# Exacerbation of Anti-Cytomegalovirus Immunity and Mobilization of γ9δ1 T Cells During the Acute Phase of Hepatitis E Virus Infection

Marion Retailleau, Fanny Martini, Florence Abravanel, Nassim Kamar, Jacques Izopet, Eric Champagne

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## To cite this version:

Marion Retailleau, Fanny Martini, Florence Abravanel, Nassim Kamar, Jacques Izopet, et al.. Exacerbation of Anti-Cytomegalovirus Immunity and Mobilization of γ9δ1 T Cells During the Acute Phase of Hepatitis E Virus Infection. Microbiology and Immunology, 2025, 70 (2), pp.113 - 124. 10.1111/1348-0421.70034. hal-05528572

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ORIGINAL ARTICLE

OPEN ACCESS

# Exacerbation of Anti-Cytomegalovirus Immunity and Mobilization of γ9δ1 T Cells During the Acute Phase of Hepatitis E Virus Infection

Marion Retailleau¹ | Fanny Martini¹ | Florence Abravanel¹,² ID | Nassim Kamar¹,³ ID | Jacques Izopet¹,² ID |
Eric Champagne¹ ID

¹Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM), UMR1291—CNRS UMR5051, Infinity, Université de Toulouse, Toulouse, France | ²CHU Toulouse, Hôpital Purpan, Laboratoire de virologie, National Reference Centre for Hepatitis E, Toulouse, France | ³CHU Toulouse, Hôpital Rangueil, Service de Néphrologie et Transplantation, Toulouse, France

**Correspondence:** Florence Abravanel (abravanel.f@chu-toulouse.fr)

**Received:** 25 February 2025 | **Revised:** 30 August 2025 | **Accepted:** 2 December 2025

**Funding:** ANRS, Grant/Award Number: ECTZ103203; Transhepate-Occitanie association, Grant/Award Number: ECTZ103206

**Keywords:** cytomegalovirus | gamma delta T cells | hepatitis E virus | innate immunity | organ transplantation

## ABSTRACT

HEV causes chronic infections that are detrimental to immunocompromised patients. Previous studies showed alterations of γδ T cell subsets at the acute phase of HEV infection. To assess a possible role of CMV, we have examined the frequencies and responses to CMV and HEV of blood γδ T cell subsets from control donors and acute-phase HEV patients with or without CMV. CMV DNA was mostly undetectable in the blood of CMV-seropositive HEV patients, and anti-CMV antibodies were only slightly elevated. However, $V_γV_δ^{neg}V_δ^1^{pos}$ cells were enriched in vivo, suggesting an increased CMV burden. In contrast, γ9δ1 cells were depleted in most HEV patients, regardless of CMV status. Culturing with IL-2 and IL-15 led to strong γ9δ1 T cell enrichment in samples from HEV patients. After IL-2/IL-15 sensitization, analysis of IFN-γ responses to CMV-infected fibroblasts or hepatocarcinoma cells (with IL-18) showed innate responsiveness to CMV in γ9δ1, γ9δ2, and $γ_9^{neg}δ_1^{pos}$ cells in some control subjects and CMV-seronegative patients. However, these responses were selectively exacerbated in CMV-seropositive HEV patients, who also showed significant $α_β$ T cell responses to CMV. This indicates reactivation of anti-CMV immunity. Responses to HEV-infected HepG2 cells remained undetected. IFN-γ responses were not associated with TCR downmodulation in γ9δ2 or $γ_9^{neg}δ_1^{pos}$ cells. However, IFN-γ-producing γ9δ1 from HEV patients were characterized by high TCR expression, which was downmodulated after stimulation with CMV-infected fibroblasts or CMV/HEV-coinfected HepG2 cells. We conclude that γ9δ1 T cells are selectively mobilized in HEV patients and can be activated by CMV.

## 1 | Introduction

Hepatitis E virus (HEV) is a single-stranded positive RNA virus and the most common agent of acute hepatitis worldwide. Four genotypes frequently infect humans. In industrialized countries, HEV3 and HEV4 infections are prevalent and typically result in

self-limiting hepatitis. However, in immunocompromised individuals, particularly in the context of organ transplantation, infections become chronic in about two-thirds of individuals and may progress to cirrhosis [1]. This progression can be prevented or treated with ribavirin therapy, though the virus

**Abbreviations:** ATCC, American Type Culture Collection; CMV, cytomegalovirus; EphA2, ephrin type A receptor 2; ffu, fluorescence focus forming unit; gc, genome copy; HEV, hepatitis E virus; LCM, lymphocyte culture medium; mfi, mean fluorescence intensity; PBMC, peripheral blood mononuclear cells; PFA, paraformaldehyde; TCR, T cell receptor.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

© 2025 The Author(s). *Microbiology and Immunology* published by The Societies and John Wiley & Sons Australia, Ltd.
persists in some patients despite treatment [2]. Better charac-
terizing the natural immune response after HEV infection is
important for understanding the pleomorphic evolution of the
disease and viral persistence.

Regarding HEV3 infections [3], we have focused our attention
on γδ T cells. This quantitatively minor population of T lym-
phocytes contributes to tissue homeostasis, anti-cancer
immunity, and responses to viral, bacterial, and parasitic
infections [4-7]. Subsets of γδ T cells are often defined by the
V-gene families that comprise their TCR. In humans, the
Vγ9posVδ2pos subset usually predominates in the blood of
healthy individuals. This subset is mobilized during bacterial
or parasitic infections due to its ability to detect cells pro-
ducing exogenous phosphoantigens. It may also be activated or
altered during viral infections through TCR-dependent detec-
tion of phosphoantigenic metabolites [6, 8, 9] or TCR-
independent mechanisms. Currently, it is accepted that this
subset displays innate reactivity, whereas other subsets col-
lectively forming a “non-Vγ9Vδ2” population have more
adaptive potential [10-12]. Non-Vγ9Vδ2 T cells predominate
in tissues [10] and are subject to in vivo oligoclonal amplifi-
cations in the course of acute CMV infections. In the context of
organ transplantation, their amplification correlates with
CMV clearance [13, 14]. Recently, an expansion of cells with a
Vγ9negVδ2pos phenotype was found to indicate severe CMV
disease in kidney transplant patients [15]. After infection,
latent CMV persists lifelong in cells of the myeloid lineage and
CD34+ precursors. It may reactivate in infectious or inflam-
matory contexts [16-20]. Carriage of CMV is responsible for
significant biases in the T cell repertoires in the elderly,
resulting in an accumulation of CMV-specific CD8+ T cells as
well as non-γ9δ2 T cells with terminally differentiated phe-
notype [14, 21-24]. The accumulation of effector memory
Vδ2neg T cells with age in healthy CMV carriers has been
shown to correlate with an increase in anti-CMV antibodies
and may thus reflect CMV burden [25].

Alterations in γδ T cell subsets have been observed in various
viral infections, including HIV, HBV, HCV, and dengue and
influenza viruses. However, the factors that determine γδ T
cell responses remain unclear. Coinfections with highly
prevalent and persistent viruses, such as Herpes family
viruses, are potential confounding factors, making viral tar-
gets for these responses elusive [26]. The current view is that
γδ T cells target stress-induced cellular determinants. Such
targets have been characterized in the case of anti-CMV
responses and include molecules related to and unrelated to
MHC [27]. However, responses appeared to be specific to
CMV, as the activated γδ subsets did not recognize fibroblasts
infected with herpes viruses other than CMV. In addition,
cross-reactivity against leukemic or intestinal cancer cells is
well documented [28].

In previous studies, we have observed alterations of γδ T cell
subsets during the acute phase of HEV infection in both
immunocompromised transplant patients and immuno-
competent individuals [29, 30]. Interestingly, in vivo amplifi-
cation of cells with a non-γ9δ2 phenotype was only observed in
CMV-seropositive (CMVpos) patients, despite the absence of
detectable CMV reactivation based on CMV genome detection
in serum or plasma. Although contact with HEV-infected cells

promoted IL-10 secretion by γδ cells, attempts to expand putative HEV-specific γδ T cells from patients have failed [30]. Therefore, it was unclear whether the γδ T cell amplifications were truly HEV-specific. This study aimed to reexamine γδ T cell subsets in HEV patients and assess their responses to HEV and CMV in vitro compared to healthy individuals.

2 | Materials and Methods

## 2.1 | Cell Samples From HEV Patients and Healthy Control Donors

PBMC and serum samples collected from HEV3-infected
patients during the first 2 weeks of clinical or biological
symptoms were obtained from the DC-2015-2450 collection,
which is hosted by the Biological Resources Center of the
Toulouse University Hospital. PBMC and plasma from healthy
adults were provided by the French National Blood Service and
processed and stored by the local Immunomonitoring platform
registered with the Ministry of Higher Education and Research
(DC-2016-2772). Patients' consent or non-opposition to the use
of samples for HEV research was registered according to
French ethical guidelines. The study was approved by the
French South-West & Overseas Ethics Committee. Experiments
were performed in accordance with the Declaration of
Helsinki guidelines. Patients' characteristics are provided in
the Supporting Information.

Serological (anti-HEV IgM and IgG) tests were performed on
blood sera or plasma using Wantai enzyme immunoassays
(Wantai Biological Pharmacy Enterprise, Beijing, China) fol-
lowing the manufacturer's recommendations. Acute HEV
infection was diagnosed in patients by positivity for anti-HEV
IgM, elevated ALT, and/or the presence of the HEV RNA in the
blood by quantitative RT-PCR (RealStar HEV RNA kit 2.0,
Altona Diagnostics). Anti-cytomegalovirus viral lysate anti-
bodies were titrated using the CMV IgG Abbott diagnostic test
(Abbott Laboratories). CMV viremia was determined by PCR as
previously reported, with a sensitivity of 300 IU/mL [31].

## 2.2 | Cells Lines and Viral Isolates

The MRC-5 fibroblasts and the HepG2/C3A hepatocarcinoma
cells were obtained from the American Type Culture Collection
(ATCC) and cultured in DMEM supplemented with 10% FCS,
penicillin, and streptomycin (culture medium).

The CMV strain AD169 (CCL-171) was also obtained from
ATCC. The virus was amplified on MRC-5 fibroblasts and col-
lected from supernatants by ultracentrifugation at 70,000 g for
40 min. The virus was then resuspended in culture medium and
frozen in aliquots. The CMV titer in the viral stock and the
supernatant from infected cultures was determined by infection
of MRC-5 monolayers with serial dilutions. Then, the fibroblast
layers were immunostained with anti-CMV-IE Ab on Day 4,
and the fluorescence focus forming unit (ffu) counts were
determined by immunofluorescence microscopy.

HEV infections were performed using the clinical isolate
HEV-3f (TLS09-0) [32]. The virus was propagated in a
MAVS-knockdown HepG2 cells (a gift from Z. Feng, Ohio State
University) [33] and purified from pooled cell lysates and
supernatants by ultracentrifugation for 1 h at 70,000 g. The virus was then resuspended in PBS and kept frozen in aliquots at -80°C. Concentrations of HEV RNA in viral stocks were determined using a one-step RT-qPCR (Taqman) targeting the overlapping region of ORF2 and ORF3, as previously described [34]. A viral stock titrating 2 × 10¹² genome copies (gc) per mL was used for experiments. To quantify HEV RNA in infected HepG2 cells, the cell layers were washed with PBS and collected in RNA lysis buffer by scraping, and the RNA was extracted using the Quick-RNA miniprep kit (Zymo Research). Reverse transcription was carried out on 1 µg of RNA using a Sensifast cDNA synthesis kit (Meridian Bioscience). PCR amplification was performed in duplicate using 1/20 of the first-strand reaction, Cybergreen mix (Roche Diagnostics), and ORF2/3 region-specific primers (0.5 µM each). The LightCycler 480 and software from Roche Diagnostics were used for fluorescence detection of PCR products and threshold cycle (Ct) determination. GAPDH cDNA amplification was used as a reference to calculate the ΔCt (Ct gene - Ct GAPDH). Results from duplicate amplifications were averaged. Primers sequences are listed in the Supporting Information.

## 2.3 | Lymphocyte Cultures

After thawing, the PBMC samples were cultivated at a concentration of 5 × 10⁶ cells/mL, for a 7-day primary culture in lymphocyte culture medium (LCM: RPMI-GlutaMAX [LifeTechnologies] supplemented with 10% FCS, penicillin, streptomycin, and sodium pyruvate) in the presence of IL-2 (50 ng/mL) and IL-15 (20 ng/mL), with half of the medium changed on Day 4.

For co-cultures with cytokine-sensitized PBMC, MRC5 and HepG2 cells were plated in 24-well plates 3 days before infection (2.5 × 10⁴ and 10⁵ cells/cm², respectively) to reach ~80% confluence on the day of infection. The cells were then infected with CMV at a multiplicity of infection of 0.3 ffu/cell, HEV (200 gc/cell), or both viruses. After 24 h, the infected and non-infected adherent cells were washed twice with DMEM. To assess IFN-γ production and TCR modulation, the sensitized PBMC were washed and incubated for 4 h in LCM containing IL-2 (20 ng/mL) and IL-18 (10 ng/mL). Then the PBMC were added to MRC-5/HepG2 cells (1.5 × 10⁶ cells/well) in a final volume of 1.5 mL, along with IL-2 (20 ng/mL) and IL-18 (10 ng/mL). Parallel control wells were coated with anti-CD3 (MEM57 clone from ExBio Praha, 16 h, 5 µg/mL in PBS) and washed with RPMI for control TCR stimulation. After a 16-h co-culture with no cytokine secretion blocker, non-adherent cells were recovered from all wells and stained as described below.

IL-2, IL-15, and IL-18 were premium-grade cytokines purchased from Miltenyi Biotech.

## 2.4 | Flow Cytometry Analysis

For the analysis of lymphocyte subsets, cells from PBMC samples or cultured cells were washed once in PBS and stained with Live Dead Yellow (Thermo Fisher Scientific, 20 min, 0.5 µM) to identify dead cells. Then, the cells were washed with PBS and suspended in PBS plus 10% human serum for 15 min to saturate the Fc receptors. Surface staining was performed in staining

buffer (PBS, 5% FCS, and 0.02% NaN₃). Anti-pan-γδ was added first on pelleted cells for 20 min, followed by the addition of a mix of anti-CD3, anti-Vδ2, anti-Vδ1, and anti-Vγ9 for 40 min. After washing, the cells were fixed with PBS plus 2% paraformaldehyde (PFA) for 15 min at 4°C. When intracellular staining was performed, a fixation and permeabilization kit for cytokine detection (Invitrogen) was used according to the manufacturer's recommendations. Anti-IFN-γ was then added for 45 min in permeabilization buffer. After washing, cells were fixed again for 10 min in 2% PFA. Fluorescence was acquired using a Fortessa X20 cytofluorometer (Becton Dickinson). Antibody details are provided in the Supporting Information.

## 2.5 | Data Treatment and Statistical Analysis

To analyze TCR modulation on T cells after lymphocyte culture, we collected the mean fluorescence intensities (mfi) of the pan-γδ-TCR staining on IFN-γ<sup>Low</sup> and IFN-γ<sup>Hi</sup> cells for each subset. The TCR mfi on IFN-γ<sup>Low</sup> cells in noninfected cultures was averaged for each patient/control group, and the resulting value was used for normalization of the corresponding dataset.

Nonparametric statistics were used in Section 3 unless specified in the figure legends. Statistics were calculated using the GraphPad Prism software.

# 3 | Results

## 3.1 | Sample Individuals and Experimental Design

Twelve patients in the acute phase of HEV infection, including six organ-transplant patients, were characterized for their CMV seropositivity and CMV-DNAemia. Among the six CMV-seropositive (CMV<sup>pos</sup>) patients, one had detectable CMV-DNA in the serum. A control panel of 16 healthy donors, characterized for their HEV and CMV seropositivity, was constituted, including an equal number of HEV-seropositive individuals and HEV-seronegative donors (Table 1 and Supporting Information). CMV<sup>pos</sup> individuals were slightly older (p = 0.10, Mann-Whitney). The experimental strategy is depicted in Figure 1 and explained further in this section.

## 3.2 | Quantification of γδ T Cell Subsets Ex Vivo

PBMC from control and patient samples were phenotyped to quantify γδ T cell subsets ex vivo by flow cytometry. Fluorescence co-staining of lymphocytes for CD3, γδ-TCR, Vδ1, Vδ2, and Vγ9 can identify six γδ T cell subsets: γ9δ1, γ9δ2, γ9δX (Vγ9<sup>pos</sup>Vδ1<sup>neg</sup>Vδ2<sup>neg</sup>), γXδ1 (Vγ9<sup>neg</sup>Vδ1<sup>pos</sup>Vδ2<sup>neg</sup>), γXδ2 (Vγ9<sup>neg</sup>Vδ1<sup>neg</sup>Vδ2<sup>pos</sup>), and γXδX (Vγ9<sup>neg</sup>Vδ1<sup>neg</sup>Vδ2<sup>neg</sup>). However, γ9δX, γXδ2, and γXδX cells were rare or undetectable in some conditions or individuals. Gating strategies for flow cytometry analysis and representative histograms are shown in the Supporting Information.

Ex vivo, γ9δ2 cells dominated non-Vγ9Vδ2 γδ T cells similarly in most CMV<sup>pos</sup> and CMV<sup>neg</sup> control individuals. This was also true for CMV<sup>neg</sup> HEV patients. In contrast, CMV<sup>pos</sup> HEV patients had increased percentages of γXδ1 T cells in their γδ T cells. γXδ1 T cells increased proportionally among CD3+ cells,
<table><caption>TABLE 1 | Control and patient groups.</caption><thead><tr><th></th><th>Cont-CMV<sup>neg</sup></th><th>Cont-CMV<sup>pos</sup></th><th>HEV-CMV<sup>neg</sup></th><th>HEV-CMV<sup>pos</sup></th></tr></thead><tbody><tr><td>N (SOT)<sup>a</sup></td><td>8 (0)</td><td>8 (0)</td><td>5 (2)</td><td>7 (4)</td></tr><tr><td>Gender M/F</td><td>5/3</td><td>3/5</td><td>3/2</td><td>4/3</td></tr><tr><td>Age (range)<sup>b</sup></td><td>40 (26–63)</td><td>49.9 (30–67)</td><td>43.4 (22–77)</td><td>53 (24–73)</td></tr><tr><td>HEV-IgG pos/neg</td><td>4/4</td><td>4/4</td><td>5/0</td><td>7/0</td></tr><tr><td>CMV IgG titer (range)<sup>c</sup></td><td>Neg. (0–0.8)</td><td>306 (28–1114)</td><td>Neg. (0–0.5)</td><td>1316 (120–5890)</td></tr></tbody></table>

<figure><img src="image_4.png" /><figcaption>FIGURE 1 | Experimental strategy. PBMC samples were examined by flow cytometry after thawing (ex vivo) and after 7 days of amplification in IL-2+IL-15. Viable cells were then transferred to medium containing IL-2+IL-18 and seeded on wells with MRC5 or HepG2 cell layers, previously infected or not. Flow cytometry was then used to monitor intracellular IFN-γ accumulation in lymphocyte subsets and the level of TCR expression.</figcaption></figure>

suggesting a moderate expansion of this subset in peripheral blood. Thus, HEV infection in CMV carriers is associated with alterations described in open CMV infections or CMV reactivation [14, 35–37]. Notably, γ9δ1 cells were consistently rare in CMV<sup>pos</sup> and CMV<sup>neg</sup> HEV individuals ex vivo, whereas they represented a significant subpopulation of the γδ T cells in controls (Figure 2).

### 3.3 | Anti-CMV IgG Titers in Seropositive Individuals

Although CMV DNA was detected in one CMV<sup>pos</sup> HEV patient, the observation of γδ T cell subset modifications, such as the gXd1 bias, in most CMV<sup>pos</sup> patients (Figure 2) could reflect occult CMV reactivation. We hypothesized that this might also result in an elevation of anti-CMV antibodies. Thus, anti-CMV IgG titers were analyzed in CMV<sup>pos</sup> individuals (Figure 3). Anti-CMV IgG titers in serum were slightly higher in CMV<sup>pos</sup> HEV patients than in CMV<sup>pos</sup> controls. Serum samples from before the acute phase were available for five HEV patients who had undergone solid organ transplantation. No consistent elevation of anti-CMV IgG was observed in these patients. Additionally, anti-CMV IgG levels did not significantly correlate with the frequency of γXδ1 cells in either controls or patients. Thus, the

CMV serological data suggest an increased CMV burden in HEV patients, but do not support systemic CMV replication.

### 3.4 | Quantification of γδ T Cell Subsets After Expansion With Cytokines

Due to the limitation in the amount of biological material available for evaluating antiviral reactivity, we aimed to amplify γδ T cells in vitro with cytokines without additional antigenic stimulation to favor amplification of T cells that are sensitized in vivo. The combination of IL-2 + IL-15 is reported to promote amplification of activated γδ T cells while preserving viability [15, 38–40]. Thus, PBMC from patients and controls were cultivated for 7 days in a medium supplemented with IL-2 and IL-15. T cell subsets quantification was assessed again by flow cytometry to evaluate the effect of culture on the relative proportions of γδ subsets (Figure 4). The total number of viable cells recovered increased slightly compared to the initial number (by a factor of 1.2–1.5; not shown). Nevertheless, the culture led to an enrichment of CD3+ cells. No significant enrichment of γδ over αβ cells occurred in patients or controls. Among the γδ T cells, the proportions of the γ9δ2, γ9δ1, and γXδ1 subsets were not consistently altered by the culture with cytokines in the controls. In contrast, in CMV<sup>neg</sup> and CMV<sup>pos</sup> HEV patients, the relative proportion of γ9δ2 cells was decreased, while a substantial population of γ9δ1 cells emerged (Figure 4B). This increase was more pronounced in cultures from transplanted patients, though the patient sample size was insufficient for statistical significance (Figure 4C). Thus, although γ9δ1 T cells are depleted ex vivo, they appear highly proliferative in the presence of IL-2 and IL-15 in HEV patients.

### 3.5 | IFN-γ Response of T Cell Subsets to Infected Target Cells

We then examined whether the expanded cells reacted to CMV- and/or HEV-infected targets. Intracellular IFN-γ induction was chosen as a readout, as this cytokine is usually produced or coproduced in human γδ T cells in response to viral infection. To monitor T cell subset reactivity to CMV, we used CMV-permissive MRC-5 cells for stimulation. We used HepG2 hepatocarcinoma cells, which are permissive for CMV and HEV, to monitor T cell reactivity to target cells infected with HEV, CMV, or both (see Section 3 and [30, 41, 42]). The cells were infected for 24 h and co-cultured with cytokine-sensitized lymphocytes in the presence of IL-2 and IL-18. This cytokine combination was used because preliminary testing indicated

<footnote>aSolid organ transplant.</footnote>

<footnote>b$p>0.9$ for all group combinations (Kruskal–Wallis test).</footnote>

<footnote>cLog IU; titers < 1 are considered negative; comparison Cont-CMV<sup>pos</sup>/HEV-CMV<sup>pos</sup>; $p=0.094$ (Mann–Whitney test).</footnote>
<figure><img src="image_5.png" /><figcaption>**FIGURE 2** | γδ T cell subsets ex vivo and after culture with IL-2+IL-15. The frequencies of γδ T cell subsets ex vivo were determined by flow cytometry on PBMC samples. (A) Relative frequency of γδ T cells and major subsets among CD3 cells. (B) Frequencies of major subsets among total γδ T cells. Controls and patients are separated according to their seropositivity for CMV (C-/C+). Bars indicate median values. Red dots represent transplanted HEV patients. Statistical analysis by Kruskal–Wallis test (n = 8/8/5/7). Only *p* values < 0.1 are indicated.</figcaption></figure>

that it allows optimal IFN-γ induction without the background response of the synergistic IL-12/IL-18 combination [43–45]. Then, intracellular IFN-γ accumulation was measured by flow cytometry in T cell subsets. TCR stimulation with plastic-coated anti-CD3 was used to assess the ability of subsets to produce IFN-γ in the presence of the same cytokines (Figure 5). Under these conditions, there was no significant background IFN-γ induction in the absence of infected cells or anti-CD3 stimulation.

For most control donors, stimulation by CMV-infected MRC-5 fibroblasts induced IFN-γ production in cytokine-sensitized total γδ T cells. This response was more significant in CMV<sup>pos</sup> donors, presumably due to in vivo clonal selection. However, this response was not statistically significant when γδ subsets were considered separately. This suggests that γ9δ2, γ9δ1, and γXδ1 subsets were not simultaneously involved in the response of controls. αβ (CD3<sup>pos</sup>pan-γδ<sup>neg</sup>) T cell responses were not detected in controls.

γδ-T cells from most HEV patients reacted to CMV-infected MRC-5 cells, and CMV<sup>pos</sup> HEV patients had highly significant γδ T cell responses. Stimulation of IFN-γ production by CMV was statistically significant in the three major subsets in this group of patients. Notably, the IFN-γ response of γδ T cells to anti-CD3 stimulation was low in CMV<sup>pos</sup> HEV patients, suggesting that Th1 differentiation is lower in this group. Most importantly, CMV<sup>pos</sup> HEV patients had a high frequency of αβ T cells that responded to CMV-infected MRC-5 cells (Figure 5A).

IFN-γ production after stimulation with CMV-infected HepG2 cells was never significant in the control group, though weak responses were detected in CMV<sup>pos</sup> controls. CMV-infected HepG2 cells most significantly induced IFN-γ expression in αβ and γδ T cells from CMV<sup>pos</sup> HEV patients. However, this induction was significant in γ9δ2 and γXδ1 cells but not in γ9δ1 cells. HEV alone or in coinfection with CMV did not influence IFN-γ production in γ9δ2, γ9δ1, or γXδ1 T cells in controls or patients (Figure 5B).

Thus, the IFN-γ response to CMV-infected cells in this setting is an innate property of a large fraction of γ9δ2 and non-γ9δ2 cells. However, their ability to respond increases during the acute phase of HEV infection in patients carrying CMV, which coincides with the activation of conventional T cell immunity to CMV. In this setting, HEV infection of target cells does not promote IFN-γ production in lymphocytes.

## 3.6 | TCR Modulation After Stimulation With Infected Targets

The increased IFN-γ response of lymphocyte subsets in the presence of CMV-infected cells may result from TCR cross-linking or co-stimulatory signals, such as NK receptor or TLR stimulation. As TCR stimulation induces TCR modulation on the cell surface, TCR levels were recorded. This was performed on IFN-γ<sup>Hi</sup> and IFN-γ<sup>Lo</sup> cells for each subset (Figure 6). TCR
<figure><img src="image_6.png" /><figcaption>**FIGURE 3** | Anti-CMV IgG titers in CMV<sup>pos</sup> controls and HEV patients. CMV-IgG antibody levels were determined in sera from CMV-seropositive control individuals and HEV patients. Red dots represent transplanted patients. (A) Comparison of CMV-IgG titers between CMV-seropositive control and HEV samples. (B) Comparison of titers before and during HEV-acute infection in five transplanted patients (Mann-Whitney test). (A) Bars indicate means; (A, B) statistics: Mann-Whitney test. (C) Simple linear regression and correlation analysis of CMV-IgG titers and γXδ1 frequency in CMV<sup>pos</sup> controls and patients (*p* value of Spearman's correlation).</figcaption></figure>

cross-linking with anti-CD3 induced significant TCR down-
modulation in all γδ subsets. However, TCR downmodulation
was not detected in γ9δ2 and γXδ1 T cells following stimulation
with infected fibroblasts. This suggests that IFN-γ production by
these T cells is, in large part, TCR-independent in this setting.
Unlike other subsets, IFN-γ-producing γ9δ1 T cells from HEV
patients exhibited high TCR expression in uninfected cultures,
which significantly decreased upon stimulation with CMV-
infected fibroblasts. Thus, this subset appears to be particularly
CMV-reactive through a process that may involve the TCR
(Figure 6A). No similar downmodulation was detected in
IFN-γLo cells upon stimulation with CMV-infected MRC5 cells;
therefore, virus-responsive cells producing another cytokine
appear to be rare (Figure 6B).

No TCR downmodulation was observed with CMV or HEV in
single infections of HepG2 cells. Nevertheless, γ9δ1 T cells from
CMV<sup>pos</sup> HEV patients displayed higher TCR expression, which
was downmodulated on IFN-γ-producing cells upon exposure
to HEV + CMV co-infected HepG2 cells (Figure 6C). Thus,
although infected HepG2 cells did not significantly increase the
frequency of γ9δ1 cells producing IFN-γ, the data suggest that
TCR triggering of this subset occurs in CMV<sup>pos</sup> HEV patients
due to the synergistic effect of CMV and HEV. Similar to MRC-5
cocultures, TCR downmodulation was not detected in the IFN-
γLo cells (Figure 6D). Overall, these results suggest that TCR
stimulation occurs in the γ9δ1 cells of CMV<sup>pos</sup> HEV patients
after triggering by virus-infected cells. This is most evident after
stimulation with CMV-infected cells.
A

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B

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C

<figure><img src="image_16.png" /></figure>

**FIGURE 4** | Expansion of γδ T cell subsets in the presence of IL-2 and IL-15. The frequencies of γδ T cell subsets were determined by flow cytometry after 7 days of PBMC culture in the presence of IL-2 and IL-15 and compared to ex vivo frequencies in controls and HEV patients. (A) Frequency of total CD3+ among lymphocytes, γδ T cells among CD3+ cells, and γδ T cell subsets among γδ cells. (B) Comparison of γ9δ1 T cell frequencies in controls and patients separated according to their CMV status. (C) Comparison of γ9δ1 T cell frequencies in immunocompetent (non-Tx) and transplanted (Tx) HEV patients. Statistics (A, B, C): Kruskal-Wallis test, \*p > 0.05; \*\*p < 0.01; ns: p > 0.05.

3.7 | Effect of Coinfection on CMV and HEV Replication in HepG2 Cells

As HEV and CMV synergize in vivo and possibly in vitro to activate γδ T cells, we tested whether coinfection alters HEV or CMV replication (Figure 7). After CMV infection in HepG2

cells, infectious CMV particles accumulated in the cell culture medium over 14 days, a process that was not significantly affected by HEV coinfection when HEV was added 24 h before or after CMV infection (Figure 7A). HEV infection of HepG2 cells resulted in progressive HEV RNA increase in cell lysates over 14 days. This was not significantly affected by CMV added
A

<figure><img src="image_17.png" /></figure>

B

<figure><img src="image_18.png" /><figcaption>FIGURE 5 | Legend on next page.</figcaption></figure>
<figure><img src="image_19.png" /><figcaption>**FIGURE 6** | TCR modulation of γδ T cells stimulated with CMV/HEV-infected cells. PBMCs from controls (Cont.) and HEV patients were primed and stimulated as described in Figure 5. (A, C) The mean fluorescence intensities (mfi) of pan-γδ TCR staining were recorded in IFN-γ<sup>Pos</sup> and IFN-γ<sup>Low</sup> cells for each subset (pan-γδ staining). Results are expressed as the percentage of variation of TCR expression relative to IFN-γ<sup>Low</sup> cells (pan-γδ staining) for each subset: relative TCR expression after stimulation with (A) uninfected or CMV-infected MRC5 fibroblasts and (C) HepG2 cells infected with CMV, HEV, or both viruses. (B, D) Relative TCR expression on IFN-γ<sup>Low</sup> cells after stimulation with (B) infected MRC5 and (D) HepG2 cells, using cocultures with uninfected cells as a reference. Data normalization was applied and is described in Section 2. Statistical analysis used a two-way ANOVA mixed effects model.</figcaption></figure>

<figure><img src="image_20.png" /><figcaption>**FIGURE 7** | HEV and CMV<sub>AD169</sub> replication in MRC-5 and HepG2 cells. (A) Determination of infectious CMV release in day-7 and day-14 supernatants of HepG2 cells in single CMV infections or coinfections with HEV, added either 24 h before (HEVpre) or after CMV (HEVpost). (B) RT-PCR quantification of HEV genome in HepG2 lysates 1, 7, or 14 days after HEV infection, alone or in coinfection with CMV, added either 24 h before (CMVpre) or after HEV (CMVpost) (n = 6 independent experiments). Statistical analysis by Friedman's ANOVA; (A) n = 4; (B) n = 6.</figcaption></figure>

<caption>**FIGURE 5** | IFN-γ response and TCR modulation of γδ T cells stimulated with CMV/HEV-infected cells. PBMCs from controls (Cont.) and HEV patients were primed for 7 days with IL-2 and IL-15 and stimulated for 16 h with (A) MRC-5 or (B) HepG2 cells, either noninfected (NI), pre-infected for 24 h with CMV (A, B) or HEV (B), or co-infected (C). Stimulation with plastic-coated anti-CD3 was used as a positive control. HEV patients are separated according to their CMV seropositivity (CMV-/CMV+). IFN-γ responses were determined by intracellular cytokine staining. The results are expressed as the percentage of IFN-γ<sup>pos</sup> cells in the indicated T cell subsets. Bars indicate median values. Red dots represent transplanted HEV patients. Statistical analysis by Kruskal-Wallis test (n = 10/9/6/8).</caption>
either 24 h before or after HEV (Figure 7B). Thus, the syner-
gistic effects of the two viruses do not simply result from
increased replication following coinfection. Additionally, HEV's
slow replication might limit the detection of responses against
HEV in the experimental setting used in the above experiments.

4 | Discussion

This study confirms that γδ T cells are mobilized during the
acute phase of HEV infection and reveals specific alterations in
individuals carrying CMV. The increase in γXδ1 and the mod-
erate decrease in γ9δ2 cells in blood are consistent with previ-
ous reports [24, 46, 47] and support the idea that they are
induced by a CMV reactivation, which remains generally occult.
This study also reveals a shift in the behavior of γ9δ1 cells
linked to HEV infection in vivo.

IFN-γ responses against CMV-infected cells are exacerbated in cells of CMV<sup>pos</sup> HEV patients, including αβ and γδ T cell subsets. This most likely results from oligoclonal or polyclonal amplification [36] while subsets engage in a CMV response, supporting the notion that anti-CMV immunity reactivates in most CMV carriers during the acute phase of HEV infection. CMV viremia was rarely detected in these patients, and CMV-specific IgG titers did not consistently increase. This may be because IgG titers are tightly linked to the level of viremia [48], or it may be due to the fact that the blood trafficking of CMV-reactive conventional and nonconventional T cells precedes the elevation of antibody titers upon viral reactivation in tissues. In line with a possible occult reactivation, previous reports have documented CMV shedding in secretions rather than in blood in CMV carriers [49–51]. Thus, CMV is most likely responsible for the relative depletion of γ9δ2 and the inflation of γXδ1 observed ex vivo. The rarity of CMV detection in serum and its lack of association with clinical manifestations, such as CMV disease, suggest that CMV infection is well controlled in most patients in our study, including those undergoing immunosuppressive treatment.

In HEV patients, no reactivity to HEV was detected through IFN-γ responses. This may be due to HEV's non-cytopathic and slow replicative properties. The experimental setting involved a short-term (24-h) infection, which induces a strong cytopathic effect with CMV but little HEV replication. This may hinder the detection of HEV effects, and alternative approaches, such as the transfection of HEV RNA replicons, could be useful [52]. Although the hepatocarcinoma line used in our study is particularly permissive for HEV [30, 33], the usage of primary hepatocytes as targets should also be considered in future work. Note that a specific reactivity to chronically infected HepG2 cells was not detected in previous studies using chronically infected cells [30].

CMV stimulation increased IFN-γ accumulation in several γδ T-cell subsets in HEV CMV<sup>pos</sup> patients. This reactivity was observed in IL-2/IL-15-sensitized cells; thus, it may be favored by the cytokine environment. Furthermore, IL-18 was included in the stimulation step and was necessary for IFN-γ induction. Interestingly, γ9δ2 responses to CMV-infected MRC5 were particularly pronounced and detectable in several control individuals, even in the absence of CMV carriage. Thus, reactivity to CMV-infected cells is an intrinsic property of many γδ T cells, including γ9δ2, γ9δ1, and γXδ1 subsets, without stringent TCR

selectivity. Furthermore, the absence of TCR modulation in
most instances suggests that this reactivity is largely TCR-
independent. In contrast to our results, Kaminsky et al. reported
selective non-γ9δ2 responses, despite using a similar sensitiza-
tion protocol [15]. The differences in virus isolates and target
fibroblasts used in the assays may explain the discrepancy: the
CMVAD169 isolate used in our experiments is particularly cyto-
pathic in MRC5 cells, which may influence recognition of viral
ligands by stimulatory or regulatory coreceptors. It is also pos-
sible that bystander cells contributed, since γδ cells were not
sorted in our setting.

The quantitatively minor subset of γ9δ1 cells exhibits a partic-
ular behavior in HEV patients. In previous reports, γ9δ1 cells
have been isolated from a patient with Lyme arthritis [53–56]
and from renal or lung transplant patients [18, 28]. Some γ9δ1 T
cell clones have been found to become activated upon recog-
nizing metabolic stress during bacterial infections [52–56] or in
the context of CMV infection [18, 28]. These cells frequently
cross-react with tumors [28, 57, 58], and one clone was found to
recognize the ephrin receptor A2 (EphA2) stress antigen in a
TCR-dependent manner [18]. In our experiments, this subset
was usually detected ex vivo in control samples but was
depleted from the periphery during the acute phase of HEV
infection, suggesting homing and sequestration in tissues,
independent of CMV. Their high proliferative potential in the
presence of IL-2 and IL-15 suggests priming in vivo during HEV
infection. If so, their activation by CMV-infected cells in vitro
may result from the recognition of a common stress antigen
induced by CMV and HEV. In contrast, the poor recognition of
HEV-infected cells could result from low expression of this
antigen or the lack of co-stimulation by HEV-infected cells.

γ9δ1 T cells may infiltrate tumors, display cytotoxic activity against tumor cells, and exhibit a Th1 phenotype [57]. In our setting, a relatively low percentage of cells in this subset produced IFN-γ after restimulation. Since the strategy and cytokine combinations used here favor the detection of Th1 cells, future investigations should assess the ability of γ9δ1 cells to express or differentiate into alternative functional types, such as Th17 or regulatory cells, in the context of antiviral responses. This differentiation might also be influenced by the viral environment, and previous investigations indicated that HEV-infected cells were able to promote IL-10 production in γδ T cells [30]. Thus, at present, it is not known whether these cells are bystanders, cytotoxic effectors, or regulatory cells. This differentiation can differ with the anti-tumor or antiviral context of activation, and our study suggests that, in the context of HEV infections, it may also differ whether CMV is present or not. For example, γ9δ1 cells from CMV<sup>neg</sup> HEV patients were not significantly reactive to CMV-infected targets. Thus, we suspect that CMV in vivo promotes clonal selection. Thus, it will be important to investigate whether this is beneficial or not to patients and if this influences the progression and persistence of HEV infection in immunocompromised individuals.

The number of patients in the present study was limited by the amount of cells required by our cellular approach. This limitation prevented us from linking CMV carriage and progression of HEV infection. Moreover, the study included transplant patients, and the real impact of transplantation and immunosuppression in vivo also deserves further attention. To overcome these limitations, further studies using single-cell analyses on more samples will be necessary.
## Acknowledgments

We thank R. Romieu-Mourez and P-E. Paulet from the human immune-monitoring platform at Inserm UMR 1291 (INFINITy) for the supervision of the PBMC and plasma biobanks from healthy adult subjects and Dr Z. Feng (Ohio State University) for providing the HepG2-MAVS knockdown cell line. We are grateful to the staff of the Flow Cytometry and Imaging Core Facilities of INSERM UMR 1043 for their help and to Dominique Rozet for administrative assistance. This work was funded by ANRS, grant number ECTZ103203, and by the Transhépate-Occitanie association. F.M. was supported by a fellowship from ANRS, grant number ECTZ103206.

## Ethics Statement

The study on patients and healthy donor samples was approved by the French South-West & Overseas Ethics Committee and was registered at the Ministry of Higher Education and Research (DC-2016-2772 and DC-2016-2772). Patients' consent or non-opposition for use in HEV research was registered as required by French ethical guidelines. Experiments were performed in agreement with the guidelines of the Declaration of Helsinki.

## Conflicts of Interest

The authors declare no conflicts of interest.

## Data Availability Statement

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

## References

1. N. Kamar, J. Izopet, N. Pavio, et al., "Hepatitis E Virus Infection," *Nature Reviews Disease Primers* 3 (2017): 17086.

2. N. Kamar, A. Del Bello, F. Abravanel, Q. Pan, and J. Izopet, "Unmet Needs for the Treatment of Chronic Hepatitis E Virus Infection in Immunocompromised Patients," *Viruses* 14, no. 10 (2022): 2116.

3. S. Lhomme, L. Bardiaux, F. Abravanel, P. Gallian, N. Kamar, and J. Izopet, "Hepatitis E Virus Infection in Solid Organ Transplant Recipients, France," *Emerging Infectious Diseases* 23, no. 2 (2017): 353-356.

4. J. C. Ribot, N. Lopes, and B. Silva-Santos, "γδ T Cells in Tissue Physiology and Surveillance," *Nature Reviews Immunology* 21, no. 4 (2021): 221-232.

5. B. Silva-Santos, S. Mensurado, and S. B. Coffelt, "γδ T Cells: Pleiotropic Immune Effectors With Therapeutic Potential in Cancer," *Nature Reviews Cancer* 19, no. 7 (2019): 392-404.

6. L. Gay, S. Mezouar, C. Cano, et al., "Role of Vγ9vδ2 T Lymphocytes in Infectious Diseases," *Frontiers in Immunology* 13 (2022): 928441.

7. C. Junqueira, R. B. Polidoro, G. Castro, et al., "γδ T Cells Suppress Plasmodium falciparum Blood-Stage Infection by Direct Killing and Phagocytosis," *Nature Immunology* 22, no. 3 (2021): 347-357.

8. C. Daguzan, M. Moulin, H. Kulyk-Barbier, C. Davrinche, S. Peyrottes, and E. Champagne, "Aminobisphosphonates Synergize With Human Cytomegalovirus to Activate the Antiviral Activity of Vγ9Vδ2 Cells," *Journal of Immunology* 196, no. 5 (2016): 2219-2229.

9. T. Herrmann and M. M. Karunakaran, "Butyrophilins: γδ T Cell Receptor Ligands, Immunomodulators and More," *Frontiers in Immunology* 13 (2022): 876493.

10. D. Vermijlen and I. Prinz, "Ontogeny of Innate T Lymphocytes—Some Innate Lymphocytes Are More Innate Than Others," *Frontiers in Immunology* 5 (2014): 486.

11. J. L. McMurray, A. von Borstel, T. E. Taher, et al., "Transcriptional Profiling of Human Vδ1 T Cells Reveals a Pathogen-Driven Adaptive Differentiation Program," *Cell Reports* 39, no. 8 (2022): 110858.

12. C. Khairallah, T. H. Chu, Z. Qiu, J. N. Imperato, D. Yang, and B. S. Sheridan, "The Accumulation of Vγ4 T Cells With Aging Is Associated With an Increased Adaptive Vγ4 T Cell Response After Foodborne *Listeria monocytogenes* Infection of Mice," *Immunity & Ageing* 19, no. 1 (2022): 19.

13. C. Khairallah, S. Netzer, A. Villacreces, et al., "γδ T Cells Confer Protection Against Murine Cytomegalovirus (MCMV)," *PLoS Pathogens* 11, no. 3 (2015): e1004702.

14. V. Pitard, D. Roumanes, X. Lafarge, et al., "Long-Term Expansion of Effector/Memory Vδ2-γδ T Cells Is a Specific Blood Signature of CMV Infection," *Blood* 112, no. 4 (2008): 1317-1324.

15. H. Kaminski, C. Menard, B. El Hayani, et al., "Characterization of a Unique γδ T Cell Subset as a Specific Marker of CMV Infection Severity," *Journal of Infectious Diseases* 223, no. 4 (2021): 655-666.

16. C. Söderberg-Nauclér, K. N. Fish, and J. A. Nelson, "Reactivation of Latent Human Cytomegalovirus by Allogeneic Stimulation of Blood Cells From Healthy Donors," *Cell* 91, no. 1 (1997): 119-126.

17. C. Söderberg-Nauclér, D. N. Streblow, K. N. Fish, J. Allan-Yorke, P. P. Smith, and J. A. Nelson, "Reactivation of Latent Human Cytomegalovirus in CD14(+) Monocytes Is Differentiation Dependent," *Journal of Virology* 75, no. 16 (2001): 7543-7554.

18. C. Harly, S. P. Joyce, C. Domblides, et al., "Human γδ T Cell Sensing of AMPK-Dependent Metabolic Tumor Reprogramming Through TCR Recognition of EphA2," *Science Immunology* 6, no. 61 (2021): eaba9010.

19. M. Jaganjac, T. Matijevic, M. Cindric, et al., "Induction of CMV-1 Promoter by 4-Hydroxy-2-Nonenal in Human Embryonic Kidney Cells," *Acta Biochimica Polonica* 57, no. 2 (2010): 179-183.

20. Y. L. Lee, C. E. Liu, W. L. Cho, et al., "Presence of Cytomegalovirus DNA in Leucocytes Is Associated With Increased Oxidative Stress and Subclinical Atherosclerosis in Healthy Adults," *Biomarkers* 19, no. 2 (2014): 109-113.

21. S. Lee, J. S. Affandi, A. B. Irish, and P. Price, "Cytomegalovirus Infection Alters Phenotypes of Different γδ T-Cell Subsets in Renal Transplant Recipients With Long-Term Stable Graft Function," *Journal of Medical Virology* 89, no. 8 (2017): 1442-1452.

22. J. Nikolich-Zugich, "Ageing and Life-Long Maintenance of T-Cell Subsets in the Face of Latent Persistent Infections," *Nature Reviews Immunology* 8, no. 7 (2008): 512-522.

23. M. J. Kallemeijn, A. M. H. Boots, M. Y. van der Klift, et al., "Ageing and Latent CMV Infection Impact on Maturation, Differentiation and Exhaustion Profiles of T-Cell Receptor Gammadelta T-Cells," *Scientific Reports* 7, no. 1 (2017): 5509.

24. S. Waters, E. Brook, S. Lee, R. Estiasari, I. Ariyanto, and P. Price, "HIV Patients, Healthy Aging and Transplant Recipients Can Reveal the Hidden Footprints of CMV," *Clinical Immunology* 187 (2018): 107-112.

25. K. Wistuba-Hamprecht, D. Frasca, B. Blomberg, G. Pawelec, and E. Derhovanessian, "Age-Associated Alterations in γδ T-Cells Are Present Predominantly in Individuals Infected With Cytomegalovirus," *Immunity & Ageing* 10, no. 1 (2013): 26.

26. F. Martini and E. Champagne, "The Contribution of Human Herpes Viruses to γδ T Cell Mobilisation in Co-Infections," *Viruses* 13, no. 12 (2021): 2372.

27. M. Deseke and I. Prinz, "Ligand Recognition by the γδ TCR and Discrimination Between Homeostasis and Stress Conditions," *Cellular & Molecular Immunology* 17, no. 9 (2020): 914-924.

28. F. Halary, V. Pitard, D. Dlubek, et al., "Shared Reactivity of Vδ2neg γδ T Cells Against Cytomegalovirus-Infected Cells and Tumor Intestinal Epithelial Cells," *Journal of Experimental Medicine* 201, no. 10 (2005): 1567-1578.

29. F. Abravanel, H. Barragué, G. Dörr, et al., "Conventional and Innate Lymphocytes Response at the Acute Phase of HEV Infection in Transplanted Patients," *Journal of Infection* 72, no. 6 (2016): 723-730.
30. H. Barrague, J. Fontaine, F. Abravanel, et al., "Mobilization of γδ T Cells and IL-10 Production at the Acute Phase of Hepatitis E Virus Infection in Cytomegalovirus Carriers," *Journal of Immunology* 206, no. 5 (2021): 1027-1038.

31. C. Mengelle, K. Sandres-Sauné, J. M. Mansuy, C. Haslé, J. Boineau, and J. Izopet, "Performance of a Completely Automated System for Monitoring CMV DNA in Plasma," *Journal of Clinical Virology* 79 (2016): 25-31.

32. F. Abravanel, K. Sandres-Saune, S. Lhomme, M. Dubois, J. M. Mansuy, and J. Izopet, "Genotype 3 Diversity and Quantification of Hepatitis E Virus RNA," *Journal of Clinical Microbiology* 50, no. 3 (2012): 897-902.

33. X. Yin, X. Li, C. Ambardekar, Z. Hu, S. Lhomme, and Z. Feng, "Hepatitis E Virus Persists in the Presence of a Type III Interferon Response," *PLoS Pathogens* 13, no. 5 (2017): e1006417.

34. S. Lhomme, S. Magne, S. Perelle, et al., "Clustered Cases of Waterborne Hepatitis E Virus Infection, France," *Viruses* 15, no. 5 (2023): 1149.

35. L. C. M. Arruda, A. Gaballa, and M. Uhlin, "Graft γδ TCR Sequencing Identifies Public Clonotypes Associated With Hematopoietic Stem Cell Transplantation Efficacy in Acute Myeloid Leukemia Patients and Unravels Cytomegalovirus Impact on Repertoire Distribution," *Journal of Immunology* 202, no. 6 (2019): 1859-1870.

36. M. S. Davey, C. R. Willcox, S. P. Joyce, et al., "Clonal Selection in the Human Vδ1 T Cell Repertoire Indicates γδ TCR-Dependent Adaptive Immune Surveillance," *Nature Communications* 8 (2017): 14760.

37. S. Ravens, C. Schultze-Florey, S. Raha, et al., "Human γδ T Cells Are Quickly Reconstituted After Stem-Cell Transplantation and Show Adaptive Clonal Expansion in Response to Viral Infection," *Nature Immunology* 18, no. 4 (2017): 393-401.

38. T. Yamaguchi, Y. Suzuki, R. Katakura, T. Ebina, J. Yokoyama, and Y. Fujimiya, "Interleukin-15 Effectively Potentiates the In Vitro Tumor-Specific Activity and Proliferation of Peripheral Blood γδT Cells Isolated From Glioblastoma Patients," *Cancer Immunology, Immunotherapy* 47, no. 2 (1998): 97-103.

39. H. Wang, X. Wang, W. Wang, et al., "Interleukin-15 Enhanced the Survival of Human γδT Cells by Regulating the Expression of Mcl-1 in Neuroblastoma," *Cell Death Discovery* 8, no. 1 (2022): 139.

40. Y. Yang and A. Lundqvist, "Immunomodulatory Effects of IL-2 and IL-15; Implications for Cancer Immunotherapy," *Cancers* 12, no. 12 (2020): 3586.

41. D. Todt, M. Friesland, N. Moeller, et al., "Robust Hepatitis E Virus Infection and Transcriptional Response in Human Hepatocytes," *Proceedings of the National Academy of Sciences of the United States of America* 117, no. 3 (2020): 1731-1741.

42. Q. Lepiller, W. Abbas, A. Kumar, M. K. Tripathy, and G. Herbein, "HCMV Activates the IL-6-JAK-STAT3 Axis in HepG2 Cells and Primary Human Hepatocytes," *PLoS One* 8, no. 3 (2013): e59591.

43. G. Kaplanski, "Interleukin-18: Biological Properties and Role in Disease Pathogenesis," *Immunological Reviews* 281, no. 1 (2018): 138-153.

44. K. Nakanishi, "Unique Action of Interleukin-18 on T Cells and Other Immune Cells," *Frontiers in Immunology* 9 (2018): 763.

45. C. Kessel, C. Rossig, and H. Abken, "Weal and Woe of Interleukin-18 in the T Cell Therapy of Cancer," *Journal for Immunotherapy of Cancer* 13, no. 1 (2025): e010545.

46. D. Vermijlen, M. Brouwer, C. Donner, et al., "Human Cytomegalovirus Elicits Fetal γδ T Cell Responses In Utero," *Journal of Experimental Medicine* 207, no. 4 (2010): 807-821.

47. J. Dechanet, P. Merville, F. Berge, et al., "Major Expansion of Gammadelta T Lymphocytes Following Cytomegalovirus Infection in Kidney Allograft Recipients," *Journal of Infectious Diseases* 179, no. 1 (1999): 1-8.

48. M. Iglesias-Escudero, M. A. Moro-García, R. Marcos-Fernández, et al., "Levels of Anti-CMV Antibodies Are Modulated by the Frequency and Intensity of Virus Reactivations in Kidney Transplant Patients," *PLoS One* 13, no. 4 (2018): e0194789.

49. R. R. Razonable and A. Humar, "Cytomegalovirus in Solid Organ Transplant Recipients-Guidelines of the American Society of Transplantation Infectious Diseases Community of Practice," *Clinical Transplantation* 33, no. 9 (2019): e13512.

50. L. Puhakka, S. Pati, M. Lappalainen, et al., "Viral Shedding, and Distribution of Cytomegalovirus Glycoprotein H (UL75), Glycoprotein B (UL55), and Glycoprotein N (UL73) Genotypes in Congenital Cytomegalovirus Infection," *Journal of Clinical Virology* 125 (2020): 104287.

51. D. Ju, X. Z. Li, Y. F. Shi, Y. Li, L. Q. Guo, and Y. Zhang, "Cytomegalovirus Shedding in Seropositive Healthy Women of Reproductive Age in Tianjin, China," *Epidemiology and Infection* 148 (2020): e34.

52. L. D. Xu, F. Zhang, L. Peng, et al., "Stable Expression of a Hepatitis E Virus (HEV) RNA Replicon in Two Mammalian Cell Lines to Assess Mechanism of Innate Immunity and Antiviral Response," *Frontiers in Microbiology* 11 (2020): 603699.

53. C. Collins, Y. Lui, A. M. Santos, et al., "Detection of Cell Surface Ligands for Human Synovial γδ T Cells," *Journal of Immunology* 203, no. 9 (2019): 2369-2376.

54. C. C. Collins, P. Hahn, Z. Jiang, K. A. Fitzgerald, T. S. Xiao, and R. C. Budd, "Regulation of Synovial γδ T Cell Ligand Expression by Mitochondrial Reactive Oxygen Species and Gasdermin-D," *Journal of Immunology* 210, no. 1 (2023): 61-71.

55. M. S. Vincent, K. Roessner, D. Lynch, et al., "Apoptosis of Fashigh CD4+ Synovial T Cells by Borrelia-Reactive Fas-Ligand(high) γδ T Cells in Lyme Arthritis," *Journal of Experimental Medicine* 184, no. 6 (1996): 2109-2118.

56. M. S. Vincent, K. Roessner, T. Sellati, et al., "Lyme Arthritis Synovial γδ T Cells Respond to *Borrelia burgdorferi* Lipoproteins and Lipidated Hexapeptides," *Journal of Immunology* 161, no. 10 (1998): 5762-5771.

57. H. W. Lee, C. Park, J. G. Joung, et al., "Renal Cell Carcinoma-Infiltrating CD3low Vγ9Vδ1 T Cells Represent Potentially Novel Anti-Tumor Immune Players," *Current Issues in Molecular Biology* 43, no. 1 (2021): 226-239.

58. M. Otto, R. C. Barfield, R. Iyengar, et al., "Human γδ T Cells From G-CSF-Mobilized Donors Retain Strong Tumoricidal Activity and Produce Immunomodulatory Cytokines After Clinical-Scale Isolation," *Journal of Immunotherapy* 28, no. 1 (2005): 73-78.

## Supporting Information

Additional supporting information can be found online in the Supporting Information section.

1) Characteristics of patients. 2) Antibodies. 3) PCR primers. 4) Supplemental figure 1. Gating Strategy for γδ T cell subset identification by flow cytometry. 5) Supplemental figure 2. IFN-γ detection in lymphocyte subsets by flow cytometry.