδ T cells will deepen our understanding of immune surveillance and regulation relevant to studies of WNV disease in humans. The current understanding of the biological significance of γδ T cell receptors during pathogen infection remains ambiguous. Future research will also aim to clarify how γδ T cells are activated or exhausted during WNV infections in humans as part of host defence strategies.

Author Contributions

E.C. conceived and wrote the first draft of the manuscript. E.T., F.C., and S.G. drew the figures and revised the manuscript. F.M. revised the manuscript and provided a critical revision. All authors read and approved the manuscript and agreed to its submission for publication.

Funding

This work was supported by grants from the Italian Ministry of Health (Ricerca Corrente, Linea 1).

Ethics Statement

The authors have nothing to report.

Consent

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Picture Licence

All the pictures in the manuscript were obtained under BioRender and modified accordingly.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.