key: cord-0758616-wusf3ckw
authors: Stankovic, Sanda; Davey, Martin S.; Shaw, Evangeline M.; von Borstel, Anouk; Cristiano, Yvonne; Levvey, Bronwyn J.; Rossjohn, Jamie; Westall, Glen P.; Snell, Gregory I.; Brooks, Andrew G.; Sullivan, Lucy C.
title: Cytomegalovirus replication is associated with enrichment of distinct γδ T cell subsets following lung transplantation: A novel therapeutic approach?
date: 2020-08-26
journal: J Heart Lung Transplant
DOI: 10.1016/j.healun.2020.08.014
sha: d169952aae4ca02593902fb8140ffab9936a7e88
doc_id: 758616
cord_uid: wusf3ckw

BACKGROUND: Antiviral treatments to control cytomegalovirus (CMV) following lung transplantation (LTx) are associated with toxicity and antiviral resistance. Cellular immunotherapy with virus-specific cytotoxic T cells have yielded promising results but requires donor/recipient matching. Gamma delta (γδ) T cells are involved in antiviral immunity and can recognize antigens independently of major histocompatibility complex (MHC) and may not require the same level of matching. We assessed the phenotype of circulating γδ T cells following LTx to identify candidate populations for CMV immunotherapy. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from LTx recipients pre-transplant and at routine bronchoscopies post-LTx. Patients were stratified by risk of CMV disease into moderate risk (MR, recipient CMV seropositive, n=15) or high-risk (HR, recipient CMV seronegative/donor CMV seropositive, n=10). CMV replication was classified as PCR positive (>150 copies/ml) in blood and/or bronchoalveolar lavage within the first 18-months. The phenotype of γδ T cells was assessed by multi-colour flow cytometry and T cell receptor (TCR) sequences were determined deep sequencing. RESULTS: In HR LTx recipients with CMV replication, we observed striking phenotypic changes in γδ T cells, marked by an increase in the proportion of effector Vδ1+ γδ T cells expressing the activating natural killer cell receptor NKG2C. Moreover, we observed a remarkable increase in TCR diversity. CONCLUSION: NKG2C+ Vδ1+ γδ T cells were associated with CMV replication and may indicate their potential to control infection. As such, we propose they could be a potential target for cellular therapy against CMV.

Lung transplantation (LTx) is a life-saving procedure for end stage lung disease. However LTx recipients have a lower long term survival when compared to other solid organ transplants, with a median survival of only 6.5 years (1) . Chronic lung allograft dysfunction (CLAD) is the major factor limiting LTx long-term survival and is the result of alloimmune and infection-induced damage to the graft, resulting in allograft failure and death. In LTx recipients, cytomegalovirus (CMV) replication is common and impacts on survival directly through end-organ infection, but is also associated with the development of CLAD (2) . Whilst antiviral drugs can effectively limit CMV replication, the dosing duration is largely empirical, side effects including neutropenia are common and indiscriminate dosing are associated with drug resistance (3) . Furthermore, prolonged antiviral use can result in inhibition of immune function, warranting an alternative approach to antiviral treatment (4) .

Host control of CMV involves both the innate and adaptive immune systems (5) , with a well-established contribution of αβ T cells, B cells and Natural Killer (NK) cells. An additional subset with possible contribution in the immune control of CMV are γδ T cells that can provide protection from CMV in murine models (6) and human γδ T cells can kill CMVinfected cells in vitro (7) . Following kidney and hematopoietic stem cell transplantation (HSCT), replication of CMV is associated with subset perturbations in the frequencies of circulating γδ T cells. In particular, CMV replication has been linked to an expansion of γδ T cells lacking the TRDV2 gene segment of the γδ T cell receptor (TCR), called Vδ2 neg γδ T cells (8) , (9) . The increased frequency of Vδ2 neg γδ T cells following CMV infection is substantial, often resulting in an expansion from 1% to more than 10% of the total circulating T cells (10) , similar to that seen for CMV-specific CD8+ T cells (11) . However, there has been minimal investigation of the contribution of these γδ T cells in CMV immunity following LTx.

In addition to their TCR, γδ T cells express several receptors that are typically associated with NK cells, including NKG2D, which engages stress-induced receptors such as the MHC class I polypeptide-related sequence (MIC)-A and -B (12) . γδ T cells can also express receptors from the CD94-NKG2 family, which recognise the non-classical MHC class I molecule HLA-E (13) . Upregulation of CD94-NKG2C (NKG2C) on NK cells has been associated with CMV seropositivity (14) , and there are a number of reports describing the contribution of NKG2C+ NK cells in the control of CMV following solid organ and HSCT (15, 16) . Our own studies (17) and those of others (18) have demonstrated expansion of NKG2C+ NK cells following CMV replication post-LTx, further implicating a role for this receptor in immunity to CMV. However, a role for NKG2C in the context of γδ T cells remains largely unexplored.

Here, we longitudinally assessed the phenotype of circulating γδ T cells in LTx recipients at risk of CMV disease and temporally correlated this with CMV replication within 18 months post-LTx. The data suggest that there are changes in the composition of γδT cell subsets associated with CMV infection. Thus, clinical monitoring of this compartment might provide a guide for establishing the optimal duration of viral prophylaxis post-LTx.

Furthermore, the dramatic increases in the proportion of NKG2C+ Vδ1+ γδ T cells observed post-infection raise the prospect that γδ T cells could be a promising target for future cellular therapy.

collected pre-LTx and at surveillance bronchoscopies (0.5, 1.5, 3, 6, 9, 12 and 18-months post-LTx), separated into peripheral blood mononuclear cells (PBMC) by Ficoll-Paque (GE Healthcare, Sydney, NSW, Australia) and then cryopreserved in 90% FCS/10% DMSO until analysis. All patients were given standard triple immunosuppressant regimen (prednisolone, tacrolimus and azathioprine or mycophenolate).

CMV prophylaxis, monitoring and treatment: The patient's risk of CMV replication was further grouped into moderate risk (MR, CMV seropositive recipient, n=15) or high risk (HR, CMV seronegative recipient with a CMV seropositive donor, n=10). Most MR patients (12/15 MR) received standard antiviral prophylaxis for 5.5 months, consisting of 2 weeks intravenous ganciclovir (5g/kg body weight), followed by 450 mg twice daily oral valganciclovir. However, only 2/10 high risk recipients were on antiviral prophylaxis for less than 6 months. In the presence of a negative QuantiFERON-CMV assay (QIAGEN, USA), 3/15 MR and 6/10 HR LTx recipients received extended valganciclovir prophylaxis to 11 months post-LTx. Two HR patients received continuous valganciclovir throughout the entire monitoring period. As per protocol, HR recipients also received CMV hyper-immune immunoglobulin (1.5 million units) on day 1, 2, 3, 7, 14, 21 and 28, whilst also on (val)ganciclovir. CMV replication was detected by COBAS Amplicor CMV monitor test (Roche Diagnostic Systems, NSW, Australia) in both the plasma and bronchoalveolar lavage (BAL), with a PCR result >150 copies per ml (>137 international units) considered positive.

Patients with high level CMV replication in either blood or BAL (>10,000 copies/ml) were treated with intravenous ganciclovir for 2 weeks (5 mg/kg). Patients with low level infection in blood or BAL (600-10,000 copies/ml) were given oral valganciclovir for 2 weeks. Figure 1A) . Vγ9/Vδ2neg γδ T cells were sorted from 2 HR recipients, one at pre LTx (15,000 cells) and 9 months post-LTx during CMV reactivation (15,000 cells) and for another Vδ1+ γδ T cells at 6 months post LTx (prior to CMV detection, 1,000 cells) and 12 months post-LTx (during CMV reactivation, 2,900 cells). Cells were sorted into RNAlater (Sigma Aldrich). RNA was extracted using an RNAmicro plus kit (Qiagen) according to the manufacturer's instructions. Extracted RNA was then used for high throughput deep sequencing of γδ TCRs, using amplicon rescued multiplex (ARM)-PCR. Following initial first-round RT-PCR using high concentrations of gene-specific primers, universal primers were used for the exponential phase of amplification (iRepertoire Inc., Huntsville, USA, patent: WO2009137255A2), allowing deep, quantitative amplification of TCR Gamma (TRG) and TCR Delta (TRD) sequences. All cDNA synthesis, amplification, NGS library preparation were performed using TRG/TRD iRprofile kits (iRepertoire, Inc.

Huntsville, USA) and subsequent libraries were pooled and deep sequenced using an Illumina MiSeq (Micromon Genomics, Monash University).

TCR repertoire data analysis: V, D and J gene usage and complementarity determining region (CDR) 3 sequences were identified and assigned and tree maps generated using iRweb tools (iRepertoire, Inc, Huntsville, AL, USA). Tree maps show each unique CDR3 as a coloured rectangle, the size of each rectangle corresponds to each CDR3s abundance within the repertoire and the positioning is determined by the V region usage. Analysis of TCR repertoire diversity was measured by D50 metric, this value indicates the percentage of clonotypes required to occupy 50% of the total TCR repertoire from either TRD and TRG repertoires.

CMV infection significantly impacts on LTx success. Therefore, a cohort of LTx recipients were assessed for the presence of actively replicating CMV at routine bronchoscopies post-LTx. Prior to transplant, donor and recipient CMV serostatus was assessed and patients were stratified into two groups at risk of CMV reactivation: MR (recipient CMV seropositive, n=15) and HR (recipient CMV seronegative, donor CMV seropositive, n=10). Following LTx, evidence of CMV replication was assessed by quantitative PCR (Table 1) . Overall, there was more CMV replication in the blood and/or BAL in the HR group and 8/10 recipients (H3-H10, Table 1 ) showed evidence of viral replication, whereas CMV replication was observed in 7/15 MR recipients (M9-M15, Table 1 ). Of the 2 HR recipients where CMV replication was absent, one was on extended antiviral prophylaxis for 338 days (H1) and the other was treated with valganciclovir for the entire monitoring period (H2). Viral replication was typically first detected at the cessation of antiviral prophylaxis, at 6 months (4 recipients), 8-9 months (5 recipients) or 11-12 months (6 recipients) post-LTx (Table 1) CMV replication was more frequently observed in the blood of individuals from the HR group (5/10 HR recipients vs 3/15 MR recipients, Table 1 ). Symptomatic CMV infection was observed in 4 individuals, one MR and 3 HR recipients. In MR recipient M12, reduced lung function was observed coincident with high level viral replication in the blood (>700,000 copies per ml). CMV pneumonitis was observed in HR recipient H7 at 8 months post-LTx, whereas CMV syndrome/gastritis was observed in HR recipients H3 and H4 at 12 months post-LTx ( Table 1) .

To determine whether there was an association between CMV infection and circulating γδ T cells, we initially compared the proportion of γδ T cells within PBMC between MR and HR recipients. The gating strategy employed to identify T cells is shown in Figure 1A . Overall, the proportion of γδ T cells did not differ between the different CMV risk groups at individual time points post-LTx ( Figure 1B ). Stratifying results from HR recipients in whom active CMV replication was detected by PCR, there was a small but significant increase in the proportion of γδ T cells over the first 18 months post-LTx ( Figure 1C ) but not in the 2 HR recipients where CMV replication was not observed (data not shown). Therefore, CMV replication in previously CMV seronegative LTx recipients was associated with relatively minor changes in the overall proportion of γδ T cells in the blood.

To determine whether there were gross changes in the γδ T cell repertoire in the LTx cohort, we initially enumerated the proportion of Vδ1+ and Vδ2+ γδ T cells and found a significantly higher proportion of Vδ1+ cells in CMV seropositive recipients prior to transplant, although there were 3 MR recipients with a notably lower proportion than others ( Figure 2A ). MR recipients maintained higher proportions of Vδ1+ γδ T cells throughout the post-transplant period ( Figure 2B and 2C) than HR recipients who remained PCR negative ( Figure 2C ). Furthermore, MR recipients with or without CMV replication both had a higher proportion of Vδ1+ γδ T cells than HR recipients without CMV replication (mean proportion Vδ1+ γδ T cells MR with CMV = 68%, MR without CMV = 65%, HR without CMV = 24%).

Without considering timing over the post-LTx period, there was no significant differences between HR recipients that experienced CMV replication to any other group (mean proportion Vδ1+ = 35%). However, in HR recipients in whom CMV replication was confirmed, the proportion of circulating Vδ1+ γδ T cells significantly increased over time post LTx, which was not observed in the two HR individuals on prolonged antiviral treatment ( Figure 2C and 2D). These changes were apparent following the cessation of antiviral prophylaxis and occurred prior to or coincident with CMV replication, such that by 18 months post-LTx, more than 60% of circulating γδ T cells were Vδ1+ in the HR recipients in which CMV replication was detected ( Figure 2D ). Therefore, CMV infection results in a skewing in the γδ T cell repertoire towards the Vδ1+ subset.

To assess post-LTx changes in γδ TCR repertoire in more detail, we performed a pilot analysis of TCR sequences prior to and during active CMV replication. Given previous findings (19) and our own observations that the archetypal blood Vγ9/Vδ2 are not involved in CMV control (20) , we excluded clones that were double positive for Vγ9 and Vδ2, and only sequenced γδ T cells lacking this specific TRG and TRD combination (called Vγ9/Vδ2neg cells, Supplementary Figure 1A) . In an HR recipient that had evidence of CMV replication (recipient H7), we used deep sequencing to assess the pre-and post-transplant γδ TCR repertoire. Analysis of the TCR repertoire of Vγ9/Vδ2neg γδ T cells by tree plot found a dramatic expansion of individual clonotypes during CMV replication ( Figure 3A ) and was not a result of differences in sequencing depth between each sample (Supplementary Figure   1B) . Moreover, the total number of clonotypes and D50 diversity index sharply increased and the accumulated frequency of the top 20 clonotypes decreased from the pre-transplant repertoire (Supplementary Figure 1C) . The flow cytometry data for this recipient indicated that the proportion of Vδ1+ γδ T cells increased from ~20% pre LTx to more than 50% of γδ T cells during CMV replication, concurrent with a decrease in the proportion of Vδ2+ γδ T cells from ~40% to ~10% of γδ T cells over the same time frame (data not shown).

Therefore, when filtering on the expanding Vδ1+ fraction for this donor, we observed that the TCR repertoire was surprisingly diverse during CMV replication ( Figure 3B ).

To exclude the possibility that the transplant itself impacted on Vδ1+ diversity rather than CMV replication, we performed deep sequencing on a second HR recipient at two post-LTx timepoints, one prior to CMV replication (6 months post LTx) and one during CMV 

Given substantial diversity in TCR repertoires during the post-LTx period in HR recipients, we investigated whether we could identify the expression of a common marker that was indicative of CMV exposure. Previous studies had shown an increase in the proportions of NK cells expressing the NKG2C activating receptor were associated with active CMV replication (17) . Therefore, the expression of NKG2C expression on γδ T cells was compared between MR and HR LTx recipients. Akin to NK cells, a higher proportion of NKG2C+ γδ T cells prior to LTx was associated with MR recipients, where the mean proportion of circulating γδ T cells that expressed NKG2C was 40% in MR recipients, in contrast to less than 20% in HR recipients ( Figure 4A) . Interestingly, the same 3 MR recipients with a lower proportion of Vδ1+ cells also had a lower proportion of NKG2C+ γδ T cells (M11, M12 and M15, Figure 4A ) and flow cytometric analysis revealed that most NKG2C+ γδ T cells were Vδ1+ ( Figure 4B ). Most MR recipients consistently maintained a high proportion of NKG2C+ γδ T cells over the post-LTx period ( Figure 4B , 4C) and similar to results for Vδ1+ analysis, HR recipients without CMV replication had significantly lower proportions of NKG2C+ γδ T cells than both MR groups (mean % NKG2C+ γδ T cells in MR without CMV replication = 32%, MR with CMV = 35% versus HR without CMV = 8%, Figure   4C ). Further analysis of HR recipients with active CMV replication indicated an associated increase in the proportion of NKG2C+ γδ T cells over the post-LTx period (p=0.003), with a mean proportion of NKG2C+ γδ T cells at 2 weeks post-LTx of 9%, increasing to 32% at 18 months post-LTx (mean overall %NKG2C+ post-LTx = 18%, Figure 4B and 4D). Importantly, this increase was observed after the cessation of antiviral prophylaxis and occurred coincident with CMV replication. Moreover, this change was not evident in two HR recipients without CMV replication who were on prolonged antiviral treatment ( Figure 4C ). Therefore, this suggests that the induction of NKG2C on γδ T cells following CMV infection is at least partially dependent on active viral replication.

To better understand whether the remodeling of the γδ T cell compartment was accompanied by changes in phenotypic marker expression post-LTx, γδ T cells were assessed for the expression of CD27 and CD45RA, two receptors used to define naïve (CD27+/CD45RA+) and effector (CD27low CD45RA+) γδ T cells (20, 21) . Prior to LTx, MR recipients had more effector γδ T cells pre-LTx than HR recipients, although the same 3 MR recipients with lower Vδ1+ and NKG2C+ γδ T cells also had a lower proportion of effector γδ T cells ( Figure 5A ). Consistent with previous data, individuals from the MR group maintained higher proportions of effector γδ T cells post LTx ( Figure 5B , 5C) and both MR groups had significantly higher proportions of effector γδ T cells than HR patients without CMV replication (MR without CMV replication = 70%, MR with CMV = 67% versus HR without CMV = 25%, Figure 5C ). In contrast, there was a dramatic proportional increase in CD27low CD45RA+ effector γδ T cells in the HR group that went on to experience an episode of CMV replication ( Figure 5B, C and D) . Overall the proportion of CD27low CD45RA+ γδ T cells in HR recipients with CMV replication was 48%, however at 2 weeks this proportion was 33%, increasing to 90% at 18 months post-LTx. Indeed, the higher proportions of effector γδ T cells was not seen in the HR recipients without CMV replication on extended prophylaxis ( Figure 5C ).

Our data suggested that CMV replication resulted in an expansion of effector γδ T cells that co-expressed Vδ1 and NKG2C. Indeed, a detailed analysis of 2 HR recipients (H6 and H9) showed similar trends prior to and following CMV replication ( Figure 6 ). Following LTx but prior to CMV replication, the proportions of γδ T cells in the blood were similar in both recipients (~2% of lymphocytes), which increased following CMV replication to 22% and 7%, in H6 and H9, respectively ( Figure 6A ). In both HR recipients prior to CMV replication, the vast majority of γδ T cells expressed Vδ2 (~80%) while the proportion that expressed Vδ1 in the circulation was only 14% ( Figure 6B ). Strikingly, following CMV replication, this ratio changed markedly, as the proportion of Vδ1+ γδ T cell increased to 87% and 65% of γδ T cells in H6 and H9 respectively, which was accompanied by a concomitant decrease in the proportion of Vδ2+ γδ T cells, to the point where they were almost absent in recipient H6 ( Figure 6B) . Notably, it was the expanded Vδ1+ γδ T cell population that expressed NKG2C at much higher levels than Vδ2+ γδ T cells and CD3+ non-γδ T cells ( Figure 6C) . Moreover, the proportion of Vδ1+ γδ T cells expressing an effector phenotype (CD27low CD45RA+) increased dramatically following CMV replication; namely from 30% to 100% in H6 and from 15% to 85% in H9 ( Figure 6D ). Although there was also an increase in proportion of effector Vδ2+ γδ T cells in recipient H9 (19% to 54%, Figure 6D ), this was not to the same extent as Vδ1+ γδ T cells. Moreover, as mentioned above, the overall proportion of Vδ2+ γδ T cells had dramatically decreased following CMV replication in recipient H9. Notably, Vδ2+ γδ T cells were virtually absent in the circulation of recipient H6 following CMV replication ( Figure   6B ). A higher proportion of NKG2C+ γδ T cells also expressed an effector phenotype following CMV replication in both recipient H6 and H9 ( Figure 6E ), likely as a result of the co-expression of NKG2C on Vδ1+ γδ T cells ( Figure 6C ).

In order to examine if subtle changes were also present in MR recipients with CMV reactivation, we performed a similar analysis of the co-expression of Vδ1, NKG2C and effector γδ T cell subsets in an MR recipient (M9). Indeed, although the proportion of γδ T cells in the blood did not change prior to and following CMV replication in this MR individual ( Figure 6A ), there was a proportional increase in the Vδ1+ and decrease in Vδ2+ populations following CMV replication ( Figure 6B) . Notably, it was the Vδ1+ subset that expressed NKG2C ( Figure 6C ) and both the Vδ1+ ( Figure 6D ) and the NKG2C+ populations ( Figure 6E ) transitioned to an effector phenotype, whereas the Vδ2+ population lacked NKG2C ( Figure 6C ) and did not possess an effector phenotype ( Figure 6D ). Indeed, an analysis of MR recipients with CMV replication indicated changes in the composition of the γδ T cell compartment. Of particular note were recipients M11 and M15 that had low proportions of pre-LTx Vδ1+/NKG2C+/effector γδ T cells (Figure 2A , 4A and 5A) but showed dramatic changes following CMV replication (Supplementary Figure 2) . Thus, taken together, the data show that CMV replication was associated with the development of effector Vδ1+ NKG2C+ γδ T cell population following LTx.

