Umbilical CB units were obtained under written informed consent of the mothers in accordance with study protocols approved by Hannover Medical School Ethics Review Board (approval Nr. 4837). All experiments involving mice were performed in accordance with the regulations and guidelines of the animal welfare of the State of Lower Saxony (“Niedersächsiches Landesamt für Verbraucherschutz und Lebensmittelsicherheit, Dezernat 33/Tierschutz,” approval Nr. 33.12-42502-04-1). Euthanasia was performed with cervical dislocation after CO₂ narcosis.

MRC-5 (human fetal lung fibroblasts) and HEK-293T cells (ATCC, Manassas, VA, USA) were expanded in DMEM (ThermoFisher, Waltham, MA) supplemented with 10% fetal bovine serum (FBS, HyClone, Logan, UT), non-essential amino acids (ThermoFisher) and penicillin/streptomycin (Merck Millipore, Billerica, MA) at 37°C with 5% CO₂. Mononuclear cells were isolated by Ficoll gradient centrifugation, CD34⁺ cells were purified after two rounds of immune-magnetic bead positive selection (Miltenyi Biotech, Bergisch-Gladbach, Germany).

HCMV/GLuc seeding stocks were generated using a TB40-BAC4-cloned HCMV variant and propagated on MRC-5 cells as described (30). Cell-free supernatants were concentrated by centrifugation at 19,000 rpm for 2 h 20 min, re-suspended in Dulbecco's modified Eagle medium (DMEM, ThermoFisher) plus 1% non-essential amino acids (ThermoFisher) and stored at −80°C. Viral batches were titered in MRC-5 cells by immunofluorescence analyses using the mouse anti-IE1 p63-27 antibody essentially as described (30). For the generation of batches of HCMV-infected carrier cells, 1.0 × 10⁷ MRC-5 cells were infected at a multiplicity of infection (MOI) of 1 overnight at 37°C, the medium was replenished and then the cells were cultivated for additional 2 days. Cells were detached with Trypsin/EDTA (Merck Millipore), centrifuged at 300 g for 10 min, washed with PBS and cryopreserved in freezing medium containing 15.5% human albumin, 10% DMSO (Braun, Melsungen, Germany), and 5% glucose (Sigma-Aldrich, St.Louis, MO). A thawed cell aliquot was stained for detection of gB on the cell surface using the mouse anti-gB p27-287 antibody and a secondary anti-mouse-Alexa647 antibody (Biolegend, San Diego, CA). The frequency of gB-positive cells was determined by flow cytometry using an LSR II cytometer (BD Bioscience, Becton Dickinson GmbH, Heidelberg, Germany).

Cell culture supernatants were collected and frozen at −80°C. Twenty microliter of thawed supernatant were mixed with 50 μl BioLux® Gaussia Luciferase Assay Kit according to the manufacturer's instructions (NEB, Frankfurt am Main, Germany) or with 50 μl 0.2 μg/ml coelenterazine (Promega, Mannheim, Germany). Bio-luminescence (light intensity per second) was measured using an Orion II Microplate Luminometer (Titertek-Berthold, Pforzheim, Germany) or a Tristar² Microplate Reader (Berthold Technologies, Bad Wildbach, Germany).

NRG mice were obtained from The Jackson Laboratory (JAX, Bar Harbor, ME) and bred in-house under pathogen-free conditions. We used only female NRG mice, as we have shown that they supported better T cell development (28) and differentiation after DC immunizations (26). Prior to HSCT, 5 weeks-old mice were sub-lethally irradiated (450 cGy) using a [137^Cs] column irradiator (Gammacell 3000 Elan; Best Theratronics, Ottawa, Canada). Four hours after irradiation, 2.0 × 10⁵ human CB-CD34⁺ cells were injected into mice through the tail vein as described (27, 29).

Humanized mice were generated and analyzed essentially as described (26–29). Frequencies of human immune cell subsets in the PBL samples were determined at 17 weeks after HSCT. Next, mice were treated with 150 ng mouse/day s.c. with hG-CSF (Granocyte, Kohlpharma GmbH, Merzig, Germany) for 5 days. On the third day of hG-CSF administration, 1.0 × 10⁶ cryopreserved/thawed viable HCMV-infected MRC-5 cells were injected i.p. 7 weeks after infection, one cohort was treated with hG-CSF for 7 days (2.5 mg/mouse/day s.c.) to stimulate viral reactivations. After optical imaging analyses and euthanasia, several tissues were collected SPL, lymph nodes (LN), mesenteric lymph nodes (mLN), BM (flushed from two femurs), salivary glands (SG), and LI, dissociated into single cell suspensions and the numbers of viable cells per tissue were quantified by trypan blue exclusion assay.

Mice were anesthetized with Ketamin/Xylazine (10 and 0.5 mg/ml, respectively, Sigma-Aldrich). Coelenterazine (Promega, Madison, WI) was solubilized in ethanol (5 mg/ml) and diluted shortly prior to administration in PBS (final dose 50 μg/mouse). Mice were injected i.v. with 100 μl of coelenterazine solution and imaged immediately. In vivo optical imaging analyses were performed with an IVIS 200 optical imaging system (PerkinElmer, Waltham, MA) or with an IVIS SpectrumCT (PerkinElmer). Different CB stem cell sources were used for R1 and R2. Pictures were taken with a field of view C, f stop 1 and medium binning for each mouse. Exposure time was set to 300 s per mouse. The anatomical region of interest (ROI) was kept constant for quantified analyses of all mice.

Cells obtained from peripheral blood or tissues were treated with a hypotonic solution (0.83% ammonium chloride/20 mM HEPES, pH 7.2, for 5 min at room temperature, followed by stabilization with cold PBS and washing) in order to remove the erythrocytes and further processed as described as fresh or cryopreserved/thawed material for immune staining and flow cytometry analyses (29, 26). The appropriate concentration of each antibody was determined in the titration assay before the main experiment. After the cells were washed to remove unbounded antibodies and immune fluorescence was assessed by LSR II cytometer (BD Biosciences). Commercially available antibodies against all markers are provided in Table S1.

Tissues were processed with the Blood and Tissue isolation kit (Qiagen, Hilden, Germany). DNA concentration was determined by spectrophotometry and stored at −20°C. Twenty microliter of the DNA sample was used for PCR analyses with an Artus® CMV TM PCR kit according to the manufacturer's instructions (Qiagen). The standard curve was determined with primers specific for HCMV sequences and internal control primers against a human gene. PCR was performed and analyzed with StepOnePlus-PCR cycler (ThermoFisher). The Ct values were used to calculate the copies of HCMV genomes per sample adjusted with the respective DNA concentrations. For detection of HCMV in human lymphocyte subsets, cells were sorted using a FACS Aria 2 (BD Bioscience).

Concentrations of human Th1/Th2 cytokines in serum were quantified by fluorescent bead–based 14-plex Luminex assay (Merck Millipore) according to the manufacturer's instructions. Following human cytokines were analyzed: GM-CSF, IL-4, TNF-α, IL-6, IL-8, MCP-1, IL-10, IL-1b, IL-5, IFN-γ, IL-7, IL-2, and IL-12(p70). The human IgG and IgM ELISA kit (eBioscience, San Diego, CA) was used corresponding to instructions. Serum samples separated from peripheral blood were thawed on ice, diluted, and samples were measured in triplicates. Plates were analyzed with Spectramax 340PC384 plate reader (Molecular Devices, Sunnyvale, CA).

Following a fixation period of 24 h in 3.7% formaldehyde buffer (Roth, Karlsruhe, Germany) the spleen was embedded in paraffin. Three micrometer-thick sections were prepared, dewaxed, and subjected to immunohistological staining as described (29). Briefly, heat-induced antigen retrieval was performed in a citrate-buffered solution. All slides were rinsed with Tris-buffered saline (pH 7.6) plus 0.01% Tween®20 (Merck, Darmstadt, Germany). Slides were incubated for 20 min at 21°C in normal goat serum (Vector Laboratories Inc., Burlingame, CA) and then incubated with the primary antibody (anti-human nuclei, CD3 and CD79a) for 1 h at 21°C. Secondary biotin-SP-conjugated antibody was applied for 30 min at 21°C. Final staining was achieved by routine method using alkaline phosphatase streptavidin-biotin (Vector Laboratories Inc., Burlingame, CA) and Fast Red as chromogen (Fast Red substrate pack, BioGenex, Freemont, CA). The slides were counterstained using Bluing Reagent (Thermo Scientific, