Inhibiting the NADase CD38 improves cytomegalovirus-specific CD8+ T cell functionality and metabolism

Cytomegalovirus (CMV) is one of the most common and relevant opportunistic pathogens in people who are immunocompromised, such as kidney transplant recipients (KTRs). The exact mechanisms underlying the disability of cytotoxic T cells to provide sufficient protection against CMV in people who are immunosuppressed have not been identified yet. Here, we performed in-depth metabolic profiling of CMV-specific CD8+ T cells in patients who are immunocompromised and show the development of metabolic dysregulation at the transcriptional, protein, and functional level of CMV-specific CD8+ T cells in KTRs with noncontrolled CMV infection. These dysregulations comprise impaired glycolysis and increased mitochondrial stress, which is associated with an intensified expression of the nicotinamide adenine dinucleotide nucleotidase (NADase) CD38. Inhibiting NADase activity of CD38 reinvigorated the metabolism and improved cytokine production of CMV-specific CD8+ T cells. These findings were corroborated in a mouse model of CMV infection under conditions of immunosuppression. Thus, dysregulated metabolic states of CD8+ T cells could be targeted by inhibiting CD38 to reverse hyporesponsiveness in individuals who fail to control chronic viral infection.


Kidney transplant recipients and healthy control group
A total of 52 kidney transplant recipients (KTRs) and 13 healthy controls (HC) were included.Main study population: Patients with CMV high-or intermediate-risk category and anti-CMV IgG positive healthy volunteers were included (Supplemental Table 1).All individuals were positive for HLA-A*02 and received at least two doses of mRNA vaccines against SARS-CoV-2 (bnt162b2 or mrna-1273).An interval of at least 14 days to last vaccine dose was required.Individuals with an acute, PCR-proofed SARS-CoV-2-infection were excluded.Further exclusion criteria for patients were: kidney transplantation within last 3 months, a rejection therapy within last 3 months and an eGFR <15ml/min/m 2 (Supplemental Table 2).Most of the patients received triple immunosuppressive therapy with tacrolimus, mycophenolate, and prednisone (Supplemental For the longitudinal analysis referring to Figure 6 and Supplemental Figure 6, a total of 16 KTRs (6 controllers, 10 non-controllers) were studied for 15 months after transplantation.Blood was taken at 6 weeks, 3 months, 6 months, and 12 months after transplantation.PBMCs were isolated and cryopreserved.Samples were thawed and analyzed on one day.The patients were clinically monitored and CMV replication in blood was assessed 10-15 times/patient during study period.All patients were HLA-A*02-positive and at CMV intermediate-risk (controllers n=6; noncontrollers n=7) or high-risk constellation (controllers n=0; non-controllers n=3).
Immunosuppression was equal for all longitudinally analyzed patients: induction therapy with basiliximab; maintenance therapy with tacrolimus, prednisone, and mycophenolate.No rejection therapy occurred during study period.

Individuals with acute SARS-CoV-2 infection
Individuals (n=7) were recruited through a cohort study, with ethical approval number NL77841.058.21.This study has been registered at clinicaltrials.govunder study number: NCT06039527.Individuals aged 18-30 years old, or over 65 years old were included.Baseline samples, including PBMCs, were collected from all participants.Additional PBMCs samples were collected from participants if they experienced symptoms of respiratory infection within 3 months after baseline sampling.These samples were collected within 7 days of symptom onset and participants with a positive result for SARS-CoV-2 (PCR or self-test) were included for this analysis.For the present study frozen PBMCs of n=7 study participants (one sample before and one sample during acute infection/patient) were used.All individuals received SARS-CoV-2 vaccination before first blood sampling.

Sample processing
Blood from patients and healthy donors was drawn at the outpatient clinic of the Department of Nephrology, University Hospital Essen.Blood from patients was drawn before the intake of immunosuppressive medication.Heparinized blood was shipped per overnight-express at room temperature to the Department of Immunology, Leiden University Medical Center.Immediately after arrival (within 24 hours after blood donation), peripheral blood mononuclear cells were isolated by Ficoll-Hypaque (GE Healthcare) density gradient centrifugation and cryopreserved in liquid nitrogen until the day of analysis.For analysis, PBMCs were thawed at 37°C, and washed with Iscove's Modified Dulbecco's Medium (IMDM, Capricorn Scientific or Lonza) supplemented with 8% FCS (Bodinco), 100 U/ml penicillin (Gibco), 100 U/ml streptomycin (Gibco), 2mM Lglutamine (Gibco).Next, PBMCs were rested in IMDM containing 30 µg/ml DNAse I (Merck) at 37°C for 2 hours to recover before subsequent procedures.

Flow cytometry Viability, cell surface and tetramer staining
To assess viability, dead cells were excluded by staining with Zombie NIR (BioLegend) or Live Dead Fixable Blue (ThermoFisher) in PBS for 15 minutes at room temperature.Tetramer staining was performed in staining buffer (PBS + 1% FCS) for 30 minutes at 4 °C.For cell surface staining, cells were incubated with fluorescently-labeled antibodies for 30 minutes at 4 °C.All used antibodies and dead cell exclusion dyes are indicated in Supplemental Table 4.

Intracellular staining for metabolic proteins, transcription factors, and histone methylation
All intracellularly stained metabolic proteins except FDFT1 were self-conjugated using the Lightning-Link antibody conjugation kits (Abcam) according to the manufacturer's protocol (Supplemental Table 4).After viability staining, cell surface and tetramer staining, cells were permeabilized and fixed for 45 minutes using the eBioscience Foxp3/Transcription Factor Staining Buffer Set (Invitrogen) according to manufacturer's instructions.Following two washes in Permeabilization Buffer, antibodies against metabolic proteins, transcription factors or Histone 3 methylated at Lysine 27 (H3K27me3) were added and cells were incubated for 1 hour at 4 °C.Before analysis, cells were washed two more times in Permeabilization Buffer.

Acquisition and analysis
All flow cytometry samples were acquired on a 5-Laser Aurora Cytek spectral analyzer with SpectroFlo acquisition software (version 3, Cytek).Data were analyzed using FlowJo software (TreeStar) and OMIQ analysis software.A representative gating strategy is shown in Figure S1A.High-dimensional analysis was performed by subsampling on equal numbers of antigen-specific CD8 + T cells followed by UMAP and FlowSOM consensus meta-clustering, Wanderlust trajectory, or clustered heatmap using default settings.

Ex vivo T cell stimulation
PBMCs were cultured in the presence of 9-mer CMV peptide pp65495-503 (NLVPMVATV) at a concentration of 10 µg/ml.PBMCs were stimulated with pp65495-503 peptide for either 20 hours (short stimulation) to assess intracellular cytokine production, cytokine secretion and functional metabolic changes (e.g., MTDR and TMRM) or 6 days (long stimulation) to assess changes in metabolic protein expression.Because peptide stimulation elicits TCR down-regulation and tetramer internalization but also activation-induced expression of CD137, we used the latter marker for the detection of antigen-reactive CD8 + T cells (1).After long stimulation, the TCR recirculates, and therefore the pp65495-503-specific CD8 + T cells were identified by MHC class I tetramers.
For intracellular cytokine staining, cells were stimulated with peptide for 20 hours of which the last 19.5 hours in presence of Brefeldin A (BioLegend, 1 µg/ml).Intracellular cytokine staining was performed as described previously (2).As the relative fraction rather than absolute numbers of cytokine producing CMV-specific CD8 + T cells correlates better with clinical course of CMV infection in transplant patients (3), we calculated the ratio between cytokine producing cells after stimulation (from total CD8 + T cells) and pp65495-503 + cells (from total CD8 + T cells) in unstimulated condition (Supplemental Figure 1H).Quantification of cytokine secretion by Legendplex (Biolegend) was performed according to the manufacturer's guidelines.

SCENITH
PBMCs were directly subjected to SCENITH (Figure 3J; Supplemental Figure 3I) or PBMCs were first stimulated with pp65495-503 peptide (NLVPMVATV) for 4 hours followed by SCENITH (Figure 4J; Supplemental Figure 4E).In case of CD38 inhibition, PBMCs were pre-incubated for 16 hours with CD38i or vehicle, and then directly subjected to SCENITH (Supplemental Figure 7E) or first stimulated for 4 hours with pp65495-503 peptide (Figure 4L).SCENITH was performed in accordance with the original SCENITH protocol (4).Briefly, cells underwent a 20minute treatment with either 100 mM 2-DG, 1 µM oligomycin (O), or a sequential combination of both drugs at 37 °C, 5% CO2.Subsequently, puromycin (10 µg/mL) was added to the culture for additional 20 minutes.Afterwards, cells were washed and flow cytometry staining was performed.
Cells were fixed and permeabilized using the eBioscience Foxp3/Transcription Factor Staining Buffer Set (Invitrogen) and stained intracellularly with the monoclonal anti-puromycin antibody for 45 min.Values for glucose dependence, mitochondrial dependence, glycolytic capacity, and FAO and AAO capacity were calculated.

Nanostring nCounter Gene expression analysis
PBMCs were thawed and rested overnight in IMDM.The next day, PBMCs were labelled with Zombie NIR to exclude dead cells, and anti-CD8 mAbs plus pp65495-503 (NLVPMVATV)/tetramers.Subsequently, pp65-specific CD8 + T cells (17000 cells/sample) were sorted on a Cytoflex SRT (Beckman Coulter) and collected in cold IMDM (2% FCS) (Figure S2A).Next, cells were washed three times in PBS and lysed in buffer containing 1 portion of RLT buffer (Qiagen) and 2 portions of UltraPure TM DNAse/RNase-free distilled water (Invitrogen).Lysates were immediately placed on dry ice and subsequently stored at -80 °C until the day of analysis.Samples were analyzed in the NanoString ® nCounter FLEX platform according to manufacturer's instructions.The nCounter ® Metabolic Pathways Panel (768 genes) was used to interrogate the metabolic profile.Briefly, 5 µl of cell lysate was mixed with reporter probes, capture probes and hybridization buffer, and proteinase K (0.45 mg/ml, Thermo Fisher Scientific), and hybridized at 65°C for 17 hours.Samples were then processed on the NanoString Prep station and cartridges were read on the NanoString Digital Analyzer.RNA count data were normalized, scores for different pathways were calculated, automated pathway analysis based on the expression of predefined genes and differential expression analysis were performed using the nSolver TM software (version 4.0) including the advanced analysis package (version 2.0.134).Volcano plot was created with GraphPad Prism (version 8, La Jolla, CA, United States).Heatmaps were created with Morpheus, https://software.broadinstitute.org/morpheus.Protein-protein interaction analysis and pathway elucidation analysis were performed using STRING (5), with a minimum required interaction score of 0.7 (high confidence).
In vivo viral infection and immunosuppressive medication C57BL/6 mice were i.p. infected with murine cytomegalovirus (MCMV-Smith, obtained from the American Type Culture Collection, ATCC VR-194; Manassas, VA) (2×10 4 PFU).Mice receiving immunosuppressive treatment were injected on day 3 post-infection with tacrolimus (3 mg/kg, s.c.) and dexamethasone (1 mg/kg, i.p.), and subsequently every 24 hours until day 7, and then every 48 hours until sacrifice.Mice were weighed 3 times per week, but no weight loss or other signs of discomfort were observed.On day 20 after MCMV infection mice were sacrificed.Single cell suspensions from the spleen were prepared by mincing the tissue through a 70 µm cell strainer.Contaminating erythrocytes were removed by ammonium chloride buffer.Salivary glands were collected from euthanized mice and snap-frozen on dry ice.Samples were stored at -80°C until the viral load was determined by quantitative real-time PCR as described previously (6).
In vivo treatment with CD38i (78c) C57BL/6 mice were infected with MCMV-Smith and received immunosuppressive treatment as described above (until sacrifice).Mice were weighed 3 times per week.On day 20 after MCMV infection the treatment group received 15 mg/kg/dose 78c twice daily i.p. for 7 days as described previously (7).Control mice received vehicle (5% DMSO, 40% PEG300, 5% Tween 80 and 50% NaCl 0.9%).On day 27 after infection mice were sacrificed.Single cell suspensions from the spleen were prepared by mincing the tissue through a 70 µm cell strainer.Contaminating erythrocytes were removed by ammonium chloride buffer.Intracellular cytokine staining was performed after 5 hours stimulation in presence of short M38 peptide (SSPPMFRV) and Brefeldin A as described previously (6).Livers were collected from euthanized mice and snap-frozen on dry ice.Samples were stored at -80°C until the viral load was determined by quantitative real-time PCR as described previously (6).

In vivo viral infection and mass cytometry analysis
Expression data of PD-1, CD38 and CD39 on viral-specific CD8 + T cells elicited by different types of infection (i.e., LCMV-Armstrong, LCMV-clone 13, MCMV-ie2-GP33, MCMV-ie2-E7, MCMV-ie2-FKBP-E7) were derived from our previous study by van der Gracht et al. (8).Briefly, mice were infected with the aforementioned viruses and at day 50 post infection the viral-specific CD8 + T cell responses were characterized by mass cytometry.A at time of blood sampling; B present or in the past; n.a.= not applicable.Numbers are given as mean and range for metric variables and percentage for categorical variables.

Supplemental Figure 1 . 2 .
Frequency and phenotype of CMV-and SARS-CoV-2-specific CD8 + T cells are unaffected by loss of viral control.(A) Representative flow cytometry gating strategy for the detection of MHC class I tetramer positive CD8 + T cells (shown for pp65 495-503 ).(B) After 20 hours stimulation with pp65 495-503 peptide, cytokine production was measured by intracellular cytokine staining.Representative flow cytometry plots from a non-controller.Plots show gated CD8 + T cells in unstimulated and stimulated condition.(C) Following UMAP analysis, FlowSOM consensus meta-clustering with 7 clusters was performed on 200 down-sampled pp65 495-503 and spike 269-277 -specific CD8 + T cells from KTR (n = 24; 15 controller; 9 non-controller).Virus-specific T cell populations (left), overlay of the 7 FlowSOM clusters (right) on the UMAP.(D) Expression intensity of cell surface markers.(E) Hierarchically clustered heatmap of phenotypes of the clusters shown in (C).The indicated marker expression is shown per cluster as z-score of median signal intensity per channel.(F) Cluster frequencies of pp65 495-503 (blue) and spike 269-277 -specific (orange) CD8 + T cells.(G) Percentage IFN-γ + CD137 + of spike 269-277 -specific CD8 + T cells from controllers and non-controllers stimulated for 20 hours with peptide (n=5/group).(H) Blood levels of tacrolimus and mycophenolic acid (MPA) at the date of blood sampling.(I) Percentage of regulatory CD4 + CD25 + FOXP3 + T cells (Tregs) of total CD4 + T cells (n=5/group).Data are presented as mean ± SEM or as box-plot.Bounds of the boxes indicate upper and lower quartile, lines indicate median, whiskers indicate min and max.Each symbol in G-H represents an individual.Statistical analysis by two-sided student's t-test or two-way ANOVA with Sidak's test for multiple comparisons.*p<0.05;**p<0.01;***p<0.001;****p<0.0001.Transcriptional analysis of metabolic pathways in CMV-specific CD8 + T cells.CMV pp65 495-503 -specific CD8 + T cells (n=3 independent donors/group) were sorted from PBMCs and subjected to nCounter Metabolic Pathways.(A) Representative flow cytometry plot of sorting pp65 495-503 + CD8 + T cells.(B) Lysosomal degradation pathway score.Scores are presented as relative expression.(C) Signature scores plots of mitochondrial respiration with glycolysis and fatty acid oxidation, respectively.Scores are presented as relative expression.Data are presented as mean ± SEM.Each symbol represents an individual.Statistical analysis by one-way ANOVA with Tukey's test for multiple comparisons.*p<0.05;**p<0.01;***p<0.001;****p<0.0001.
2-deoxy-d-glucose (2-DG), oligomycin (O) or media controls (Co) (n=4/group).TMRM = Tetramethylrhodamine methyl ester.MTDR = MitoTracker Deep Red.Data are presented as mean ± SEM or as box-plot (bounds of the boxes indicate upper and lower quartile, line indicates median, whiskers indicate min and max).Each symbol represents an individual.Statistical analysis by two-sided student's t-test, paired t-test one-way

Table 1 )
. Kidney transplant recipients were further divided into two groups, based on CMV-PCR results (threshold defined as a result > 65 IU/ml) and clinical data: 1. KTR CMV non-controllers: CMV-replication (> 300 IU/ml blood) without spontaneous clearance by the host and/or symptomatic infection (CMV syndrome or disease) in last 6 months.Requirement of anti-viral treatment to stop viral replication.2. KTR CMV controllers: No CMV-replication or asymptomatic, low level (<300 IU/ml blood) CMV replication in last 6 months.No requirement of anti-viral treatment to stop viral replication. n.a.