Our study has found marked changes in circulating γδ T cells following LTx with a profound expansion of Vδ1+ NKG2C+ γδ T cells strongly correlating with CMV replication post-LTx. γδ T cells have previously been shown to expand in response to CMV infection in kidney transplant patients and change the proportion of γδ TCR subtypes (22), from Vδ2+ to Vδ2-, with Vδ1+ cells specifically correlated with CMV immunity (20) . Our results suggest this to also be the case in HR LTx recipients following CMV exposure, in keeping with what has been observed in other solid organ transplantation (9) . Interestingly, most CMV seropositive recipients had a stable, elevated frequencies of effector Vδ1+ cells, that were similar in the pre-and post LTx period, irrespective of subsequent episodes of CMV replication. In contrast, in HR LTx recipients, there was a marked change in the composition of the γδ T cell population with CMV replication towards a cytotoxic effector phenotype, marked by the loss of CD27 (20) . However, 3 CMV seropositive recipients (M11, M12 and M15) had lower frequencies of effector γδ cells and all 3 of these experienced CMV replication. Notably, 2 of these (M11 and M15) showed subsequent changes in composition of γδ T cells following CMV replication, similar to those seen in HR recipients. Interestingly, M11 showed the highest CMV replication in the BAL, whereas for M15 it was in the blood ( Table 1 ), indicating that the site of CMV replication did not impact on whether this subset was observed in the circulation. In contrast, recipient M12 failed to show such alterations in the γδ T cell compartment and experienced multiple recurrences of CMV replication at 6, 9 and 11 months post-LTx. It is tempting to speculate that CMV replication not only initiates the expansion of effector γδ cells, but that high levels of this subset protect from further replication. A larger cohort from several transplant centers will verify these findings.

In contrast to previous studies, we did not observe expansion of selected clones in post-LTx period (8, 20, 23) but rather we found a substantial increase in TCR diversity with CMV replication post-LTx. Our observations may reflect expanded minor clones or novel thymic emigrants recruited into the immune repertoire in response to CMV. Indeed, a polyclonal CD8+ T cell response to CMV has been observed following umbilical cord blood transplantation (24). It is also possible that this diversity is due to immunological 'space' created by immunosuppression post-transplant or inflammatory cytokines allowing for nonspecific expansion of unique clones. This however is unlikely, as it does not explain preferential increased diversity in Vδ1+ cells and moreover Vδ1+ cells are reportedly refractory to expansion by key inflammatory cytokines (IL-12 or IL-18) (20) . However, we acknowledge that our sequencing analyzes only 2 HR recipients and requires more HR and MR recipients to draw solid conclusions, which will be a focus of future studies.

Although the Vδ1+ γδ T cell population following CMV replication in LTx had incredibly diverse TCRs, we found the co-expression of NKG2C. Intriguingly, and similar to that observed for NK cells (16) , the proportion of NKG2C+ Vδ1+ γδ T cells appeared to remain high and stable following the clearance of actively replicating virus. Although NKG2C expression on Vδ1+ γδ T cells has also been associated with cytotoxicity against HIVinfected CD4+ αβ T cells (13) , to our knowledge this is the first study associating the expression of NKG2C on γδ T cells is linked with CMV replication and suggest their role in anti-viral immunity. NKG2C recognizes a monomorphic non-classical MHC class I molecule human leukocyte antigen (HLA)-E. Moreover, NKG2C binds HLA-E presenting the CMVderived UL40 peptide with a high affinity (25) . NKG2C expression on lymphocytes has been directly associated with CMV seropositivity (26) and a higher incidence of CMV replication in the LTx cohort in whom NKG2C gene deletion exists (27) . Although TCR ligand(s) for Vδ1+ γδ T cell remain poorly defined, they have not been explicitly linked to CMV infection, but include molecules induced by cellular stress. Although cells can be activated via NKG2C without engagement of TCR (28), their action is likely to be classical HLA-independent, favoring them as good candidates for cellular therapy sourced clinically from a third-party without being HLA-matched to the recipient (29) . Moreover, although NKG2C has been largely associated with CMV immunity, it is possible Vδ1+ γδ T cells are effective against other diseases where HLA-E is overexpressed such as EBV-associated cancers (30) .

Intriguingly, a recent report found an association between the genetic lack of NKG2C and more severe coronavirus 2019 disease (COVID-19) (31) . Future research will be required to investigate the functional potential of NKG2C+ γδ T cells in these settings. Moreover, investigations of this subset in the lung allograft itself will be of great benefit in pinpointing their contribution to local CMV immunity.

One drawback of our study was that all the recipients were on (val)ganciclovir post-LTx and that the withdrawal of antiviral prophylaxis could have been responsible for initiating the expansion of this subset rather than active CMV replication. However, most MR and 2 HR recipients ceased antiviral prophylaxis prior to 6 months post LTx, yet the enrichment of 

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