WO2021016652A1

WO2021016652A1 - METHODS AND COMPOSITIONS FOR MONITORING, TREATING AND PREVENTING CMV INFECTION OR GRAFT REJECTION USING γδ T-CELLS

Info

Publication number: WO2021016652A1
Application number: PCT/AU2020/050145
Authority: WO
Inventors: Lucy Catherine SULLIVAN; Sanda STANKOVIC; Andrew Geoffrey BROOKS
Original assignee: The University Of Melbourne
Priority date: 2019-07-29
Filing date: 2020-02-20
Publication date: 2021-02-04

Abstract

The present invention relates to methods, systems and compositions for the monitoring and treatment and prevention of CMV infection using γδ T-cells.The present invention also relates to methods of decreasing graft rejection or increasing graft survival post transplantation using γδ T-cells. The activation of NKG2C+ γδ T-cells with an antibody or fragment thereof that binds to NKG2C is also disclosed.

Description

METHODS AND COMPOSITIONS1FOR MONITORING, TREATING AND PREVENTING CMV INFECTION OR GRAFT REJECTION USING gd T-CELLS

FIELD OF THE INVENTION

The present invention relates to methods, systems and compositions for the monitoring and treatment and prevention of CMV infection.

BACKGROUND OF THE INVENTION

Cytomegalovirus (CMV) is a double stranded DNA virus of the beta subfamily of herpesviruses. It is ubiquitous amongst the human population with 50-90% of people seroconverting by adulthood with higher rates of seroprevalence correlating closely to socioeconomic level and race.

During primary CMV infection, a strong adaptive immune response is stimulated. B cells are activated and produce antibodies against CMV proteins e.g. structural tegument proteins (IE-1 and pp65) and envelope glycoproteins although whether these antibodies provide protection is debated. Risk of CMV reactivation is prevented through cellular immune control with the pp65 and IE-1 -specific response being particularly well characterised. CMV-specific memory T cells are generated which may constitute 10% of both CD8 and CD4 memory compartments in the peripheral blood of CMV seropositive individuals.

In lung transplant recipients, immunosuppressive therapy diminishes the immune response leading to primary CMV infection or viral reactivation in CMV seropositive recipients. Detection of CMV antigenemia is associated with reduced frequency of cytokine producing CMV-specific CD8+ or CD4+ T cells. In these immunocompromised patients, CMV can cause invasive disease such as pneumonitis or colitis. Patients may also develop CMV syndrome which causes fever, pancytopenia and liver enzyme derangement from hepatosplenomegaly. The inflammatory nature of CMV infection may also accelerate atherosclerosis and lead to chronic lung allograft dysfunction (CLAD).

The consensus guidelines for CMV management in solid organ transplantation (SOT) includes viral load testing and risk stratification based on donor and recipient CMV serostatus to guide prophylactic treatment. Patients at highest risk are donor (D) seropositive and recipient seronegative (R) (D+/R-) followed by moderate risk, where the recipient is seropositive at the time of transplant. Both high and moderate CMV risk groups receive extended antiviral prophylaxis although the duration varies among centres (e.g. from 6-12 months for lung transplant recipients). Ganciclovir and valganciclovir are the mainstays of antiviral therapy. In patients not receiving antiviral prophylaxis therapy, viral load is measured in the blood and bronchoalveolar lavage (BAL) fluid by quantitative PCR at the time of follow-up appointments to monitor for actively replicating virus. While antiviral therapies have reduced the incidence of CMV infection post-transplant, widespread use of antivirals in SOT is associated with side effects, including neutropenia, bone marrow toxicity, antiviral resistance and late- onset CMV disease. Due to the complications of antiviral treatment and uncertainty in antiviral prophylaxis duration, there is interest in developing CMV immune monitoring tests to guide antiviral prophylaxis and individualise CMV treatment.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a method for determining if a subject is at increased risk of CMV infection, the method comprising: a) determining the presence of a gdT cell response in a sample from the subject, wherein the presence of a gdT cell response in the sample indicates that the subject has an increased risk of CMV infection.

In another embodiment, the present invention provides a method as described herein, wherein the method further comprises b) providing a recommendation that the subject will have an increased risk of CMV infection.

In one embodiment, the subject is a transplant recipient.

In one embodiment, the present invention provides a method of determining if a transplant recipient is at increased risk of graft rejection post-transplant, the method comprising: a) determining the presence of a gdT cell response in a sample from the subject, wherein the presence of a gdT cell response in the sample indicates that the subject has an increased risk of graft rejection post-transplant. In another embodiment, the present invention provides a method as described herein, wherein the method further comprises b) providing a recommendation that the subject will have an increased risk of graft rejection post-transplant.

In one embodiment, the present invention provides a method of determining if a transplant recipient has a decreased prospect of graft survival post-transplant, the method comprising: a) determining the presence of a gdT cell response in a sample from the subject, wherein the presence of a gdT cell response in the sample indicates that the subject has a decreased prospect of graft survival post-transplant.

In another embodiment, the present invention provides a method as described herein, wherein the method further comprises b) providing a recommendation that the subject will have a decreased prospect of graft survival post-transplant.

In one embodiment, the sample is obtained from the subject post-transplantation.

In another embodiment, the present invention provides a method as described herein, wherein the gdT cell response in the sample obtained from the subject post transplantation is compared to a gdT cell response in a sample obtained from the subject pre-transplantation.

In another embodiment, the present invention provides a method as described herein, wherein the gdT cell response in the sample from obtained from the subject post transplantation is compared to a threshold level of a gdT cell response.

In one embodiment, the present invention provides a method of decreasing graft rejection post-transplant in a subject, the method comprising: a) determining the presence of a gdT cell response in a sample from the subject, wherein the presence of a gdT cell response in the sample indicates that the subject has an increased risk of graft rejection post-transplant; and b). administering to a subject with an increased risk of graft rejection post-transplant a therapeutically effective amount of a composition comprising a gdT cell or a population thereof and/or a therapeutically effective amount of an antiviral agent.

In one embodiment, the present invention provides a method of increasing graft survival post-transplant in a subject, the method comprising: a) determining the presence of a gdT cell response in a sample from the subject, wherein the presence of a gdT cell response in the sample indicates that the subject has an increased risk of CMV reactivation post-transplant; and b). administering to a subject with a decreased risk of graft survival post-transplant a therapeutically effective amount of a composition comprising a gdT cell or a population thereof and/or a therapeutically effective amount of an antiviral agent.

In one embodiment, the present invention provides a method of treating and/or preventing CMV infection in a subject, the method comprising: a) determining the presence of a gdT cell response in a sample from the subject, wherein the presence of a gdT cell response in the sample indicates that the subject has an increased risk of CMV infection post-transplant; and b) administering to a subject with an increased risk of CMV infection post-transplant a therapeutically effective amount of a composition comprising a gdT cell or a population thereof and/or a therapeutically effective amount of an antiviral agent.

In another embodiment, the present invention provides a method as described herein, wherein the gdT cell response is an increase in gdT cells as a proportion of T cells.

In another embodiment, the present invention provides a method as described herein, wherein the gdT cell response is a post-transplant increase in gdT cells as a proportion of T cells. In another embodiment, the present invention provides a method as described herein, wherein the gdT cell response is a V61 gdT cell response. In another embodiment, the present invention provides a method as described herein, wherein the gdT cell response is an increase in V61 gdT cells as a proportion of gdT cells. In another embodiment, the present invention provides a method as described herein, wherein the gdT cell response is a post-transplant increase in V61 gdT cells as a proportion of gdT cells. In another embodiment, the present invention provides a method as described herein, wherein the gdT cell response is a NKG2C+ gdT cell response. In another embodiment, the present invention provides a method as described herein, wherein the gdT cell response is an increase in NKG2C+ gdT cells as a proportion of gdT cells. In another embodiment, the present invention provides a method as described herein, wherein the gdT cell response is a post-transplant increase in NKG2C+ gdT cells as a proportion of gdT cells. In another embodiment, the present invention provides a method as described herein, wherein the gdT cell response is NKG2C activation. In another embodiment, the present invention provides a method as described herein, wherein the gdT cell response is an increase in NKG2C activation.

In another embodiment, the present invention provides a method as described herein, wherein the gdT cell response is a post-transplant increase in NKG2C activation.

In another embodiment, the present invention provides a method as described herein, wherein the gdT cell response is TNF-a and/or IFN-g production by NKG2C+ gdT cells. In another embodiment, the present invention provides a method as described herein, wherein the gdT cell response is an increase in TNF-a and/or IFN-g production by NKG2C+ gdT cells. In another embodiment, the present invention provides a method as described herein, wherein the gdT cell response is a post-transplant increase in TNF-a and/or IFN-g production by NKG2C+ gdT cells.

In another embodiment, the present invention provides a method as described herein, wherein the gdT cell response further comprises a V62 gdT cell response. In another embodiment, the present invention provides a method as described herein, wherein the V62 gdT cell response is a decrease in V62 gdT cells as a proportion of gdT cells. In another embodiment, the present invention provides a method as described herein, wherein the V62 gdT cell response is a post-transplant decrease in V62 gdT cells as a proportion of gdT cells.

In another embodiment, the present invention provides a method as described herein, wherein the gdT cell response is a Vd1+NKG2C+ CD45RA+ CD27- gdT cell response. In another embodiment, the present invention provides a method as described herein, wherein the gdT cell response is an increase in Vd1+NKG2C+ CD45RA+ CD27- gdT cells as a proportion of gdT cells. In another embodiment, the present invention provides a method as described herein, wherein the gdT cell response is a post-transplant increase in Vd1+NKG2C+ CD45RA+ CD27- gdT cells as a proportion of gdT cells.

In another embodiment, the present invention provides a method as described herein, wherein the sample is a blood, whole blood or a sample comprising blood-derived cells. In another embodiment, the present invention provides a method as described herein, wherein the subject is a transplant recipient. In another embodiment, the present invention provides a method as described herein, wherein the subject is a lung transplant recipient.

In another embodiment, the present invention provides a method as described herein, wherein the CMV infection is CMV reactivation. In another embodiment, the present invention provides a method as described herein, wherein the CMV infection is CMV viraemia in the blood and/or CMV infection of the graft.

In another embodiment, the present invention provides a method as described herein, further comprising administering to the subject an effective amount of an antiviral agent. In another embodiment, the present invention provides a method as described herein, wherein the antiviral agent is selected from the group consisting of ganciclovir (GCV), valganciclovir (VGCV), foscarnet (FOS), and cidofovir (CDV).

In another embodiment, the present invention provides a method as described herein, further comprising administering to the subject a therapeutically effective amount of a composition comprising a gdT cell or a population thereof.

In one embodiment, the present invention provides a method of treating and/or preventing CMV infection in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising a gdT cell or a population thereof.

In one embodiment, the present invention provides a method of treating and/or preventing CMV reactivation in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising a gdT cell or a population thereof.

In one embodiment, the present invention provides a method of increasing graft survival in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising a gdT cell or a population thereof. In another embodiment, the present invention provides a method as described herein, wherein the gdT cell is a V61 gdT cell or a population thereof.

In another embodiment, the present invention provides a method as described herein, wherein the gdT cell is a NKG2C+ gdT cell or a population thereof.

In another embodiment, the present invention provides a method as described herein, wherein the gdT cell is a V61+ NKG2C+ CD45RA+ CD27- gdT cell or a population thereof.

In another embodiment, the present invention provides a method as described herein, wherein the gdT cell or a population thereof is derived from a gdT cell or a population thereof obtained from a subject seropositive for CMV.

In another embodiment, the present invention provides a method as described herein, wherein the subject administered a therapeutically effective amount a composition comprising a gdT cell or a population thereof is not the subject seropositive for CMV.

In another embodiment, the present invention provides a method as described herein, wherein the composition comprises a Vb1 gdT cell or a population thereof. In another embodiment, the present invention provides a method as described herein wherein the composition comprises a NKG2C+ gdT cell or a population thereof. In another embodiment, the present invention provides a method as described herein, wherein the composition comprises a V61 + NKG2C+ CD45RA+ CD27- gdT cell or a population thereof. In another embodiment, the present invention provides a method as described herein, wherein the composition comprises a V61 + NKG2C+ CD45RA+ CD27- CD16- gdT cell or a population thereof.

In one embodiment, the present invention provides a composition comprising a gdT cell or a population thereof and a pharmaceutically acceptable excipient.

In one embodiment, the present invention provides a composition comprising a gdT cell or a population thereof obtained from a subject seropositive for CMV and a pharmaceutically acceptable excipient. In another embodiment, the present invention provides a composition as described herein, wherein the composition comprises a V61 gdT cell or a population thereof.

In a further embodiment, the present invention provides a composition as described herein, wherein the composition comprises a NKG2C+ gdT cell or a population thereof. In a further embodiment, the present invention provides a composition as described herein, wherein the composition comprises a V61+ NKG2C+ CD45RA+ CD27- gdT cell or a population thereof. In a further embodiment, the present invention provides a composition as described herein, wherein the composition comprises a V61+ NKG2C+ CD45RA+ CD27- CD16- gdT cell or a population thereof.

In another embodiment, the present invention provides a use of a composition as described herein in the manufacture of a medicament for treating and/or preventing CMV infection or CMV reactivation.

In one embodiment, the present invention provides a method of activating a NKG2C+ gdT cell or a population thereof, the method comprising contacting a NKG2C+ gdT cell or a population thereof with an antibody or a fragment thereof that binds to NKG2C.

In one embodiment, the present invention provides a method of producing an activated NKG2C+ gdT cell or a population thereof, the method comprising contacting a NKG2C+ gdT cell or a population thereof with an antibody or a fragment thereof that binds to NKG2C.

In one embodiment, the present invention provides a use of an antibody or a fragment thereof that specifically binds to NKG2C for the manufacture of a composition for activating a NKG2C+ gdT cell or a population thereof.

In one embodiment, the present invention provides a method of measuring a gdT cell response, the method comprising; a) contacting a sample from a subject, with an NKG2C activating agent, wherein the sample from the subject comprises a gdT cell or a population thereof; and b). measuring a gdT cell response.

In one embodiment, the present invention provides a method for determining if a subject is at increased risk of CMV infection, the method comprising: a) contacting a sample from a subject, with an NKG2C activating agent, wherein the sample from the subject comprises a gdT cell or a population thereof; and b). measuring a gdT cell response.

In one embodiment, the present invention provides a method as described herein wherein the NKG2C activating agent is an anti-NKG2C antibody or a fragment thereof.

In one embodiment, the present invention provides a method as described herein wherein the NKG2C activating agent is an HLA-E-peptide complex.

In one embodiment, the present invention provides a method as described herein wherein the gdT cell response is TNF-a and/or IFN-g expression.

In one embodiment, the present invention provides a method as described herein wherein the gdT cell response is intracellular TNF-a and/or intracellular IFN-g expression. In one embodiment, the present invention provides a method as described herein wherein the gdT cell response is an increase in expression of TNF-a and/or IFN-g. In one embodiment, the present invention provides a method as described herein wherein the gdT cell response is an increase in expression of TNF-a and/or IFN-g relative to a control.

In one embodiment, the present invention provides a method as described herein wherein the presence of a gdT cell response the sample indicates that the subject has an increased risk of CMV infection.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 A. shows the flow cytometry gating strategy to identify for gamma delta T cells (Y6TCR+ CD3+ cells) within peripheral blood mononuclear cells (PBMC) or lymphocytes, dead cells, CD14+ and CD19+ (‘Dump’ gate). Cryopreserved PBMC from lung transplant recipients were thawed, stained with antibodies and analysed by flow cytometry. Figure 1 B shows a box and whiskers plot of all values for the proportion of gd T cells of total lymphocytes (based on fcs/ssc gate, excluding Live/dead+CD14+CD19+ cells) between moderate and high risk recipients from all time-points post-transplant (n=13 patients and total of 54 time-points for high risk recipients; n=12 patients and total of 77 time-points for moderate risk recipients). Lines indicate mean +- SEM and individual symbols represent different time-points. Statistical significance was tested using Mann-Whitney test. ^* p=0.02. . Figure 1 C shows gd T cell proportions segregated on time-points of sample collection following lung transplant. Each symbol represents a sample within assigned risk group (dark circles-moderate risk; open triangles-high risk).. Figure 1 D shows linear regression analysis of the proportion of gd T cells post-transplant in high risk recipients who were CMV viral DNA+ve with p and R2 values shown.

Figure 2A shows the proportion of NKG2C+ gdT cells (%NKG2C+ gdT cells of total blood gdT cells) in moderate risk patients (CMV -i-ve recipient) (circles) and high risk patients (CMV -i-ve donor CMV -ve recipient) (triangles) pre-transplant. Each symbol represents one recipient with mean ± SEM shown. Statistical significance was tested using two-way Mann-Whitney test ^** p=0.0085. Figure 2B shows a flow cytometry plot of moderate and high risk LTx recipients at 2 weeks and 12 months post-LTx in whom CMV active replication was detected. Numbers show the proportion of cells within a given gate (gated on gd T cells). Figure 2C shows proportion of NKG2C+ve gdT cells (% of gdT cells) in moderate risk patients (CMV +ve recipient;“Moderate”) (circles) and high risk patients (CMV+ donor CMV -ve recipient;“High”) (triangles) following lung transplant, stratified for CMV replication post-transplant (“Neg” = no replication, “Pos” = CMV replication). Pooled values for the proportion of NKG2C+ gd T cells from moderate risk and high risk recipients with individual time-points designated by discrete groups with mean ± SEM are shown for each (2wk, 6wk, 3mo, 6mo, 9mo, 12mo and 18mo post-transplant). Statistical significance between moderate and high risk groups was tested using Mann-Whitney two-tailed test. ^**** p<0.0001. Figure 2D shows linear regression analysis of NKG2C+ gd T cells over time post-transplant in high risk recipients that acquired active CMV infection post-LTx; the proportion of NKG2C+ gdT cells (% NKG2C+ve gdT cells of total gdT cells) in high risk patients with CMV replication following lung transplant is shown.

Figure 3A shows the proportion of Vb1 gdT cells (%Vd1 gdT cells of total gdT cells) in moderate risk patients (CMV -i-ve recipient) (circles) and high risk patients (CMV -i-ve donor CMV -ve recipient) (triangles) pre-transplant. Figure 3B shows a flow cytometry plot of moderate and high risk LTx recipients at 2 weeks and 12 months post-LTx in whom CMV active replication was detected. Numbers show the proportion of cells within a given gate (gated on gd T cells). Figure 3C shows proportion of Vb1 gdT cells (%nd1 gdT cells of total gdT cells) in moderate risk patients (CMV -i-ve recipient; “Moderate”) (circles) and high risk patients (CMV+ donor CMV -ve recipient;“High”) (triangles) following lung transplant, stratified for CMV replication post-transplant (“Neg” = no replication,“Pos” = CMV replication). Data shown is from all timepoints post-transplant (wk=weeks; mo=months) divided by CMV risk group (n=13moderate and n=12 high risk recipients). Figure 3D shows linear regression analysis of proportion of V61 + gd T cells post-transplant in high risk recipients who were CMV viral DNA+ve with p and R2 values shown; the proportion of V61 gdT cells (%V61 -i-ve gdT cells of total gdT cells) in high risk patients with CMV replication, following lung transplant is shown.

Figure 4A shows the proportion of TEMRA (CD27low CD45RA+ gdT cells) (%CD27- CD45RA+ gdT cells of total gdT cells) in the blood of moderate risk patients (CMV -i-ve recipient) (circles) and high risk patients (CMV -i-ve donor CMV -ve recipient) (triangles) pre-transplant, each donor is represented by individual symbol. Statistical significance was tested using Mann-Whitney test ^* p=0.02. Figure 4B shows the proportion of TEMRA gd T cells; data from all timepoints post-transplant (2wk, 6wk, 3mo, 6mo, 9mo, 12mo, 18mo) divided by CMV risk group. Figure 4B shows proportion of CD27- CD45RA+ gdT cells (% CD27- CD45RA+ of gdT cells in moderate risk patients (CMV +ve recipient; “Mod”) (circles) and high risk patients (CMV+ donor CMV -ve recipient;“High”) (triangles) following lung transplant, stratified for CMV replication post-transplant (“NO” = no replication,“YES” = CMV replication). Figure 4D shows linear regression analysis of TEMRA gd T cells over time post transplant in high risk recipients that acquired active CMV infection post-LTx; the proportion of CD27- CD45RA+ gdT cells (%CD27- CD45RA+ gdT cells of total gdT cells) in high risk patients with CMV replication is shown.

Figure 5 shows the proportion of V61 + and V62 + gdT cells as a proportion of gdT cells pre- and 12 months post-transplant in patients with CMV reactivation, the proportion of V61+ gdT cells that are NKG2C+ post-transplant, and the proportion of nd1+ gdT cells that are CD45RA+ CD27- post-transplant. Shown is a flow cytometry plot of a high-risk recipient prior to transplant and at 12 months post-transplant coincident with a positive PCR result for CMV. Expression of NKG2C at 12 months post LTx is shown in as a solid line for V61+ gd T cells and dashed line for V62+ gd T cells. Expression of CD27 and CD45RA at 12 months post LTx is shown in black for V61+ gd T cells and grey for V62+ gd T cells. Figure 6 shows proportion of V62 gdT cells (%V62 gdT cells of total gdT cells) in moderate risk patients (CMV +ve recipient;“R+”) (green, left hand side) and high risk patients (CMV+ donor CMV -ve recipient;“D+/R-”) (red, right hand side) following lung transplant (“LTx”).

Figure 7A shows QuantiFERON-CMV does not predict protective immunity against CMV infection in the serum or lung allograft. QuantiFERON-CMV results were obtained at 5 months post-transplant and recipients with an indeterminate result were considered QuantiFERON negative. Recipients were followed up for 18 months post transplant. Measured QuantiFERON level separated by R+ and D+/R- serostatus. Kaplan-Meier survival curves of CMV infection with threshold of >0 copies/ml in the lung allograft. For Kaplan-Meier survival curves, the log-rank test was used to calculate the significance in difference between the two curves. Figure 7B shows T- Track-CMV (ELISPOT) does not predict protective immunity against CMV infection in the serum or lung allograft. Frozen PBMCs collected post-transplant were thawed and cultured and then tested using the ELISPOT assay provided in the T-Track-CMV kit.. Kaplan Meier survival curves of incidence of CMV infection in the lung allograft with threshold of >0 copies/ml. The log-rank test was used to calculate the significance in difference between the two curves. Figure 7C shows NKG2C ligation using anti- NKG2C antibody results in IFN-gamma production from NK cells, gd, CD4+ and CD8+ T cells. Figure 7D shows that using NKG2C based assays recipients without CMV in the lung allograft have significantly higher IFN-g responses.

Figure 8 shows that the percentage of NKG2C+ NK cells does not predict CMV infection risk. Frozen PBMCs were thawed and cultured overnight and stained for NKG2C surface marker expression. Data collected over six independent experiments a) The gating strategy used to find the proportion of NK cells expressing NKG2C. Percentage of NK cells expressing NKG2C in b) different serostatus groups, c) patients who did and did not develop CMV viraemia, d) patients with different levels of viraemia, e) patients who did and did not develop CMV in the lung allograft and f) patients with different levels of CMV in the lung allograft. Unpaired t-tests were used to calculate significance in difference between the groups analysed.

Figure 9A shows NKG2C ligation activates NKG2C+ gdT cells. PBMC from a healthy donor were incubated overnight on a plate bound anti-NKG2C (‘anti-2C’), no antibody (‘no mAb’) or anti-NKG2A (‘anti-2A’) in the presence of brefeldin A and CD107a mAb, prior to surface staining for CD3 and gd TCR. Figure 9B shows PBMC from a healthy donor were incubated in the absence of target cells (no target, left panel) or presence of B cell line 721.221 . Wild type 721 .221 (+721 .221 , middle panel) expresses low levels of HLA-E. 721 .221 transfected with HLA-G (721 221 -HLA-G, right panel) surface expresses higher levels of HLA-E in complex with a peptide ligand from HLA- G, a strong activator of NKG2C. Cells were surface stained for gd TCR, NKG2C and CD107a (degranulation marker). Figure 9B shows NKG2C+ gamma delta T cells can be activated using HLA-E, and that CD107A degranulation/cell killing is increased when NKG2C+ gamma delta T cells are activated with HLA-E or HLA-E/G complex. The B cell line 721 .221 as a target cell for cytotoxicity assays, and expresses very low levels of HLA in general but also low levels of HLA-E. 721 .221 cells are transfected with HLA-G; HLA-G contains the peptide ligand for HLA-E, and the cells also express HLA-E at the cell surface. This HLA-E/G complex is a strong activator of NKG2C. Panel 1 shows PBMCs from a healthy donor incubated with no target cells. Panel 2 shows PBMCs from a healthy donor incubated with 721 .221 wild type cells (low HLA- E), Panel 3 shows PBMCs from a healthy donor incubated with 721.221 -HLA-G (HLA- E/G). Cells are surface stained for gamma delta TCR, NKG2C and CD107A as a marker of degranulation as marker of gamma delta T cell functional activity, to indicate killing of cells by gamma delta T cells.

Figure 10 shows NKG2C+ gamma delta T cells are activated by CMV infected cells and that CD107A degranulation/cell killing is increased when NKG2C+ gamma delta T cells are activated by CMV infected cells. Figure 10A shows a flow cytometry plot of PBMC from a healthy donor incubated for 24h in the presence of IL2 and IL15 before incubating overnight with CMV-infected human foreskin fibroblasts (HFF). Cells are surface stained for gamma delta TCR, NKG2C and CD107A as a marker of degranulation as marker of gamma delta T cell functional activity, to indicate killing of cells by gamma delta T cells. Figure 10B shows a comparison of cytotoxicity (CD107a levels) between NKG2C+ and NKG2C- V61+ gd T cells in the presence of uninfected HFF or infected HFF at 2 different dilutions of the AD169 strain of human cytomegalovirus.

Figure 1 1 shows in vitro expansion of NKG2C+ V61+ cells. In brief, PBMC from a healthy donor were stained with antibodies to CD3, gd TCR, V61 and NKG2C and sorted by flow cytometry into the V61+ subset. V61+ cells were then labelled cell trace violet (CTV, to assess proliferation) and incubated in the conditions indicated above. Following 7 days of culture, cells were stained with antibodies to CD3, gd TCR, V61 , NKG2C and NKG2A.

DETAILED DESCRIPTION

The present invention in one or more embodiments relates in part to the finding that gdT cells are associated with CMV reactivation and can be used as a marker for CMV reactivation, including post organ transplant CMV reactivation.

Accordingly, in one embodiment, the present invention provides a method for determining if a subject is at increased risk of CMV infection, the method comprising: determining the presence of a gdT cell response in a sample from the subject, wherein the presence of a gdT cell response in the sample indicates that the subject has an increased risk of CMV reactivation. In one embodiment, the method further comprises b) providing a recommendation that the subject will have an increased risk of CMV infection.

As used herein the term“sample”, refers to a composition that is obtained or derived from a subject and/or individual of interest that contains a cellular and/or other molecular entity, or response that is to be characterized and/or identified, for example based on physical, biochemical, chemical and/or physiological characteristics, or response (including the gdT cell responses) described herein. For example, a sample or disease sample and variations thereof refers to any sample obtained from a subject of interest that would be expected or is known to contain the cellular and/or molecular entity, or exhibit a gdT cell response, that is to be characterized. Samples include, but are not limited to cells derived from a subject, for example from whole blood, blood derived cells, or tissue derived cells but are not limited to, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, bronchoalveolar lavage fluid, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, perspiration, mucus, tumour lysates, and tissue culture medium, tissue extracts such as homogenized tissue, tumour tissue, cellular extracts, and combinations thereof. In one embodiment, the methods described herein are performed with gdT cells, and accordingly, the sample comprises gdT cells. In a preferred embodiment, the sample is selected from the group consisting of tissue, whole blood, and combinations thereof. In some embodiments, the sample is a tissue sample. In some embodiments, the sample comprises tumour cells, tumour infiltrating immune cells or any combinations thereof.

Preferably, the sample is whole blood, or blood-derived cells.

In a preferred embodiment, the sample comprises peripheral blood mononuclear cells.

As used herein the term“determining” includes any means of detecting, including direct and indirect detection, and is used interchangeably with “measuring”. For example, a gdT cell response or biomarker described herein can be detected using an antibody, for example an anti- ybTCR, CD3, NKG2C, V61 , V62, CD27, CD45RA, CD107a, TNF-a, IFN-g and/or CD16. Presence and/or expression level/amount of a gdT cell response or biomarker in a sample can be analysed by a number of methodologies, many of which are known in the art and understood by the skilled artisan, including, but not limited to, immunohistochemistry (“IHC”), Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, ELIFA, flow cytometry, fluorescence activated cell sorting (“FACS”), MassARRAY, proteomics, quantitative blood based assays (as for example Serum ELISA), biochemical enzymatic activity assays, in situ hybridization, Southern analysis, Northern analysis, whole genome sequencing, polymerase chain reaction (“PCR”) including quantitative real time PCR (“qRT-PCR”) and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like), RNA-Seq, FISH, microarray analysis, gene expression profiling, and/or serial analysis of gene expression (“SAGE”), as well as any one of the wide variety of assays that can be performed by protein, gene, and/or tissue array analysis. Typical protocols for evaluating the status of genes and gene products are found, for example in Ausubel et al., eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Multiplexed immunoassays such as those available from Rules Based Medicine or Meso Scale Discovery (“MSD”) may also be used.

The present inventors have demonstrated that IFN-g and TNF-a responses to anti- NKG2C stimulation are associated with CMV reactivation. Accordingly, in one embodiment, the present invention provides the methods and systems described herein, wherein cytokine responses following contacting a cell or a cell population, such as cells in whole blood, with an NKG2C activating agent, are determined.

Aspects of the invention pertain to identifying or quantifying risk, such as a decreased risk or an increased risk or a decreased chance or an increased chance. In some variations, the risk can be quantified numerically as an absolute risk. For instance, based on comparison to patient data sets obtained according to techniques described herein, it is possible to assign a numerical risk score, such as a probability of experiencing an event (e.g., virus reactivation, viremia, allograft infection, graft rejection, graft survival etc.) within a certain period of time, including post-transplant in embodiments where the patient is a transplant recipient.

In some embodiments, risk assessment is a relative risk assessment. For instance, a subject has a baseline risk for an event (e.g., virus reactivation, viremia, allograft infection, graft rejection, graft survival etc.) by virtue of belonging to a recognized class (e.g., a patient that has received an organ transplant within the past month). The materials and methods described herein are useful for stratifying individuals within such a class as having elevated or increased risk, compared to the baseline risk of the organ transplant group of patients as a whole; or a reduced or decreased risk, compared to the baseline risk. Relative risk can be expressed quantitatively also (e.g., percent greater risk than the baseline or an odds ratio). Generally speaking, increased susceptibility or increased risk is descriptive of a relative risk or odds ratio greater than 1 , compared to the baseline population. The techniques described herein permit stratification of subjects to reveal and calculate a relative risk. Standard statistical techniques can be used to verify the statistical significance of the risk assessment.

The methods described herein can be used to treat and/or prevent, monitor or determine a risk of CMV infection. As used herein CMV infection includes CMV reactivation, which includes the development of viremia from a previously latent CMV infection, such as CMV viremia (in the blood), or allograft infection post-transplant. CMV is a ubiquitous human herpes virus that infects approximately 50% of normal individuals. In the majority of cases the immune response is able to control acute infection by recognising CMV derived antigens. The virus then persists for the life of the host in a latent state. Reactivation is prevented by immune system effector mechanisms including neutralising antibodies to virus membrane proteins, FILA- restricted CMV-specific helper and cytotoxic T cells, and MFIC-unrestricted effectors. CMV infection is important to certain high-risk groups. Major areas of risk of infection include pre-natal or postnatal infants and immunocompromised individuals, such as organ transplant recipients, persons with leukaemia, or those infected with human immunodeficiency virus (HIV). In HIV infected persons, CMV is considered an AIDS- defining infection, indicating that the T-cell count has dropped to low levels. CMV in immunocompromised persons (for instance, people who have had organ transplants or who have HIV) results in an increased risk for difficult eye infections (CMV retinitis), gastrointestinal CMV, and encephalitis.

CMV infection is the most significant viral complication in solid organ transplant (SOT) recipients. In the absence of antiviral therapy transplant recipients with CMV disease have rates of allograft loss. Different mechanisms of CMV-induced vascular or tissue damage in chronic allograft dysfunction have been proposed. Without wishing to be bound by theory, systemic inflammation induced by CMV replication may alter the state of allograft tolerance in the transplant recipient and subsequently trigger acute allograft rejection mediated by cytokines and other chemical mediators, which in turn will increase the risk for chronic allograft dysfunction. Persistent CMV infection measured by immunohistochemistry in the allograft biopsy of kidney transplant recipients was associated with increased expression of TGF-b and PDGF in the tubule and vascular endothelium, these molecules being directly involved in the pathogenesis of chronic allograft nephropathy.

Importantly, as is discussed herein, a critical effect of CMV on allograft function is the association between CMV serostatus of the donor and the recipient and transplant outcomes. Patients at the highest risk for CMV replication and disease (the seropositive donor/seronegative recipient) have consistently been associated with lower allograft function and survival compared with the control group of low-risk patients. Accordingly, the methods and compositions described herein are relevant to graft rejection and graft survival, CLAD, GVHD etc.

As used herein the term subject relates to an individual who is at risk of CMV reactivation, CMV viremia, allograft infection with CMV, graft rejection, decreased graft survival, GVHD, CLAD etc. The present invention provides methods for stratifying patients at risk, as demonstrated in the Examples. In one embodiment, the subject is a human CMV seropositive subject (a patient that has been previously infected with CMV). For example, Figures 1 to 6 demonstrate that the gdT cell populations and gdT cell responses described herein associate with CMV infection and immunity, and Figure 7 demonstrates that increased expression of IFN-g produced by a cell population comprising gdT cells contacted with anti-NKG2C antibody predicts CMV viremia post transplant and allograft infection post-transplant and is associated with CMV infection risk.

In one aspect the subject is immunocompromised. In another aspect the subject is immunocompromised as a result of medication, such as immunosuppressant therapies prescribed to prevent allograft rejection or graft versus host disease (GVHD) in a transplant recipient, especially an allogeneic transplant recipient. Exemplary therapies include Humira, Etanercept, Infliximab, other anti-TNF therapies, and other anti-inflammatory cytokine therapies.

Aspects of the invention relate to gdT cell subsets that are identifiable from expression of molecules including T cell receptor chains (V61 , V62 etc), molecules such as NKG2C, CD27, CD16, CD45RA, CD107a etc, and/or a cytokine expression profile of the cytokines interferon gamma (IFN-g) and tumour necrosis factor alpha (TNF-a). In some variations, the identifying or determining is by direct measuring of expression of these molecules including cytokines. As used herein a“gdT cell” as used herein refers to a T cell expressing a T cell receptor (TCR) comprising a g (gamma) chain and a d (delta) chain.

Accordingly, a“gdT cell response” as used herein includes change in levels (e.g. proportions) of gdT cells in a subject, for example a change in levels of gdT cells or a population of gdT cells in a sample obtained from subject, relative to the levels of gdT cells or a population of gdT cells in another sample obtained from subject. For example, levels of V61 gdT cells, V62 gdT cells, NKG2C+ V61 gdT cells, NKG2C+ V61 gdT cells, CD45RA+ cells. CD45RA+ CD27^l0W cells, NKG2C+ CD45RA+ cells, NKG2C+ CD45RA+ CD27^l0W cells. A“gdT cell response” as used herein includes change in levels of TNF-a and/or IFN-g in a sample, for example a change in levels of TNF-a and/or IFN-g in a sample obtained from subject, relative to the levels of TNF-a and/or IFN-g another sample obtained from subject. Without wishing to be bound by theory, gdT cells act as a bridge between the innate and adaptive immune systems. gdT cells display broad functional abilities, interacting with both adaptive and innate immune compartments. Compared to abT cells, the repertoire of gd V and J gene segments are restricted, with the gamma (TRG) locus containing 6 functional V segment genes and the delta (TRD) locus containing 8 functional V region genes. Three of these TRD genes are frequently used - V61 , V62 and V63. This is in comparison to ab T cells, which have 52 \/b genes and 70 Va genes. gdT cells have high junctional diversity due to their unique D segment rearranging ability. gdT cells belong to the non-conventional or innate lymphocyte family. They differ from conventional ab T cells, since most of gdT cells do not express the CD4 and CD8 co-receptors and antigen recognition by gd TCR is not restricted to major histo-compatibility complex (MHC) molecules. Thus, while ab TCR interact with peptides bound to MHC class I or class II molecules, gd TCR recognize a diverse array of self and non-self antigens, such as small peptides, soluble or membrane proteins, phospholipids, prenyl pyrophosphates, and sulfatides. Because gd T cell activation does not require antigen processing and presentation by antigen- presenting cells (APC), gdT cells can be rapidly activated and act during the early phase of the immune response. Like natural killer (NK) cells, gdT cells also respond to stimulation by stress- and/or infection-induced ligands.

Different gd TCR subtypes localise to anatomically distinct locations. For example, gdT cells with a /gd /dI TCR are mostly found in skin epidermis, and ng6\/d1 TCR expressing gdT cells are mostly localised in the lung, peritoneum, tongue and reproductive organs. Epithelia contain mostly gdT cells using V61 , V63 or V65 segments. These cells are collectively referred to as V62neg gdT cells’ and recognise various stress related antigens. The majority of peripheral blood gdT cells use the ng9 and V62 chains and mostly recognise phosphoantigens.

In addition to their TCR, gdT cells express receptors in common with natural killer cells, and respond to cell stress markers. They possess the activating NK receptor NKG2D, which engages the markers of cellular stress MICA and MICB, and can also express the activating NK cell receptors CD94-NKG2C and CD16. NKG2C linked with CD94 recognises the non-classical MHC class I molecule HLA-E.

CD16 is a low-affinity receptor for the constant region of IgG, allowing gdT cells to recognise IgG opsonised target cells. They can then produce interferon gamma (IFN- g) without any activation through their TCR (15), leading to release of cytolytic contents and death of target cells. In the context of transplantation, this response may be advantageous by enhancing immunity to CMV. Through both recognition of ligands by the TCR and/or the activating receptors, gdT cells are able to produce a range of inflammatory cytokines (IFN-g, TNF-a, IL-17) and directly lyse infected or transformed cells using perforin and granzymes.

Importantly, the “gdT cell response” is used as an indicator or biomarker e.g., predictive, diagnostic, and/or prognostic, which can be detected in a sample.

In one embodiment, the evaluation of risk for viral infection (e.g. CMV viremia, allograft infection) or risk of graft rejection, GVFID etc, comprises identification and quantification of subclasses of gdT cells from a sample. The subclasses are identifiable by biological markers expressed by the cells. In some variations, the biological markers to be evaluated are protein markers. In some variations, the markers are mRNAs expressed in the cells that encode the protein markers. Generally speaking, a marker-specific detection agent is used to detect each marker of interest. For mRNAs, oligonucleotide probes can be used for detection. For proteins, antibodies are exemplary marker-specific agents. For antibody detection, both polyclonal and monoclonal antibodies are suitable, with monoclonal providing a more consistent or reproducible result. Antigen binding fragments of antibodies are equivalent to whole antibodies for the purposes described herein. Marker-specific agents that comprise a detectable label are specifically contemplated.

As described herein the production of interferon gamma (IFN-g) and/or tumour necrosis factor alpha (TNF-a) and/or the surface expression of CD107a by a population of gdT cells can be used to identify and quantify a gdT cell response that are informative of a subject's risk for virus reactivation, viremia, allograft infection, graft rejection, graft survival, cytolytic activity etc. For example, TNF-a and/or IFN-g production by a cell population comprising gdT cells following contacting the cells with an NKG2C activating agent, such as an anti-NKG2C antibody, is informative of a subject's risk for virus reactivation, viremia, allograft infection, graft rejection, graft survival etc. CD107 expression or target cell death is informative of a cytotoxic (e.g. cytolytic) T cell response. In another embodiment, TNF-a and/or IFN-g production by a cell population comprising gdT in addition to NK, CD4+ T cells and or CD8+ cells following contacting the cells with an NKG2C activating agent, such as an anti- NKG2C antibody, is informative of a subject's risk for virus reactivation, viremia, allograft infection, graft rejection, graft survival etc. CD107 expression or target cell death is informative of a cytotoxic (e.g. cytolytic) T cell response.

Accordingly, a gdT cell response may serve as an indicator of a particular subtype of a disease or disorder (e.g., CMV reactivation, graft rejection) characterized by certain, molecular, pathological, histological, and/or clinical features. In some embodiments, the gdT cell response is expression of, or a level of expression of, or a change in expression of a protein or a gene. For example, a change in the level of expression of a protein (e.g. in or on a cell, or secretion from a cell), or a change in proportion of cells in a population of cells that express a protein, or a change in the level of expression of a protein and a change in proportion of cells in a population of cells that expressing a protein etc.

The terms “level of expression” or “expression level” in general are used interchangeably and generally refer to the amount of a biomarker such as a gdT cell response in a sample. “Expression” generally refers to the process by which information (e.g., gene-encoded and/or epigenetic) is converted into the structures present and operating in the cell. Therefore, as used herein,“expression” may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and/or polypeptide modifications (e.g., posttranslational modification of a polypeptide). Fragments of the transcribed polynucleotide, the translated polypeptide, or polynucleotide and/or polypeptide modifications (e.g., posttranslational modification of a polypeptide) shall also be regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a post-translational processing of the polypeptide, e.g., by proteolysis. “Expressed genes” include those that are transcribed into a polynucleotide as mRNA and then translated into a polypeptide, and also those that are transcribed into RNA but not translated into a polypeptide (for example, transfer and ribosomal RNAs).

“Increased expression,”“increased expression levels,” or“elevated levels” refers to an increased expression or increased levels of a gdT cell response (e.g. an increase in a cell population, and/or increased expression of TNF-a and/or IFN-y) in a sample or an individual relative to a control, such as an individual or individuals who are not an organ transplant recipient and/or suffering from the disease or disorder (e.g., CMV infection or CMV reactivation), or internal control (e.g., a control response). “Decreased expression,”“decreased expression levels,” or“reduced levels” refers to a decrease in expression or decreased levels gdT cell response (e.g. an increase in a cell population, and/or increased expression of TNF-a and/or IFN-g) in a sample or an individual relative to a control, such as an individual or individuals who are not an organ transplant recipient and/or not suffering from the disease or disorder (e.g., CMV infection or CMV reactivation) or an internal control (e.g., a control response). In some embodiments, decreased expression is little or no expression, as is discussed in further detail below.

In one embodiment, gdT cells of an informative phenotype(s) are quantified. In another embodiment, an absolute quantity is determined. In a further embodiment, a relative quantity is determined. Relative quantity is expressed relative to a suitable denominator. For instance, gdT cells of a phenotype of interest can be expressed as a total percentage of gdT cells, or a total percentage of T cells.

As described herein, gdT cells that are V61 positive, NKG2C positive, and/or TEMRA positive (CD45RA+ CD27-), and the gdT cell responses herein are an informative phenotype and/or response insofar as modified (e.g. increased) quantities of the relevant cells and/or responses correlate with decreased CMV infection (e.g. reactivation), viraemia, and/or allograft infection etc. in transplant patients. Accordingly, the gdT cell responses described herein can be used to stratify patients. Importantly, because a number of the gdT cell responses described herein include increased numbers (e.g. an increased proportion) of cells that correlate with a response to CMV infection (e.g. CMV viremia, allograft infection etc), uses of the gdT cells described herein is specifically contemplated, as will be discussed below.

As used herein the term “TEMRA+” is used to denote gdT cells with an effector memory TEMRA phenotype (CD45RA+ CD27-, also referred to herein as CD45RA+ CD27IOW).

In one embodiment, the gdT cell response is a post-transplant increase in gdT cells as a proportion of T cells.

In one embodiment, of the methods the gdT cell response is a V61 gdT cell response. In another embodiment, the gdT cell response is an increase in ndΐgdϊ cells as a proportion of gdT cells. In a further embodiment, the gdT cell response is a post transplant increase in /dIgdT cells as a proportion of gdT cells. In a further embodiment, the gdT cell response is a NKG2C+ gdT cell response. In a further embodiment, the gdT cell response is an increase in NKG2C+ gdT cells as a proportion of gdT cells. In a further embodiment, the gdT cell response is a post transplant increase in NKG2C+ gdT cells as a proportion of gdT cells. In a further embodiment, the gdT cell response is NKG2C activation. In a further embodiment, the gdT cell response is an increase in NKG2C activation. In a further embodiment, the gdT cell response is a post-transplant increase in NKG2C activation. In a further embodiment, the gdT cell response is TNF-a and/or IFN-g production by a cell population comprising NKG2C+ gdT cells. In a further embodiment, the gdT cell response is an increase in TNF-a and/or IFN-g production by a cell population comprising NKG2C+ gdT cells. In a further embodiment, the gdT cell response is a post-transplant increase in TNF-a and/or IFN-g production by a cell population comprising NKG2C+ gdT cells. In a further embodiment, the gdT cell response further comprises a V62 gdT cell response. In a further embodiment, the V62 gdT cell response is a decrease in V62 gdT cells as a proportion of gdT cells. In a further embodiment, the V62 gdT cell response is a post-transplant decrease in V62 gdT cells as a proportion of gdT cells. In a further embodiment, the gdT cell response is a Vd1+NKG2C+ TEMRA+ (e.g. CD45RA+ CD27-) gdT cell response. In a further embodiment, the gdT cell response is an increase in Vd1+NKG2C+ TEMRA+ (e.g. CD45RA+ CD27-) gdT cells as a proportion of gdT cells. In a further embodiment, the gdT cell response is a post-transplant increase in Vd1+NKG2C+ TEMRA+ (e.g. CD45RA+ CD27-) gdT cells as a proportion of gdT cells.

As described herein, in one embodiment, a gdT cell response as described herein can be used as a marker for increased risk of CMV infection, increased risk of graft rejection, decreased prospect of graft survival, CMV reactivation, CMV viraemia, and/or CMV allograft infection. gdT cells that are Vb1+, NKG2C+, and/or TEMRA+ are an informative phenotype when increased quantities of these cells are present in a subject, since increased quantities of these cells in a patient are associated with CMV infection (e.g. CMV reactivation). Accordingly, greater proportions of these cells are associated with a decreased risk of CMV infection, such as a decreased risk of CMV infection post transplant. For example, an increased proportion of these cells in a patient pre- transplant are associated with less CMV replication post-transplant. Increased proportions of these cells are seen during CMV replication, indicating their endeavour to control the virus.

In another embodiment, the gdT cell response refers to one or more gdT cell response (including to one or more biomarker other biomarkers) whose expression is an indicator, e.g., predictive, diagnostic, and/or prognostic.

Increased expression of TNF-a and/or IFN-g by a cell population comprising gdT cells contacted with an NKG2C activating agent (such as an anti-NKG2C antibody or a fragment thereof, or a ligand able to engage NKG2C) are an informative phenotype when increased quantities of TNF-a and/or IFN-g present in a sample, since increased quantities of TNF-a and/or IFN-g associate with CMV viremia post transplant and allograft infection post-transplant.

Increased expression of TNF-a and/or IFN-g by a cell population comprising gdT cells also comprising NK cells, CD4+ T cells and/or CD8+ T cells contacted with an NKG2C activating agent (such as an anti-NKG2C antibody or a fragment thereof, or a ligand able to engage NKG2C) are an informative phenotype when increased quantities of TNF-a and/or IFN-g present in a sample, since increased quantities of TNF-a and/or IFN-g associate with CMV viremia post-transplant and allograft infection post transplant.

Expression of TNF-a and/or IFN-g by a cell population comprising gdT cells is also used as an indicator or biomarker e.g., predictive, diagnostic, and/or prognostic, which can be detected in a sample.

In another embodiment, the expression of TNF-a and/or IFN-g by a cell population comprising gdT cells includes expression of TNF-a and/or IFN-g by one more NK cells, CD4+ T cells and/or CD8+ T cells, and which is also used as an indicator or biomarker e.g., predictive, diagnostic, and/or prognostic, which can be detected in a sample.

In one embodiment, the“amount” or“level” of expression of a gdT cell response or a biomarker associated with an increased clinical benefit to an individual is a detectable level of the gdT cell response and/or the biomarker in a biological sample. These can be measured by methods known to one skilled in the art and also disclosed herein. The expression level or amount of biomarker assessed can be used, for example, to determine the response to a treatment.

In another embodiment, the“amount” or“level” of expression of a gdT cell response or a biomarker associated with an increased clinical benefit to an individual is an absence of a detectable level of the gdT cell response or the biomarker in a biological sample. These can be measured by methods known to one skilled in the art and also disclosed herein. The expression level or amount of biomarker assessed can be used to determine the response to a treatment.

A recommendation as used herein refers to indicating a level or risk, to inform decisions and/or to inform proposing a course of action, such as commencing, maintaining, increasing, decreasing or ceasing a course of action, including monitoring and/or treatment.

In one embodiment, the present invention provides a method of determining if a transplant recipient is at increased risk of graft rejection post-transplant, the method comprising: a) determining the presence of a gdT cell response in a sample from the subject, wherein the presence of a gdT cell response in the sample indicates that the subject has an increased risk of graft rejection post-transplant. In another embodiment, the method further comprises b) providing a recommendation that the subject will have an increased risk of graft rejection post-transplant.

In one embodiment, the present invention provides a method of determining if a transplant recipient has a decreased prospect of graft survival post-transplant, the method comprising: a) determining the presence of a gdT cell response in a sample from the subject, wherein the presence of a gdT cell response in the sample indicates that the subject has a decreased prospect of graft survival post-transplant. In another embodiment, the method further comprises b) providing a recommendation that the subject will have a decreased prospect of graft survival post-transplant.

In one embodiment, the subject is a transplant recipient. The transplant recipient can be an organ transplant recipient, a tissue transplant recipient, or a cell transplant recipient. In a preferred embodiment, the subject is a lung transplant recipient. In a preferred embodiment, the subject is a kidney transplant recipient. The transplant recipient can be diagnosed with cancer, tissue injury and/or an infection by a pathogen. The methods and compositions of this invention can be used to monitor and/or enhance engraftment of transplanted tissue and/or treat CMV infection.

The present inventors have demonstrated that the gdT cell responses described herein can be determined in samples obtained from subject’s pre and/or post transplantation and be used to determine a level of risk of CMV infection, graft rejection, graft survival.

The methods described herein can also be used to guide and modulate therapeutic intervention. For example, antiviral therapy is initiated more quickly in a subject determined to be at higher risk for CMV reactivation, graft rejection etc, according to the methods described herein. For instance, therapy might be initiated at a lower absolute viral load measurement or more quickly upon detection of an upward trend in viral load measurements for subjects stratified in a high risk group based on a gdT cell response as described herein.

In some variations, the invention further comprises administering, to a subject stratified as having high risk of CMV reactivation, graft rejection etc according to the techniques described herein, a prophylaxis that comprises an antiviral agent or an antiviral cellular therapy, including those described herein.

In some variations, the prophylactic course of treatment is the same dose and duration as the therapeutic course of treatment.

In one embodiment, wherein the subject is a transplant recipient, a sample is obtained from the subject pre-transplantation. In one embodiment, a sample is obtained from the subject post-transplantation. In another embodiment, a sample is obtained from a subject pre-post-transplantation.

Accordingly, in one embodiment, the present invention provides a method described herein wherein the gdT cell response in the sample from obtained from the subject post-transplantation is compared to a gdT cell response in a sample obtained from the subject pre-transplantation. In one embodiment, the present invention provides a method described herein wherein the gdT cell response in the sample from obtained from the subject post transplantation is compared to a threshold level of a gdT cell response.

The present inventors have demonstrated that the gdT cell responses described herein are associated with CMV replication, in lung transplant recipients. In immunocompromised lung transplant recipients CMV infection is a major cause of morbidity and mortality. Lung transplants have a much lower long term survival rate compared to other solid organ transplants, with a 50% survival rate at 5 years compared to heart, kidney and liver transplants, which reach the same rate at about 10 years. Lung transplant survival continues to deteriorate over the long term - at 1 year, survival is at about 78%, with following rates of 62% at 3 years, 50% at 5 years, and 26% at 10 years.

CMV is the predominant opportunistic infection in lung transplant patients, and CMV replication damages the graft via direct manifestations of disease such as pneumonitis, but is more importantly associated with indirect long-term effects such as chronic lung allograft dysfunction (CLAD). CLAD is the major factor limiting long-term survival of lung transplants, and presents as multiple phenotypes: bronchiolitis obliterans syndrome, restrictive allograft syndrome and neutrophilic reversible allograft dysfunction. CLAD is due to recurrent alloimmune and infectious insults to the graft, and results in transplant failure and death, with CMV being the most important infectious insult.

Accordingly, the ability to determine a risk of CMV replication in a subject using a gdT cell response as described herein, allows for the monitoring, treatment and/or prevention of CMV infection in a subject, to decrease the morbidity and mortality caused by CMV infection, for example in lung transplant recipients. For example, the methods and compositions described herein can be used for identifying if a subject has an increased risk of CMV replication which will increase the risk of graft rejection post-transplant and decrease graft survival post-transplant

A frequent complication of organ transplantation is recognition of the transplanted organ as foreign by the immune system resulting in rejection. Graft rejection can be detected using biopsy and use of methods such as histology, but other criteria can be used. Other disease criteria correspond to the biopsy results and other criteria, such as the results of organ function tests, presence of CMV, outcome (such as graft failure, re-transplantation, hospitalisation, immunosuppressive intervention). Importantly, the present inventors have demonstrated the gdT cell responses described herein associate with the disease state of the graft.

In one aspect the present invention provides a method of decreasing graft rejection post-transplant in a subject, the method comprising: a) determining the presence of a gdT cell response in a sample from the subject, wherein the presence of a gdT cell response in the sample indicates that the subject has an increased risk of graft rejection post-transplant; and b) administering to a subject with an increased risk of graft rejection post-transplant a therapeutically effective amount of a composition comprising a gdT cell or a population thereof and/or a therapeutically effective amount of an antiviral agent.

In another aspect the method of increasing graft survival post-transplant in a subject, the method comprising: a) determining the presence of a gdT cell response in a sample from the subject, wherein the presence of a gdT cell response in the sample indicates that the subject has an increased risk of CMV reactivation post-transplant; and b) administering to a subject with a decreased risk of graft survival post-transplant a therapeutically effective amount of a composition comprising a gdT cell or a population thereof and/or a therapeutically effective amount of an antiviral agent.

In another aspect the method treating and/or preventing CMV infection in a subject, the method comprising: a) determining the presence of a gdT cell response in a sample from the subject, wherein the presence of a gdT cell response in the sample indicates that the subject has an increased risk of CMV infection post-transplant; and b) administering to a subject with an increased risk of CMV infection post-transplant a therapeutically effective amount of a composition comprising a gdT cell or a population thereof and/or a therapeutically effective amount of an antiviral agent.

As used herein the term “treatment” includes therapeutic treatment as well as prophylactic treatment (either preventing the onset of a disorder or a symptom of a disorder (including CMV infection/replication) altogether or delaying the onset of a symptom of a disorder (including CMV infection and/or GVHD), or a pre-clinically evident stage of a disorder in an individual. The term“prevention” includes either preventing the onset of a disorder or a symptom of a disorder altogether or delaying the onset of disorder or a symptom of a disorder, or a pre-clinically evident stage of a disorder in an individual. This includes prophylactic treatment of those at risk of developing a disease, such as a CMV infection, CLAD and/or GVHD, for example. “Prophylaxis” is another term for prevention.

A composition (including a composition comprising gdT cells) as described herein is typically administered in an effective amount. By the term "effective amount" (for example a“therapeutically effective amount” or a“pharmaceutically effective amount”) as used herein refers to an amount composition that allows an effective response to treatment. Said "effective amount" will vary from subject to subject, depending on the age and general condition of the individual and with the factors such as the particular condition being treated or prevented, the duration of the treatment, previous treatments and the nature and pre-existing duration of the condition. An“effective response” of a patient or a patient's“responsiveness” to treatment with a medicament and similar wording refers to the clinical or therapeutic benefit imparted to a patient at risk for, or suffering from, a disease or disorder, such as CMV infection, CLAD and/or GVHD. In one embodiment, such benefit includes any one or more of: extending survival (including overall survival); resulting in an objective response (including a complete response or a partial response); or improving signs or symptoms of the disease.

The present inventors have demonstrated in Example 1 that the proportion of gdT cells in total T cells is associated with CMV serology. For example, Figure 1 shows that gdT cells increase following lung transplantation in high risk patients with CMV replication. This data demonstrates that the proportion of gdT cells post-transplant can be used as a marker for post-transplant CMV reactivation, and that gdT cells are associated with immune control of CMV.

Accordingly, in one embodiment, the present invention provides a method as described herein wherein the gdT cell response is an increase in gdT cells as a proportion of total lymphocytes. In another embodiment, the present invention provides a method as described herein wherein the gdT cell response is a post-transplant increase in gdT cells as a proportion of total lymphocytes.

The present inventors have demonstrated in Example 2 that the proportion of NKG2C+ gdT cells pre-transplant is associated with pre-transplant CMV serology, and that NKG2C+ve gdT cells increase following lung transplantation in high risk patients with CMV replication, but not in high risk patients without CMV replication. Example 2 also demonstrates that the proportion of NKG2C+ gdT cells post-transplant can be used as a marker for post-transplant CMV reactivation/replication, that the proportion of NKG2C+ gdT cells pre-transplant can be used as a marker for post-transplant CMV reactivation.

Accordingly, in one embodiment, the present invention provides a method as described herein wherein the gdT cell response is a NKG2C+ gdT cell response. In another embodiment, the present invention provides a method as described herein wherein the gdT cell response is an increase in NKG2C+ gdT cells as a proportion of gdT cells. In a further embodiment, the present invention provides a method as described herein wherein the gdT cell response is a post-transplant increase in NKG2C+ gdT cells as a proportion of gdT cells.

The present inventors have demonstrated in Example 3 that Vb1 gdT cells are associated with CMV serology pre-transplant and the proportion of Vb1 gdT cells pre- and/or post-transplant can be used as a marker for post-transplant CMV reactivation. For example, Figure 3 demonstrates that the proportion of Vb1 gdT cells pre transplant is associated with pre-transplant CMV serology, that Vb1 gdT cells increase following lung transplantation in high risk patients with CMV replication, and that the proportion of Vb1 gdT cells post-transplant can be used as a marker for post transplant CMV reactivation.

Accordingly, in one embodiment, the present invention provides a method as described herein wherein the gdT cell response is a Vb1 gdT cell response. In another embodiment, the present invention provides a method as described herein wherein the gdT cell response is an increase in Vb1 gdT cells as a proportion of gdT cells. In a further embodiment, the present invention provides a method as described herein wherein the gdT cell response is a post-transplant increase in V61 gdT cells as a proportion of gdT cells.

Importantly, the present inventors have demonstrated in Example 10 that, unexpectedly, contacting a cell population comprising gdT cells with an antibody that binds NKG2C results in an increase in cytokine production relative to control. In particular, contacting a cell population comprising gdT cells with an antibody that binds NKG2C results in an increase in IFN-g and TNF-a production.

Accordingly, in one embodiment, the present invention provides a method as described herein wherein the gdT cell response is NKG2C activation. In another embodiment, the present invention provides a method as described herein wherein the gdT cell response is an increase in NKG2C activation. In a further embodiment, the present invention provides a method as described herein wherein the gdT cell response is a post-transplant increase in NKG2C activation.

As discussed briefly above, NKG2C, also known as killer cell lectin-like receptor, subfamily C, member 2, is a type II transmembrane protein with extracellular C-type lectin domain. This protein heterodimerizes with CD94 to form a surface receptor that recognizes FILA-E as ligand. The NKG2C/CD94 complex provides an activating signal due to its association with the ITAM-containing DAP12 adapter protein. NKG2C gene and protein sequences are available in published databases (Flomo sapiens NKG2C gene, GenBank accession number AJ001684.1 , Version AJ001684.1 Gl: 2989858, PRI 14-NOV-2006; Protein accession CAA04922, version CAA04922.1 . Gl:2980859, PRI 14-NOV-2006).

Importantly, the present inventors have demonstrated that NKG2C+ gdT cells can be activated using a NKG2C activating agent, such as an antibody that binds to NKG2C, or using FILA-E.

In one embodiment, the FILA-E is a FI LA- E-peptide complex.

For example, the present inventors have demonstrated in Examples 10, 1 1 and 12 that NKG2C+ gdT cells can be activated using an anti-NKG2C antibody, or using FILA-E (e.g. FILA-E on cells), and that activated NKG2C+ gdT cells have increase cytotoxic activity. As used herein the term NKG2C activation includes increasing a function of NKG2C such as association of NKG2C with its binding partners, NKG2C mediated signalling or other function of NKG2C, or a downstream effect of NKG2C activation, such as IFN-g and/or TNF-a production as demonstrated herein, gdT cell proliferation, cytolytic activity by gdT cells etc as demonstrated herein in Example 12.

Gamma delta T cells may exhibit cytolytic phenotypes. The terms cytotoxic and cytolytic are used herein interchangeably. That is, they may target and/or lyse target cells. For example, as discussed above, Example 12 demonstrates that activated NKG2C+ gdT cells have increased cytolytic activity.

Accordingly, in one embodiment, the present invention provides a method as described herein wherein the gdT cell response is TNF-a and/or IFN-g production by a cell population comprising NKG2C+ gdT cells.

In another embodiment, the present invention provides a method as described herein wherein the gdT cell response is TNF-a and/or IFN-g production by a cell population comprising NKG2C+ gdT cells, and wherein the cell population further comprises NK cells, CD4+ T cells and/or CD8+ T cells. gdT cells produced by the methods disclosed herein may produce granzyme A, granzyme B, perforin and/or granulysin. The gdT cells may be able to target and/or lyse target cells. For example, the gamma delta T cells with increased killing described herein may be useful for targeting and/or lysing viral infected cells.

In another embodiment, the present invention provides a method as described herein wherein the gdT cell response is an increase in TNF-a and/or IFN-g production by a cell population comprising NKG2C+ gdT cells.

In another embodiment, the present invention provides a method as described herein wherein the gdT cell response is an increase in TNF-a and/or IFN-g production by a cell population comprising NKG2C+ gdT cells, and wherein the cell population further comprises NK cells, CD4+ T cells and/or CD8+ T cells. In a further embodiment, the present invention provides a method as described herein wherein the gdT cell response is a post-transplant increase in TNF-a and/or IFN-g production by a cell population comprising NKG2C+ gdT cells.

In a further embodiment, the present invention provides a method as described herein wherein the gdT cell response is a post-transplant increase in TNF-a and/or IFN-g production by a cell population comprising NKG2C+ gdT cells, and wherein the cell population further comprises NK cells, CD4+ T cells and/or CD8+ T cells.

In a further embodiment, the present invention provides a method as described herein wherein the gdT cell response is an increase in cytotoxic activity of NKG2C+ gdT cells.

In a further embodiment, the present invention provides a method as described herein wherein the gdT cell response is an increase in cytotoxic NKG2C+ gdT cells.

The present inventors have demonstrated in Example 5 that V62 gdT cells are associated with CMV serology pre-transplant, the proportion of V62 gdT cells pre- and/or post-transplant can be used as a marker for post-transplant CMV reactivation. For example, Figure 5 demonstrates that the proportion of V62 + gdT cells decreased post-transplant, while the proportion of Vb1+ gdT cells increased post-transplant. Figure 6 demonstrates that the proportion of V62 gdT cells pre-transplant is associated with pre-transplant CMV serology and that the proportion of V62 gdT cells post-transplant can be used as a marker for post-transplant CMV reactivation to determine if a patient is at moderate risk of CMV reactivation or at high risk of CMV reactivation post-transplant.

Accordingly, in one embodiment, the present invention provides a method as described herein wherein the gdT cell response is a V62 gdT cell response. In another embodiment, the present invention provides a method as described herein wherein the gdT cell response is a decrease in V62 gdT cells as a proportion of gdT cells. In a further embodiment, the present invention provides a method as described herein wherein the gdT cell response is a post-transplant decrease in V62 gdT cells as a proportion of gdT cells The present inventors have demonstrated in Example 4 that CD27- CD45RA+ gdT cells are associated with CMV serology pre-transplant and the proportion of CD27- CD45RA+ gdT cells (e.g. in combination with NKG2C+ and/or nd1+ etc) pre and/or post-transplant can be used as a marker for post-transplant CMV reactivation. For example, Figure 4 demonstrates that the proportion of CD27- CD45RA+ gdT cells pre transplant is associated with pre-transplant CMV serology, and that the proportion of CD27- CD45RA+ gdT cells post-transplant can be used as a marker for post transplant CMV reactivation.

Accordingly, in one embodiment, the present invention provides a method as described herein wherein the gdT cell response is a V61 + NKG2C+ TEMRA+ (e.g. CD45RA+ CD27-) gdT cell response. In another embodiment, the present invention provides a method as described herein wherein the gdT cell response is an increase in Vd1+NKG2C+ TEMRA+ (e.g. CD45RA+ CD27-) gdT cells as a proportion of gdT cells. In a further embodiment, the present invention provides a method as described herein wherein the gdT cell response is a post-transplant increase in Vd1+NKG2C+ TEMRA+ (e.g. CD45RA+ CD27-) gdT cells as a proportion of gdT cells.

As discussed above, in one embodiment, the present invention provides a method described herein wherein CMV reactivation is CMV infection of the graft. In another embodiment, the present invention provides a method described herein wherein the CMV reactivation is CMV viraemia in the subject.

In another embodiment, the CMV infection is in a non-transplant subject.

Methods of screening blood or plasma samples from subjects for viral load are known See, e.g., Kraft et al., "Interpreting Quantitative Cytomegalovirus DNA Testing: Understanding the Laboratory Perspective." Clinical Infections Diseases, 54(12) 1793- 97 (2012).

As discussed above, in one embodiment, the present invention provides a method described herein further comprising administering to the subject an effective amount of an antiviral agent.

In one embodiment, the antiviral agent is selected from the group consisting of ganciclovir (GCV), valganciclovir (VGCV), foscarnet (FOS), and cidofovir (CDV). The present inventors have demonstrated in Example 12 that activated NKG2C+ gdT cells have increased cytotoxic activity. In Example 13, the present inventors have demonstrated increased cell killing of CMV infected cells by activated NKG2C+ gdT cells.

Pitard et al. 2008 (Long-term expansion of effector/memory Vdelta2-gammadelta T cells is a specific blood signature of CMV infection. Blood, 1 12, 1317-24) demonstrated that an increased level of effector-memory V62- gd T cells (which includes Vb1 gd T cells) in the peripheral blood is a specific signature of an adaptive immune response to CMV infection of both immunocompetent and immunosuppressed patients, and that V62- gd T cells from CMV+ donors expressed CD2107a - a cytotoxicity marker - in comparison to cells from CMV- donors when incubated with CMV infected cells. It is also known that T cell responses are increased in transplant recipients that have low CMV reactivation.

Therefore, the gdT cells described herein are suitable for use in the treatment and/or prophylaxis of CMV infection, graft infection, CLAD, GVHD, for increasing graft survival, decreasing graft rejection and other therapeutic interventions that would benefit from the use of gdT cells.

Accordingly, in one embodiment, the present invention provides a method as described herein further comprising administering to the subject a therapeutically effective amount of gdT cells, such as the gdT cells described herein.

The present inventors have demonstrated in Example 14 and Figure 1 1 that V61 + NKG2C+ gd T cells can be expanded ex vivo.

Accordingly, in one embodiment, a method of producing a cell composition of the present embodiments is provided. The method may comprise obtaining a sample of cells comprising a gdT-cell population; and culturing the gdT cell population in vitro/ex- vivo. In some aspects, the culturing may occur ex vivo for a limited period of time in order to expand the gdT cell population.

For example, methods of producing gdT cells or populations thereof may comprise stimulating a cell or a population of cells with a gdT cell stimulating agent. As used herein, a‘gdT cell stimulating agent’ refers to any agent which selectively stimulates the proliferation and/or survival of gdT cells from a mixed starting population of cells. Thus, the resulting cell population is enriched with an increased number of gdT cells— for example particular gdT cells expressing a particular gd TCR receptor— compared with the starting population of cells.

The gd T cell stimulating agent may be used in combination with a general T cell mitogen, for example a mitogenic cytokine such as IL-2, IL-7, IL-15 or combination of these.

Without wishing to be bound by theory, the present inventors propose that V61+ gd T cells can be expanded from peripheral blood using artificial antigen presenting cells, T cell mitogens such concanavalin A (ConA), anti-CD3 antibody, enrichment by cell separation techniques, or culturing in the presence of IL-2 and IL-7.

It has been demonstrated previously that IL-7 can drive the expansion of gd T cells. Surprisingly, the present inventors have demonstrated that IL-7 can drive the expansion of NKG2C+ nd1 + gd T cells. It has also been demonstrated previously that purified CD56+ populations, containing NK and T cells, divide in response to IL-15. Surprisingly, the present inventors have demonstrated that IL-15 can drive the expansion of NKG2C+ V61+ gd T cells.

In particular, the present inventors have demonstrated in Example 14 that IL-2, IL-7 and IL-15 can be used to expand V61+ NKG2C+ gd T cells.

As used herein, "interleukin 2" or "IL-2" refers to human IL-2 and functional equivalents thereof. Functional equivalents of IL-2 include relevant substructures or fusion proteins of IL-2 that remain the functions of IL-2.

As used herein, "interleukin 15" or "IL-15" refers to human IL-15 and functional equivalents thereof. Functional equivalents of IL-7 include relevant substructures or fusion proteins of IL-15 that remain the functions of IL-15. As used herein, "interleukin 7" or "IL-7" refers to human IL-7 and functional equivalents thereof. Functional equivalents of IL-7 include relevant substructures or fusion proteins of IL-7 that remain the functions of IL-7.

Alternatively, other cytokines can be used in addition to a combination of IL-2, IL-15 and/or IL-7 to promote expansion of V61 + NKG2C+ gd T cells.

In one embodiment, the method comprises obtaining a sample of cells comprising a gd T cell population; and culturing the sample comprising gd T cell population in the presence of IL-2, and culturing cells derived from the sample cultured in the presence of IL-2 in the presence of IL-15 and/or IL-7. Optionally, the cells cultured in the presence of IL-15 and/or IL-7 are contacted with an additional molecule able to engage NKG2C, for example an anti-NKG2C antibody or a fragment thereof, FILA-E, or a functional equivalent.

In some embodiments, the culture conditions (also referred to herein as expansion conditions) include incubation of the sample, or cells ultimately derived from the sample, in culture medium with a T cell mitogen (such as IL-2, IL-15 and/or IL-7) for 1 hour, 2 hours, 5 hours, 10 hours, 12 hours, 15 hours, 20 hours, or days such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 25, 30, 35, 40, 45,50, 55 or 60 or more days or weeks such as about 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks.

In certain aspects, the population of cells with an expanded gdT cell population (e.g. an expanded nd1+ NKG2C+ gd T cell population), obtained by the methods provided herein, contains 10%, 15%, 20%, 25%, 26.4%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 68.7%, 70%, 75%, 80%, 85%, 90%, 95% or more V61+ NKG2C+ gd T cells.

Additional methods of stimulating gdT cells are known in art and include, for example, the use of Concanavalin A (Siegers, G. M. et al. PLoS ONE 6, e16700 (201 1 )), anti-gd TCR antibodies immobilized on plastic; engineered artificial antigen presenting cells as feeders and engineered artificial antigen presenting cells coated in anti-ybTCR antibody (Fisher, J. et al.; Clin. Cancer Res. (2014)), or using anti-NKG2C antibodies or HLA-E expressing cells, as demonstrated herein.

In one embodiment an NKG2C antibody is not used to stimulate stimulating gdT cells. The gdT cell stimulating agent may be isopentenyl pyrophosphate (IPP); an analog of IPP (e.g. bromohydrin pyrophosphate or (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate); an inhibitor of farnesyl pyrophosphate synthase (FPPS) or aminobisphosphonates such as zoledronate or pamidronate, for example. gdT cells or populations thereof produced in accordance with the present invention may be enriched with gd T cells, including those described herein.

In one aspect, the sample of cells may be a peripheral blood sample or an umbilical cord blood sample. In another aspect, the sample of cells may be obtained from tissues such as the lung. In another aspect, the sample of cells may be obtained from a single subject. The subject may be a donor or a patient. In some aspects, gdT cells generated from a single donor may be infused into one or more allogeneic recipients. In one aspect, the gdT cells may be human gdT cells.

In some aspects, purification of the initial population of gdT cells prior to culturing may comprise isolation/enrichment, such as with paramagnetic bead selection or flow cytometry methods as described herein, for example to purify or enrich for NKG2C+, V61+, TEMRA+ (e.g. CD45RA+ CD27-) and/or CD27^IOW Y6T cells. Such selection may comprise depleting the sample of certain cell types. The purity of the relevant gdT cells may be based on the presence of markers that are bound by a monoclonal antibody specific for one or more markers.

In one embodiment, a method of producing a cell composition of the present embodiments is provided. The method may comprise obtaining a sample of cells comprising a gd T cell population; and culturing gd T cell population in the presence of at least one anti-CD3 antibody clone, such as, for example, OKT3 and/or UCHT1 , and/or at least one anti-NKG2C antibody or a fragment thereof, and/or a ligand able to engage NKG2C and/or HLA-E expressing targets, and/or further in the presence of cytokines. In some aspects, the anti-CD3 antibody clone may be expressed on the surface of a cell. In other aspects, the anti-CD3 antibody clone may be on the surface of a microbead.

In another embodiment, the method may comprise obtaining a sample of cells comprising a gd T cell population; and culturing gd T cell population in the presence of IL-2, IL-7 and/or IL-15, and/or at least one anti-NKG2C antibody or a fragment thereof, and/or a ligand able to engage NKG2C and/or HLA-E expressing targets, and optionally further in the presence of cytokines.

The present inventors have demonstrated in Example 14 that HLA-E can be used to expand V61 + NKG2C+ gd T cells.

In some aspects, the culturing may occur ex vivo for a limited period of time in order to expand the NKG2C+ gd T-cell population.

In another aspect, the sample of cells may be obtained from a single subject. The subject may be a donor or a patient. In one aspect, the NKG2C+, gdT cells may be human NKG2C+ gd T cells. In some aspects, the NKG2C+ gdT cells may be derived from stem cells, such as embryonic stem cells, hematopoietic stem cells, or induced pluripotent stem cells.

In one aspect, the disease may be CMV infection.

In one aspect, the cell composition may be allogeneic to the patient. In various aspects, an allogeneic cell composition may or may not share HLA with the patient. In another aspect, the cell composition may be autologous to the patient.

Accordingly, in one embodiment, the present invention provides a method of treating and/or preventing CMV infection in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of gdT cells. In another embodiment, the present invention provides a method of treating and/or preventing CMV reactivation in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of gdT cells. In another embodiment, the present invention provides a method increasing graft survival in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of gdT cells.

In one embodiment, the gdT cell is a Vb1 gdT cell or a population thereof. In another embodiment, the gdT cell is a NKG2C+ gdT cell or a population thereof. In another embodiment, the gdT cell is a V61+ NKG2C+ TEMRA+ (e.g. CD45RA+ CD27-) gdT cell or a population thereof. In one embodiment, the gdT cell or a population thereof is derived from a gdT cell or a population thereof obtained from a subject seropositive for CMV.

In another embodiment, the subject administered a therapeutically effective amount a composition comprising a gdT cell or a population thereof is not the subject seropositive for CMV (e.g. the cell composition is autologous to the patient). In one embodiment, the composition comprises a V61 + gdT cell or a population thereof. In another embodiment, the composition comprises a NKG2C+ gdT cell or a population thereof. In another embodiment, the composition comprises a V61 + NKG2C+TEMRA+ (e.g. CD45RA+ CD27-) gdT cell or a population thereof. In another embodiment, the composition comprises a V61+ NKG2C+TEMRA+ (e.g. CD45RA+ CD27-) CD16- gdT cell or a population thereof.

In another embodiment, the composition comprises a V61+ NKG2C+ TEMRA+ (e.g. CD45RA+ CD27-) CD16+ gdT cell or a population thereof.

As is demonstrated in the Examples, the present inventors have prepared gdT cell populations, including gdT cell populations associated with resistance to CMV infection, activated gdT cell populations (e.g. gdT cell populations contacted with anti- NKG2C antibody or a fragment thereof, or a ligand able to engage NKG2C or HLA-E or HLA-E/G complex), and gdT cell populations with cytotoxic activity.

Accordingly, in one embodiment, the present invention provides a composition comprising a gdT cell or a population thereof and a pharmaceutically acceptable excipient.

The pharmaceutically acceptable excipient can be a pharmaceutically acceptable carrier, diluent or excipient, and the composition can comprise optionally one or more further pharmaceutically active polypeptides and/or compounds. Such a formulation may, for example, be in a form suitable for intravenous infusion.

A composition of the present invention may be administered in combination with a gdT cell stimulating agent.‘In combination’ may refer to administration of the additional therapy or gd T cell stimulating agent before, at the same time as or after administration of the composition according to the present invention. As discussed above, the gdT cell or a population thereof according to the present invention may either be created ex vivo either from a subject's own peripheral blood or from another subject.

In another embodiment, the present invention provides a gdT cell or a population thereof obtained from a subject seropositive for CMV and a pharmaceutically acceptable excipient.

The gdT cell or a population thereof may be derived from ex-vivo differentiation of inducible progenitor cells or embryonic progenitor cells to T-cells.

In another embodiment, the present invention provides a composition as described herein wherein the composition comprises a nd1 gdT cell or a population thereof.

In another embodiment, the present invention provides a composition as described herein wherein the composition comprises a NKG2C+ gdT cell or a population thereof.

In another embodiment, the present invention provides a composition as described herein wherein the composition comprises a V61+ NKG2C+TEMRA+ (e.g. CD45RA+ CD27-) gdT cell or a population thereof.

In another embodiment, the present invention provides a composition as described herein wherein the composition comprises a V61+ NKG2C+TEMRA+ (e.g. CD45RA+ CD27-) CD16- gdT cell or a population thereof.

In another embodiment, the present invention provides a use of a composition described herein in the manufacture of a medicament for treating and/or preventing CMV infection or CMV reactivation.

In another embodiment, the present invention provides a method of activating a NKG2C+ gdT cell or a population thereof, the method comprising contacting a NKG2C+ gdT cell or a population thereof with an antibody or fragment thereof that binds to NKG2C. In another embodiment, the present invention provides a method of activating a NKG2C+ gdT cell or a population thereof, the method comprising contacting a NKG2C+ gdT cell or a population thereof with a ligand that binds to NKG2C. In one embodiment, the ligand is HLA-E. In another embodiment, the present invention provides a method of activating a NKG2C+ gdT cell or a population thereof, the method comprising contacting a NKG2C+ gdT cell or a population thereof with an antibody that binds to NKG2C. In another embodiment, the present invention provides a method of producing an activated NKG2C+ gdT cell or a population thereof, the method comprising contacting a NKG2C+ gdT cell or a population thereof with an antibody that binds to NKG2C. In a further embodiment, the present invention provides a use of an antibody or fragment thereof that specifically binds to NKG2C for the manufacture of a composition for activating a NKG2C+ gdT cell or a population thereof.

Anti-NKG2C antibodies are known in the art. As used herein the term "anti-NKG2C antibody or a fragment thereof" refers to any immunoglobulin or fragment thereof able to bind NKG2C. It includes monoclonal and polyclonal antibodies. The term "fragment thereof" encompasses any part of an antibody having the size and conformation suitable to bind an epitope of NKG2C. Suitable fragments include F(ab), F(ab') and Fv. An "epitope" is the part of the antigen being recognized by the immune system (B- cells, T-cells or antibodies).

As discussed above, the present inventors have demonstrated that IFN-g and TNF-a responses to anti-NKG2C stimulation are associated with CMV infection, and CMV immunity. Accordingly, in one embodiment, the present invention provides the methods and systems described herein, wherein cytokine responses are determined following contacting a cell or a cell population, such as cells in whole blood, with an NKG2C activating agent.

There are two commercially available CMV immune monitoring assays. The QuantiFERON-CMV assay (Qiagen, Germantown, USA) measures the strength of the cell-mediated immune response to CMV, specifically from CD8+ T cells following ex- vivo stimulation with various T-cell CMV epitopes. A study of kidney transplant patients showed that a negative QuantiFERON-CMV result is a better predictor of post-prophylaxis CMV development than pre-transplant CMV serostatus, however few studies have elucidated the assay’s utility in lung transplant recipients. The present inventors have demonstrated in Example 7 that QuantiFERON-CMV does not predict protective immunity against CMV infection in the serum or lung allograft. The second commercial assay is T-Track-CMV (Lophius Biosciences GmbH, Regensburg, Germany) which is an ELISPOT assay using whole CMV antigens IE-1 and pp65. A single centre study of lung transplant recipients has shown that ELISPOT non-responders have earlier onset and longer duration of CMV infection and in a cohort study of kidney transplant patients, ELISPOT has been shown to be better than the QuantiFERON assay at stratifying CMV disease risk. The present inventors have demonstrated in Example 8 that T-Track-CMV (ELISPOT assay) analysis predicts immunity against high level CMV viraemia, but does not predict patients who experienced any detectable virus in blood, or CMV replication in the lung allograft.

As discussed above, the present inventors have characterised gdT cells responses described herein that can be used to monitor CMV reactivation and immunity, including CD94/NKG2C expressed on gd T cells. Importantly, the present inventors have demonstrated in Examples 9 and 10 that NKG2C functional assays predict CMV immunity.

In one embodiment, the present invention provides a method of measuring a gdT cell response, the method comprising; a) contacting a sample from a subject, with an NKG2C activating agent, wherein the sample from the subject comprises a gdT cell or a population thereof; and b). measuring a gdT cell response. In another embodiment, the present invention provides a method for determining if a subject is at increased risk of CMV infection, the method comprising: a) contacting a sample from a subject, with an NKG2C activating agent, wherein the sample from the subject comprises a gdT cell or a population thereof; and b). measuring a gdT cell response.

As used herein the term NKG2C activating agent refers to a molecule that can activate NKG2C:“activation” and“activate” include increasing a function of NKG2C such as association of NKG2C with its binding partners, NKG2C mediated signalling or other function of NKG2C, or a downstream effect of NKG2C activation, such as IFN-g and/or TNF-a production as demonstrated herein, gdT cell proliferation, cytolytic activity by gdT cells etc as demonstrated herein in Example 12. The present inventors have demonstrated that HLA-E can be used in the methods and systems described herein as an NKG2C activating agent.

Accordingly, in another embodiment, the present invention provides method as described herein wherein the NKG2C activating agent is an anti-NKG2C antibody or a fragment thereof. In another embodiment, the present invention provides a method as described herein wherein the NKG2C activating agent is an HLA-E-peptide complex. In another embodiment, the present invention provides a method as described herein wherein the NKG2C activating agent is a ligand able to engage NKG2C.

In another embodiment, the present invention provides a method as described herein wherein the gdT cell response is TNF-a and/or IFN-g expression.

In another embodiment, the present invention provides a method as described herein wherein the gdT cell response is intracellular TNF-a and/or intracellular IFN-g expression. For example, in one embodiment, the sample from the subject comprising a gdT cell or a population thereof is permeabilised.

In another embodiment, the present invention provides a method as described herein wherein the gdT cell response is an increase in expression of TNF-a and/or IFN-g. In another embodiment, the present invention provides a method as described herein wherein the gdT cell response is an increase in expression of TNF-a and/or IFN-g relative to a control.

In another embodiment, the present invention provides a method as described herein wherein the presence of a gdT cell response the sample indicates that the subject has an increased risk of CMV infection.

In one embodiment, the sample comprising a gdT cell or a population thereof further comprises a NK cell or a population thereof, a CD4+ T cell or a population thereof and/or a CD8+ T cell or a population thereof. In one embodiment, the sample is whole blood, blood derived cells, or a sample comprising peripheral blood mononuclear cells.

In another embodiment, the present invention provides a method as described herein further comprising c). measuring a NK cell, CD4+ T cell and/or CD8+ T cell response.

For example, a method of measuring a gdT cell response, the method comprising; a) contacting a sample from a subject, with an NKG2C activating agent, wherein the sample from the subject comprises a gdT cell or a population thereof; and b). measuring a gdT cell response is performed on a sample comprising peripheral blood mononuclear cells, the method further comprises the step of c). measuring a NK cell, CD4+ T cell and/or CD8+ T cell response following contacting the sample with the NKG2C activating agent.

In a preferred embodiment, the NK cell, CD4+ T cell and/or CD8+ T cell response is intracellular TNF-a and/or intracellular IFN-g expression. For example, the sample from the subject is permeabilised.

In another embodiment, the present invention provides a method as described herein wherein the NK cell, CD4+ T cell and/or CD8+ T cell response is an increase in expression of TNF-a and/or IFN-g. In another embodiment, the present invention provides a method as described herein wherein the NK cell, CD4+ T cell and/or CD8+ T cell response is an increase in expression of TNF-a and/or IFN-g relative to a control.

In another embodiment, the present invention provides a method as described herein wherein the presence of a gdT cell response in combination with a NK cell, CD4+ T cell and/or CD8+ T cell response in the sample indicates that the subject has an increased risk of CMV infection.

In another aspect, the present invention provides kits and systems for performing the methods described herein.

The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

EXAMPLES

EXAMPLE 1 : gdT cells are associated with CMV serology pre-transplant and the proportion of gdT cells post-transplant can be used as a marker for posttransplant CMV reactivation.

Ethics

All patients gave written informed consent. The study was approved by The University of Melbourne Human Research Ethics Committee, The Alfred Hospital Ethics Committee, and the Australian Red Cross Blood Service.

Participants

Healthy donors: Australian Red Cross Blood Service by Ficoll-Paque (GE Healthcare, Sydney, NSW, Australia) supplied peripheral blood mononuclear cells (PBMC). These were isolated from buffy packs of healthy donors supplied by the density gradient. They were then cryopreserved in 90% FCS/10% DMSO until analysis.

Clinical Cohort: Adult patients recruited to this study received a bilateral lung transplant between March 2014 to October 2016 at The Alfred Hospital. Lung transplant recipients enrolled in the study did not need to make any additional trips to the Alfred Hospital nor undertake any additional investigations, as collection of the research samples was at bronchoscopies that coincided with routine blood collections.

All patients were given the standard triple immunosuppressant regimen (prednisolone, tacrolimus and azathioprine or mycophenolate). Patients that had a positive donor- or recipient-CMV serostatus at the time of transplant received antiviral prophylaxis for 5 months. This consisted of 2 weeks intravenous ganciclovir (5g/kg body weight), followed by 450 mg/kg body weight oral valganciclovir for another 4 months. As per protocol, CMV hyper-immune immunoglobulin (1 .5 million units) was given at day 1 , 2, 3, 7, 14, 21 and 28. Surveillance bronchoscopies were performed at 2 and 6 weeks and 3, 6, 9 and 12-months post-transplant. Peripheral blood was collected at each surveillance bronchoscopy. CMV reactivation was detected by COBAS Amplicor CMV monitor test (Roche Diagnostic Systems, NSW, Australia). Our final clinical cohort comprised of 31 lung transplant recipients with serial blood samples obtained from pre-transplant to 18-months post-transplant. Sample processing

PBMC were isolated from the whole blood of lung transplant recipients using Ficoll- Paque. They were cryopreserved in 90% FCS 10% DMSO for subsequent analysis by flow cytometry. PBMC from healthy controls were isolated and cryopreserved as per transplant samples.

Procedure

Longitudinal samples from a single recipient were analysed on the same day.

Samples were rapidly thawed in 1 mL warmed complete Roswell Park Memorial Institute (RPMI) media (RF10). Samples were then centrifuged at 1800rpm/4 degrees/5mins. They were then topped up to 1.5 mL and the numbers of viable cells were counted by trypan blue exclusion using a haemocytometer. Samples were next centrifuged at previously mentioned settings, supernatant removed and then transferred to a 96 well plate. The 96 well plate was centrifuged and the supernatant again removed. 50uL antibody cocktail (as below) was added to each sample well and incubated on ice for 30 minutes. Cells were then rinsed with 150uL of phosphate buffered saline containing 1% FCS and 5 mM EDTA (FACS wash), centrifuged as previously, supernatant removed and then rinsed again in 200uL FACS wash. Following centrifugation and removal of supernatant, samples were fixed in 100uL cytofix (BD Biosciences), and incubated overnight at 4°C. Before analysis by flow cytometry, samples were centrifuged, supernatant removed, and resuspended in 100uL FACS wash.

Anti-mouse Ig compensation beads (BD Biosciences) were used for compensation flow cytometry analysis using the antibodies below.

Antibodies used in this study included: TCRy/d-FITC (Becton Dickinson), CD314 (NKG2D)-PE (Miltenyi Biotec), TCR V62-VioGreen (Miltenyi Bitotech), CD159a (NKG2A)-PECy7 (Beckman Coulter), TCR V61-APC (Miltenyi Biotech), CD14 APC- Cy7, CD19 APC-Cy7, CD16 (FcyRIII)-BV605 (BD Horizon), CD56 BV421 (Biolegend), CD3 BUV 737 (BD Horizon), CD8 BUV 395, CD45RA PerCP-Cy5.5, CD27 BV785, CD4 BV650, NKG2C (CD159c)-AF700 (R&D Systems In Vitro Technologies). A viability dye (Live/Dead Fixable Near-IR, Thermofisher) was also used for all samples. Flow cytometry analysis was performed using a Becton Dickinson LSRFortessa (NJ, USA). Samples were gated on single, live lymphocytes, followed by gating on CD3+, ydTCR+ and analyzed using FlowJo software (Treestar, San Carlos, USA).

Patients were stratified into 2 groups based on recipient/donor CMV serostatus at the time of transplant: moderate (CMV seropositive (+) recipient) and high (CMV- recipient/CMV+ donor). These groups are summarised in the table below (Table 1 ).

Table 1

Table 1 : The two lung transplant patient groups analysed. Patients were stratified according to risk of CMV reactivation into moderate (CMV R+/D-) or high (CMV R- /D+). Moderate and high risk patients were further separated into those where CMV was detected in the allograft or those without reactivated CMV (moderate risk not reactivated n=6, moderate risk reactivated n = 7, high risk not reactivated n =5, high risk reactivated n = 7).

Medium and high risk patients were further split into CMV reactivation status: patients where there was detection of actively replicating CMV (reactivated) or where it was not detected (not reactivated) following the cessation of antiviral prophylaxis. The gd T cell phenotype was compared between these groups, as well as the group of healthy donors.

Between group comparisons were assessed using ANOVA and Mann-Whitney tests (GraphPad Prism).

To examine the role of gd T cells, the frequency of gdT cells as a percentage of total lymphocytes was compared between healthy donors and moderate and high CMV risk patients. Figure 1 A shows the flow cytometry gating strategy to select for gamma delta T cells (ybTCR+ CD3+ cells) selected from lymphocytes. Figure 1 C shows the proportion of gdT cells in moderate risk patients (CMV +ve recipient) (circles) and high risk patients (CMV +ve donor CMV -ve recipient) (triangles). This data shows that high risk patients (CMV +ve donor CMV -ve recipient) have lower levels of gdT cells than CMV -i-ve recipients. This data demonstrates that the proportion of gdT cells is associated with CMV serology.

Figure 1 D shows proportion of gdT cells gdT cells (% gdT cells) in high risk patients with CMV replication, as determined by CMV PCR, following lung transplant. This data demonstrates that gdT cells increase following lung transplantation in high risk patients with CMV replication. This data demonstrates that the proportion of gdT cells post-transplant can be used as a marker for post-transplant CMV reactivation.

EXAMPLE 2: NKG2C+ gdT cells are associated with CMV serology pretransplant and the proportion of NKG2C+ gdT cells pre- and/or post-transplant can be used as a marker for post-transplant CMV reactivation.

To further examine the role of gd T cells, the frequency of NKG2C+ gdT cells as a percentage of gdT cells was compared between moderate and high CMV risk patients.

Figure 2A shows the proportion of NKG2C+ gdT cells (%NKG2C+ gdT cells of total gdT cells) in moderate risk patients (CMV -i-ve recipient) (circles) and high risk patients (CMV -i-ve donor CMV -ve recipient) (triangles) pre-transplant. This data shows that high risk patients (CMV -i-ve donor CMV -ve recipient) patients have lower levels of NKG2C+ gdT cells than CMV -i-ve recipients pre-transplant. This data demonstrates that the proportion of NKG2C+ gdT cells pre-transplant is associated with pre transplant CMV serology.

Figure 2B shows a flow cytometry plot of moderate and high risk LTx recipients at 2 weeks and 12 months post-LTx in whom CMV active replication was detected.

Figure 2C shows the proportion of NKG2C+ve gdT cells (% of gdT cells) in moderate risk patients (CMV -i-ve recipient;“Mod”) (circles) and high risk patients (CMV+ donor CMV -ve recipient; “High”) (triangles) following lung transplant, stratified for CMV replication post-transplant (“Neg” = no replication,“Pos” = CMV replication). Figure 2D shows the proportion of NKG2C+ gdT cells (% NKG2C+ve gdT cells of total gdT cells) in high risk patients with CMV replication following lung transplant. This data demonstrates that NKG2C+ve gdT cells increase following lung transplantation in high risk patients with CMV replication.

This data demonstrates that NKG2C+ gdT cells increase following lung transplantation in high risk patients with CMV replication. This data demonstrates that the proportion of NKG2C+ gdT cells post-transplant can be used as a marker for post transplant CMV replication and indicates a role in viral control.

Combined with Figure 2A, this data also demonstrates that the proportion of NKG2C+ gdT cells pre-transplant can be used as a marker for post-transplant CMV reactivation, for example, a risk of post-transplant CMV reactivation. In particular, this data demonstrates that the proportion of NKG2C+ gdT cells pre-transplant can be used as a marker for post-transplant CMV reactivation, to determine if a patient is at moderate risk of CMV reactivation or at high risk of CMV reactivation.

EXAMPLE 3: V51 gdT cells are associated with CMV serology pre-transplant and the proportion of V51 gdT cells pre- and/or post-transplant can be used as a marker for post-transplant CMV reactivation.

To further examine the role of gd T cells, the frequency of Vb1 gdT cells as a percentage of gdT cells was compared between healthy donors and moderate and high CMV risk patients.

Figure 3A shows the proportion of Vb1 gdT cells (%Vd1 gdT cells of total gdT cells) in moderate risk patients (CMV -i-ve recipient) (circles) and high risk patients (CMV -i-ve donor CMV -ve recipient) (triangles) pre-transplant. This data shows that high risk patients (CMV -i-ve donor CMV -ve recipient) patients have lower levels of Vb1 gdT cells than CMV -i-ve recipients pre-transplant. This data demonstrates that the proportion of Vb1 gdT cells pre-transplant is associated with pre-transplant CMV serology.

Figure 3C shows proportion of Vb1 gdT cells (%Vd1 gdT cells of total gdT cells) in moderate risk patients (CMV +ve recipient;“Moderate”) (circles) and high risk patients (CMV+ donor CMV -ve recipient;“High”) (triangles) following lung transplant, stratified for CMV replication post-transplant (“Neg” = no replication,“Pos” = CMV replication).

Figure 3D shows proportion of V61 gdT cells (%Vd1 -i-ve gdT cells of total gdT cells) in high risk patients with CMV replication, following lung transplant. This data demonstrates that V61 gdT cells increase following lung transplantation in high risk patients with CMV replication.

This data demonstrates that V61 gdT cells increase following lung transplantation in high risk patients with CMV replication. This data also demonstrates that the proportion of V61 gdT cells post-transplant can be used as a marker for post transplant CMV reactivation.

EXAMPLE 4: CD27- CD45RA+ gdT cells are associated with CMV serology pretransplant and the proportion of CD27- CD45RA+ gdT cells pre and/or posttransplant can be used as a marker for post-transplant CMV reactivation.

To further examine the role of CD27- CD45RA+ gd T cells, the frequency of CD27- CD45RA+ gdT cells as a percentage of gdT cells was compared between healthy donors and moderate and high CMV risk patients.

Figure 4A shows the proportion of CD27- CD45RA+ gdT cells (%CD27- CD45RA+ gdT cells of total gdT cells) in moderate risk patients (CMV -i-ve recipient) (circles) and high risk patients (CMV -i-ve donor CMV -ve recipient) (triangles) pre-transplant. This data shows that high risk patients (CMV -i-ve donor CMV -ve recipient) patients have lower levels of CD27- CD45RA+ gdT cells than CMV -i-ve recipients pre-transplant. This data demonstrates that the proportion of CD27- CD45RA+ gdT cells pre transplant is associated with pre-transplant CMV serology.

Figure 4C shows proportion of CD27- CD45RA+ gdT cells (% CD27- CD45RA+ of gdT cells in moderate risk patients (CMV +ve recipient; “Mod”) (circles) and high risk patients (CMV+ donor CMV -ve recipient;“High”) (triangles) following lung transplant, stratified for CMV replication post-transplant (“No” = no replication, “YES” = CMV replication). Figure 4D shows proportion of TEMRA (CD27low CD45RA+ gdT cells) (%CD27low CD45RA+ gdT cells of total gdT cells) in high risk patients with CMV replication, following lung transplant. This data demonstrates that CD27- CD45RA+ gdT cells increase following lung transplantation in high risk patients with CMV replication.

This data demonstrates that CD27- CD45RA+ gdT cells increase following lung transplantation in high risk patients with CMV replication. This data demonstrates that the proportion of CD27- CD45RA+ gdT cells post-transplant can be used as a marker for post-transplant CMV reactivation.

The present inventors have also demonstrated the V61 proportion remains high and stable in CMV R+ recipients over 18 months post lung transplant in moderate risk patients with and without CMV replication (data not shown).

EXAMPLE 5: V62 gdT cells are associated with CMV serology pre-transplant, the proportion of V52 gdT cells pre- and/or post-transplant can be used as a marker for post-transplant CMV reactivation and V61+ TEMRA+ (e.g. CD45RA+) gdT cells can be used as a marker for post-transplant CMV reactivation.

To further examine the role of V62 + CD45RA+ gd T cells, the frequency of Vb1 +, V62+, CD27-, CD45RA+ and NKG2C+ gdT cells as a percentage of gdT cells was compared between healthy donors and moderate and high CMV risk patients.

Figure 5 shows the proportion of V61 + and V62 + gdT cells as a proportion of gdT cells pre- and 12 months post-transplant in patients with CMV reactivation. The proportion of V62+ gdT cells decreased post-transplant, while the proportion of V61 + gdT cells increased post-transplant. Figure 5 also demonstrates that the proportion of nd1+ gdT cells that are NKG2C+ increased post-transplant, and that the proportion of nd1+ gdT cells that are CD45RA+ CD27- increased post-transplant.

This data demonstrates that V62 gdT cells decrease following lung transplantation in high risk patients with CMV reactivation. This data demonstrates that the proportion of V62 gdT cells post-transplant can be used as a marker for post-transplant CMV reactivation. This data also demonstrates that V61 gdT cells increase following lung transplantation in high risk patients with CMV reactivation. This data demonstrates that the proportion of V61 gdT cells post-transplant can be used as a marker for post-transplant CMV reactivation.

This data also demonstrates that V61 NKG2C+ gdT cells increase following lung transplantation in high risk patients with CMV reactivation. This data demonstrates that the proportion of V61 NKG2C+ gdT post-transplant can be used as a marker for post-transplant CMV reactivation.

This data also demonstrates that Vb1 CD45RA+ CD27- gdT cells increase following lung transplantation in high risk patients with CMV reactivation. This data demonstrates that the proportion of Vb1 CD45RA+ CD27- gdT post-transplant can be used as a marker for post-transplant CMV reactivation.

To further examine the role of gd2 T cells, the frequency of V62 gdT cells as a percentage of gdT cells was compared between healthy donors and moderate and high CMV risk patients.

Figure 6 shows proportion of V62 gdT cells (%V62 gdT cells of total gdT cells) in moderate risk patients (CMV +ve recipient;“R+”) (black, left hand side) and high risk patients (CMV+ donor CMV -ve recipient;“D+/R-”) (grey, right hand side) following lung transplant (“TX”).

This data demonstrates that the proportion of V62 gdT cells pre-transplant is associated with pre-transplant CMV serology.

This data demonstrates that V62 gdT cells decrease following lung transplantation in high risk patients. This data demonstrates that the proportion of V62 gdT cells post transplant can be used as a marker for post-transplant CMV reactivation. EXAMPLE 6: Comparison of assays using gdT cells contacted with anti-NKG2C antibody with commercial diagnostic methods

METHODS

Ethics

All patients gave written informed consent. The study was approved by The University of Melbourne Human Research Ethics Committee (Project 1953932), The Alfred Hospital Ethics Committee (Project 401/13), and the Australian Red Cross Blood Service (Material Supply Deed 18-03VIC-21 ).

Study Population

Our study population consisted of 18 adult lung transplant recipients from Alfred Hospital who in 2014-2015 participated in a randomised study comparing standard five-month antiviral prophylaxis to QuantiFERON-CMV directed antiviral prophylaxis and consented to samples being used for additional research. Lung transplant recipients excluded from the study included recipients who were deemed low risk for CMV infection (D-/R-). Recipients were chosen based on their QuantiFERON-CMV results: 6 CMV R+ recipients who were QuantiFERON-CMV positive, 6 D+/R- recipients who were QuantiFERON-CMV negative and 6 R+ recipients who were either QuantiFERON-CMV negative or indeterminant. Each participant had at least 1 vial of cryopreserved peripheral blood mononuclear cells (PBMC) for future analysis.

Detection of CMV Infection

Prior to lung transplant, participants’ CMV serology as determined by ELISA to CMV IgG. Serum and BAL samples were tested for actively replicating virus by quantitative PCR using the Cobas Ampliprep/Cobas Taqman assay (Roche), which has a lower limit of detection of 150 copies/ml. Patient serum samples were tested every month from the time of study inclusion for 6 months and then a further 2 tests at 15 months and 18 months. BAL samples were analysed for CMV replication at 6, 9, 12 and 18 post-transplant. Additional samples were collected for quantitative CMV PCR if the patient developed clinical features of CMV infection.

QuantiFERON-CMV Assay

The results of the QuantiFERON-CMV assay were obtained from the randomised study performed at the Alfred. The QuantiFERON-CMV assay uses whole blood samples collected in 3 specialised tubes containing phytohemagglutinin (PHA) as the mitogen or positive control, heparin as a negative control and CMV CD8+ T-cell synthetic epitopes respectively. Following overnight incubation at 37°, IFN- levels in the plasma is measured using enzyme-linked immunosorbent assay (ELISA). The manufacturer has set a value of ³0.2 lU/ml as a positive result. If the mitogen control is less than 0.5 lU/ml, the result is considered indeterminate (ID). A lack of response to the mitogen control is considered ID by the manufacturer and has been demonstrated to occur early in the post-transplant period due to strong immunosuppression (20). Therefore, in this study ID results were grouped with negative assay results.

Preparation of Frozen Peripheral Blood Mononuclear Cells (PBMCs)

PBMCs were retrieved from liquid nitrogen storage, thawed and cultured overnight at 37°C/5% CO2 in media containing RPMI supplemented with 10% human AB serum and 50U/ml recombinant human IL-2.

T-Track-CMV (ELISPOT) Assay

PBMCs, following overnight culture, were adjusted to a dilution of 200,000 cells per 100pL and added to the 96-well ELISPOT plate provided in the T-Track-CMV kit (Lophius Biosciences, Regensburg, Germany). The experiment was completed in duplicate with a media only negative control, stimulation with IE-1 and pp65 whole antigen, a PHA positive control and an operator control containing IFN-y bound to capture antibodies. The plate was incubated for 17-21 hours at 37°C/5% CO2, washed, incubated with alkaline phosphatase monoclonal antibody followed by staining substrate for 6-7 minutes. The spots were counted using the AID ELISPOT reader and the results interpreted by the provided T-Track-CMV calculator.

Monoclonal Antibodies (mAbs) and Flow Cytometry Analysis

All antibodies were sourced from BD Biosciences unless otherwise indicated. The following mAbs were used for surface marker detection via flow cytometry: anti-CD8 BUV395, anti-CD4 BV650, anti-ydTCR FITC, anti-CD56 BV421 , anti-CD16 BV605, anti-CD3 BUV, anti-NKG2A PE-Cy7 (Beckman Coulter), anti-NKG2C Alexa700 (R & D Systems), CD107a-PE and CD107a PECy5. Monocytes, macrophages and B cells, as well as dead cells were excluded by using a dump channel, with the following mAbs: anti-CD14 APC-Cy7, anti-CD19 APC-Cy7, Live/Dead fixable near infra-red (ThermoFisher). For the intracellular cytokine staining, anti-IFN-g PerCP Cy5.5 and TNF-a APC was used. Data was acquired on a BD LSRFortessa flow cytometer (BD Biosciences) and analysed using FlowJo Software (TreeStar, OR, USA).

Anti-NKG2C Based Assay with Intracellular Cytokine Staining

A 96 well plate was prepared as follows and repeated for every patient sample being analysed. 50mI_ of phosphate buffered saline (PBS) (negative control) and 50mI_ each of anti-NKG2A mAb (clone Z199, Beckman Coulter), anti-NKG2C mAb (clone 134522, R & D systems) or anti-CD3 mAb (clone OKT3, generated in-house) at 10pg/ml were added to separate wells and the plate was incubated overnight at 4°C. PBMCs were adjusted to a dilution of 250,000 cells per 100mI_ and added to the plate. PMA/ionomycin stimulation served as a positive control. The plate was incubated for one hour at 37°C/5% CO2 before 1 mI_ each of GolgiStop™ and GolgiPlug™ were added to allow for intracellular accumulation of cytokines. The plate was then incubated for 15 hours overnight at 37°C/5% CO2, after which the cells were fixed, permeabilised, stained for intracellular cytokines and analysed using flow cytometry.

Statistical Analysis

Descriptive statistics were used such as median, mean and standard deviation depending on the data distribution. Proportions or counts were used to describe categorical variables. The student’s t-test was used for group comparisons. Kaplan- Meier survival curve analysis was used to show CMV infection incidence over the follow-up period and the log-rank test was used to determine whether the difference between curves was significant. Statistical analysis was performed using GraphPad Prism8 software.

Patient Characteristics

18 adult lung transplant recipients were selected from a 2014-2015 randomised study conducted at Alfred Hospital comparing outcomes following QuantiFERON-CMV directed and standard care antiviral treatment. Their baseline characteristics are shown in Table 2. Out of the 18 patients, 8 received standard care 5 months antiviral prophylaxis and 10 received QuantiFERON-directed care (Table 2). Of the 10 who received QuantiFERON-directed care, 8 had a negative or indeterminate QuantiFERON result at 5 months post-transplant and received extended antiviral prophylaxis. Table 2 nts 70)

Sex, no., male/female 13/5

QuantiFERON-CMV Directed Care 10 (56)

QFN-CMV assay, median days post-transplant

(range)

Antivirals ceased, median days post-transplant

(range) 185.5 (147-363)

Antivirals ceased at 5 months post-transplant

T-Track-CMV (ELISPOT), median days posttransplant (range) 187 (91 -217)

Anti-NKG2C assay, median days post-transplant

CMV viraemia >600 copies/ml

CMV in BAL >600 copies/ml 7 (38) Cytomegalovirus Outcomes Post-Transplant

No patients developed detectable CMV viral load in the serum or BAL while on antiviral prophylaxis. 12/18 (67%) patients developed detectable CMV viraemia with 6/18 (33%) developing viraemia with CMV viral load >600 copies/ml (Table 2). 1 1/18 (61%) patients developed CMV infection in the lung allograft with 7/18 (38%) developing CMV >600 copies/ml in the BAL (Table 2). 5/6 D+/R- patients developed high level viraemia compared to only 1/12 R+ patients.

EXAMPLE 7: QuantiFERON-CMV does not predict protective immunity against CMV infection in the serum or lung allograft.

QuantiFERON-CMV data of the 18 patients obtained as part of the 2014-2015 randomised control study was analysed. The QuantiFERON-CMV assay results were obtained at a median of 154 days post-transplant (Table 2)..

The QuantiFERON-CMV assay was not able to predict development of CMV viraemia in the 18-month post-transplant period. There was no difference when stratifying by incidence of any viraemia (data not shown) or higher level viraemia (data not shown). Of those who were QuantiFERON-CMV negative/ID, 8 received extended antiviral prophylaxis which may have reduced the incidence of CMV viraemia in that group. Accordingly, recipients receiving extended prophylaxis were excluded from further analysis.

When recipients with on standard prophylaxis and for which a sample was available prior to any detection of CMV were examined, the QuantiFERON-CMV assay was also not able to predict development of CMV viraemia in the 18-month post-transplant period (Figure 7A).

EXAMPLE 8: T-Track-CMV (ELISPOT assay) analysis predicts immunity against high level CMV viraemia, but does not predict patients who experienced any detectable virus in blood, or CMV replication in the lung allograft.

The ELISPOT assay was performed on thawed, cryopreserved PBMCs collected at a median of 187 days post-transplant (Table 2). One patient’s ELISPOT result was excluded from the analysis due to an episode of resolved viraemia prior to the time of sample collection. The ELISPOT assay failed to predict patients who experienced any detectable CMV replication in the lung allograft.

When recipients with on standard prophylaxis and for which a sample was available prior to any detection of CMV, were examined, the T-Track-CMV (ELISPOT assay) analysis was also not able to predict development of CMV viraemia in the 18-month post-transplant period (Figure 7B).

This data indicates T-Track-CMV (ELISPOT assay) analysis does not predict patients who experienced any detectable CMV replication in the lung allograft.

EXAMPLE 9: The percentage of NKG2C+ NK cells does not predict CMV infection risk

Frozen PBMCs collected at a median of 183 days post-transplant (Table 2) were thawed, cultured and stained for cell surface markers. Flow cytometric analysis was used to find the proportion of NKG2C+ CD3- CD56+ lymphocytes (Figure 8a).

As the aim of this study was to predict the risk of CMV reactivation, three patients’ results were removed from the analysis either because of active disease or resolved episodes of CMV infection prior to sample collection.

No significant difference in the proportion of NKG2C+ NK cells was found based on CMV serostatus (Figure 8b). Similarly, no significant difference could be found in proportion of NKG2C+ NK cells based on development of viraemia in the blood (p=0.41 ) (Figure 8c) nor when separating patients into those who developed low and high level viraemia, although the highest mean %NKG2C+ NK was observed in patients who developed high level viraemia (Figure 8d). Likewise, a significant difference was not found in %NKG2C+ NK cells based on development of CMV in the lung allograft (Figure 8e-f). Flowever, similar to blood, the mean %NKG2C+ NK cells was highest in patients with high level CMV replication in the lung allograft.

This data demonstrates the percentage of NKG2C+ NK cells does not predict CMV infection risk EXAMPLE 10: Increased expression IFN-g produced by gdT cells contacted with anti-NKG2C antibody predicts allograft infection post-transplant.

To examine the ability of gdT cell responses to predict CMV infection and allograft disease, NKG2C functional assays were used, in which gdT cell responses were measured following contacting blood samples from patients with an NKG2C activating agent.

Thawed PBMCs were incubated overnight with an anti-NKG2C monoclonal antibody, stained for cell surface markers, intracellularly stained for IFN-y, and the proportion of live cells producing cytokines was analysed using flow cytometry. When pooling data from all patients, stimulation with anti-NKG2C antibody led to an increase in cytokine production compared to the PBS control Figure 7C shows NKG2C ligation results in increased IFN-g production from NK cells, gd, CD4+ and CD8+ T cells.

When recipients on standard prophylaxis and for whom a sample was available prior to any detection of CMV (and when recipients on extended prophylaxis were excluded) were examined, patients who later developed CMV reactivation had an increased IFN-g cytokine response to anti-NKG2C stimulation. The proportion of cells producing IFN-g following NKG2C stimulation was higher in patients who developed detectable virus in the serum, (Figure 7D).

This data demonstrates that NKG2C functional (e.g IFN-g expression) assays predict allograft infection post-transplant. This data also surprisingly demonstrates that gdT cells contacted with an antibody that binds to NKG2C are activated, and the NKG2C activation leads to IFN-g expression.

EXAMPLE 12: Increased cell killing by activated NKG2C+ gdT cells

To examine the ability of NKG2C+ gdT cells to be activated for use in therapy, the present inventors examined the ability of an antibody that binds to NKG2C to activate NKG2C+ gamma delta T cells, using CD107A as a marker of degranulation as marker of gamma delta T cell functional activity.

Figure 9A shows that an antibody that binds to NKG2C (anti-2C) is able to activate NKG2C+ gamma delta T cells, as measured by an increase in CD107A (a marker of degranulation) expression. This data demonstrates that gdT cells contacted with anti- NKG2C antibody are activated. To examine the ability of NKG2C+ gdT cells to kill cells (e.g. CMV infected cells) for use in therapy, the present inventors examined the ability of the natural ligand for NKG2C (HLA-E) to activate NKG2C+ gamma delta T cells.

In brief, the B cell line 721.221 as a target cell for cytotoxicity assays. This cell line expresses very low levels of HLA Class I in general but also low levels of HLA-E. 721.221 cells are transfected with HLA-G, to HLA-G which contains the peptide ligand for HLA-E (and the cells also express HLA-E at the cell surface). This HLA-E/G complex is a strong activator of NKG2C.

PBMCs from a healthy donor were incubated with 1. No target cells 2. 721.221 wild type cells (low HLA-E), 3. 721 .221 -HLA-G (HLA-E/G), then surface stained for gamma delta TCR, NKG2C and CD107A. CD107A was used as a marker of degranulation as marker of gamma delta T cell functional activity, to indicate cell killing by gamma delta T cells.

Figure 9B shows higher CD107A from NKG2C+ gamma delta T cells when incubated with the 721 -221 -HLA-G cell line. This data indicates that CD107A degranulation/cell killing is increased when NKG2C+ gamma delta T cells are activated with HLA-E, and that CD107A degranulation/cell killing is increased when NKG2C+ gamma delta T cells are activated with HLA-E/G complex.

This data demonstrates that gdT cells contacted with HLA-E are activated, and that NKG2C activation leads to increase cytolytic activity.

EXAMPLE 13: Increased cell killing of CMV infected cells by activated NKG2C+ gdT cells

To examine the ability of NKG2C+ gdT cells to kill CMV cells for use in therapy, the present inventors examined the ability of NKG2C+ gamma delta T cells.

In brief, PBMC from a healthy donor were incubated for 24h in the presence of IL-2 and IL-15 before incubating overnight with uninfected or CMV-infected human foreskin fibroblasts (HFF). Cells were surface stained for gd TCR, V61 , NKG2C and CD107a. CD107A was used as a marker of degranulation as marker of gamma delta T cell functional activity, to indicate cell killing by gamma delta T cells.

Figure 10 shows NKG2C+ gamma delta T cells are activated by CMV infected cells and that CD107A degranulation/cell killing is increased when NKG2C+ gamma delta T cells are activated by CMV infected cells.

This data demonstrates that gdT cells contacted with CMV infected cells are activated, and that NKG2C activation leads to increase cytolytic activity of CMV infected cells.

EXAMPLE 14: In vitro expansion of V61+ NKG2C+ gd T cells

The ability of V61+ NKG2C+ gd T cells to be expanded ex vivo/in vitro was examined.

In brief, PBMC cells were thawed from liquid nitrogen storage and cultured in complete tissue culture medium (RPMI1640 supplemented with non-essential amino acids, HEPES, streptomycin, beta-mecaptoethanol, L-glutamate, 10% human serum) and 10U/ml IL-2 overnight. The next day gdT cells were sorted into corresponding subsets, labelled with celltrace violet proliferation dye (CTV) and cultured for 7 days in complete tissue culture medium at 37C and 5% C02. Additional cytokines, IL-7 and IL-15 were added as indicated at the final concentration of 25ng/ml. Coating of wells with HLA-E protein was performed using 30ug/ml at 37C for 2h, after which wells were washed with PBS before cell culture. PHA was added at the final concentration of 1 ug/ml. Media was changed on day 5 and analysis performed on day 7.

Figure 1 1 shows V61+ NKG2C+ gd T cells can be expanded ex vivo.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method for determining if a subject is at increased risk of CMV infection, the method comprising:

a). determining the presence of a gdT cell response in a sample from the subject, wherein the presence of a gdT cell response in the sample indicates that the subject has an increased risk of CMV infection.

2. A method according to claim 1 wherein the method further comprises b) providing a recommendation that the subject will have an increased risk of CMV infection.

3. A method according to claim 1 or claim 2; wherein the subject is a transplant recipient.

4. A method of determining if a transplant recipient is at increased risk of graft rejection post-transplant, the method comprising:

a). determining the presence of a gdT cell response in a sample from the subject, wherein the presence of a gdT cell response in the sample indicates that the subject has an increased risk of graft rejection post-transplant.

5. A method according to claim 4 wherein the method further comprises b) providing a recommendation that the subject will have an increased risk of graft rejection post-transplant.

6. A method of determining if a transplant recipient has a decreased prospect of graft survival post-transplant, the method comprising:

a). determining the presence of a gdT cell response in a sample from the subject, wherein the presence of a gdT cell response in the sample indicates that the subject has a decreased prospect of graft survival post-transplant.

7. A method according to claim 6 wherein the method further comprises b) providing a recommendation that the subject will have a decreased prospect of graft survival post-transplant.

8. A method according to any one of claims 3 to 7 wherein sample is obtained from the subject post-transplantation.

9. A method according to claim 8 wherein the gdT cell response in the sample from obtained from the subject post-transplantation is compared to a gdT cell response in a sample obtained from the subject pre-transplantation.

10. A method according to claim 8 wherein the gdT cell response in the sample from obtained from the subject post-transplantation is compared to a threshold level of a gdT cell response.

1 1 . A method of decreasing graft rejection post-transplant in a subject, the method comprising:

a) determining the presence of a gdT cell response in a sample from the subject, wherein the presence of a gdT cell response in the sample indicates that the subject has an increased risk of graft rejection post transplant; and

b) administering to a subject with an increased risk of graft rejection post transplant a therapeutically effective amount of a composition comprising a gdT cell or a population thereof and/or a therapeutically effective amount of an antiviral agent.

12. A method of increasing graft survival post-transplant in a subject, the method comprising:

a) determining the presence of a gdT cell response in a sample from the subject, wherein the presence of a gdT cell response in the sample indicates that the subject has an increased risk of CMV reactivation post transplant; and

b) administering to a subject with a decreased risk of graft survival post transplant a therapeutically effective amount of a composition comprising a gdT cell or a population thereof and/or a therapeutically effective amount of an antiviral agent.

13. A method treating and/or preventing CMV infection in a subject, the method comprising: a) determining the presence of a gdT cell response in a sample from the subject, wherein the presence of a gdT cell response in the sample indicates that the subject has an increased risk of CMV infection post transplant; and

b) administering to a subject with an increased risk of CMV infection post transplant a therapeutically effective amount of a composition comprising a gdT cell or a population thereof and/or a therapeutically effective amount of an antiviral agent.

14. A method according to any one of claims 1 to 13 wherein the gdT cell response is an increase in gdT cells as a proportion of T cells.

15. A method according to any one of claims 1 to 14 wherein the gdT cell response is a post-transplant increase in gdT cells as a proportion of T cells.

16. A method according to any one of claims 1 to 15 wherein the gdT cell response is a V61 gdT cell response.

17. A method according to any one of claims 1 to 16 wherein the gdT cell response is an increase in V61 gdT cells as a proportion of gdT cells.

18. A method according to any one of claims 1 to 17 wherein the gdT cell response is a post-transplant increase in V61 gdT cells as a proportion of gdT cells.

19. A method according to any one of claims 1 to 18 wherein the gdT cell response is a NKG2C+ gdT cell response.

20. A method according to any one of claims 1 to 19 wherein the gdT cell response is an increase in NKG2C+ gdT cells as a proportion of gdT cells.

21 . A method according to any one of claims 1 to 20 wherein the gdT cell response is a post-transplant increase in NKG2C+ gdT cells as a proportion of gdT cells.

22. A method according to any one of claims 1 to 21 wherein the gdT cell response is NKG2C activation.

23. A method according to any one of claims 1 to 22 wherein the gdT cell response is an increase in NKG2C activation.

24. A method according to any one of claims 1 to 23 wherein the gdT cell response is a post-transplant increase in NKG2C activation

25. A method according to any one of claims 1 to 24 wherein the gdT cell response is TNF-a and/or IFN-y production by a NKG2C+ gdT cells

26. A method according to any one of claims 1 to 35 wherein the gdT cell response is an increase in TNF-a and/or IFN-g production by NKG2C+ gdT cells

27. A method according to any one of claims 1 to 26 wherein the gdT cell response is a post-transplant increase in TNF-a and/or IFN-g production by NKG2C+ gdT cells

28. A method according to any one of claims 1 to 27 wherein the gdT cell response further comprises a V62 gdT cell response.

29. A method according to any one of claims 1 to 28 wherein the V62 gdT cell response is a decrease in V62 gdT cells as a proportion of gdT cells.

30. A method according to any one of claims 1 to 29 wherein the V62 gdT cell response is a post-transplant decrease in V62 gdT cells as a proportion of gdT cells.

31 . A method according to any one of claims 1 to 31 wherein the gdT cell response is a Vd1+NKG2C+ CD45RA+ CD27-ybT cell response.

32. A method according to any one of claims 1 to 32 wherein the gdT cell response is an increase in Vd1+NKG2C+ CD45RA+ CD27- gdT cells as a proportion of gdT cells.

33. A method according to any one of claims 1 to 33 wherein the gdT cell response is a post-transplant increase in Vd1+NKG2C+ CD45RA+ CD27- gdT cells as a proportion of gdT cells.

34. A method according to any one of claims 1 to 33 wherein the sample is a blood, whole blood or a sample comprising blood-derived cells.

35. A method according to any one of claims 1 to 34 wherein the subject is a transplant recipient.

36. A method according to any one of claims 1 to 35 wherein the subject is a lung transplant recipient.

37. A method according to any one of claims 1 , 2, 3, or 14 to 37 wherein the CMV infection is CMV reactivation.

38. A method according to any one of claims 1 to 37 wherein the CMV infection is CMV viraemia in the blood and/or CMV infection of the graft.

39. A method according to any one of claims 1 to 38 further comprising administering to the subject an effective amount of an antiviral agent.

40. A method according to claim 39 wherein the antiviral agent is selected from the group consisting of ganciclovir, valganciclovir, foscarnet, and cidofovir.

41 . A method according to any one of claims 1 to 40 further comprising administering to the subject a therapeutically effective amount of a composition comprising a gdT cell or a population thereof.

42. A method of treating and/or preventing CMV infection in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising a gdT cell or a population thereof.

43. A method of treating and/or preventing CMV reactivation in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising a gdT cell or a population thereof.

44. A method of increasing graft survival in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising a gdT cell or a population thereof.

45. A method according to any one of claims 39 to 44, wherein the gdT cell is a V61 gdT cell or a population thereof.

46. A method according to any one of claims 39 to 45, wherein the gdT cell is a NKG2C+ gdT cell or a population thereof.

47. A method according to any one of claims 39 to 46, wherein the gdT cell is a V61+ NKG2C+ CD45RA+ CD27- gdT cell or a population thereof.

48. A method according to any one of claims 39 to 47, wherein the gdT cell or a population thereof is derived from a gdT cell or a population thereof obtained from a subject seropositive for CMV.

49. A method according to claim 48 wherein the subject administered a therapeutically effective amount a composition comprising a gdT cell or a population thereof is not the subject seropositive for CMV.

50. A method according to any one of claims 39 to 49 wherein the composition comprises a V61 gdT cell or a population thereof.

51 . A method according to any one of claims 39 to 50 wherein the composition comprises a NKG2C+ gdT cell or a population thereof.

52. A method according to any one of claims 39 to 51 wherein the composition comprises a V61+ NKG2C+ CD45RA+ CD27- gdT cell or a population thereof.

53. A method according to any one of claims 39 to 52 wherein the composition comprises a V61+ NKG2C+ CD45RA+ CD27- CD16- gdT cell or a population thereof.

54. A composition comprising a gdT cell or a population thereof and a pharmaceutically acceptable excipient.

55. A composition comprising a gdT cell or a population thereof obtained from a subject seropositive for CMV and a pharmaceutically acceptable excipient.

56. A composition according to claim 54 or 55 wherein the composition comprises a V61 gdT cell or a population thereof.

57. A composition according to any one of claims 54 to 56 wherein the composition comprises a NKG2C+ gdT cell or a population thereof.

58. A composition according to any one of claims 54 to 57 wherein the composition comprises a V61+ NKG2C+ CD45RA+ CD27- gdT cell or a population thereof.

59. A composition according to any one of claims 54 to 58 wherein the composition comprises a V61+ NKG2C+ CD45RA+ CD27- CD16- gdT cell or a population thereof.

60. A use of a composition according to any one of claims 54 to 59 in the manufacture of a medicament for treating and/or preventing CMV infection or CMV reactivation.

61 . A method of activating a NKG2C+ gdT cell or a population thereof, the method comprising contacting a NKG2C+ gdT cell or a population thereof with an antibody or a fragment thereof that binds to NKG2C.

62. A method of producing an activated NKG2C+ gdT cell or a population thereof, the method comprising contacting a NKG2C+ gdT cell or a population thereof with an antibody or a fragment thereof that binds to NKG2C.

63. Use of an antibody or a fragment thereof that specifically binds to NKG2C for the manufacture of a composition for activating a NKG2C+ gdT cell or a population thereof.

64. A method of measuring a gdT cell response, the method comprising; a) contacting a sample from a subject, with an NKG2C activating agent, wherein the sample from the subject comprises a gdT cell or a population thereof; and

b). measuring a gdT cell response.

65. A method for determining if a subject is at increased risk of CMV infection, the method comprising:

a) contacting a sample from a subject, with an NKG2C activating agent, wherein the sample from the subject comprises a gdT cell or a population thereof; and

b). measuring a gdT cell response.

66. A method according to claim 64 or claim 65 wherein the NKG2C activating agent is an anti-NKG2C antibody or a fragment thereof.

67. A method according to any one of claims 64 to 66 wherein the NKG2C activating agent is an HLA-E-peptide complex.

68. A method according to any one of claims 64 to 67 wherein the gdT cell response is TNF-a and/or IFN-g expression.

69. A method according to any one of claims 64 to 68 wherein the gdT cell response is intracellular TNF-a and/or intracellular IFN-g expression.

70. A method according to any one of claims 64 to 69 wherein the gdT cell response is an increase in expression of TNF-a and/or IFN-g.

71 . A method according to any one of claims 64 to 70 wherein the gdT cell response is an increase in expression of TNF-a and/or IFN-g relative to a control.

72. A method according to any one of claims 64 to 71 wherein the presence of a gdT cell response the sample indicates that the subject has an increased risk of CMV infection.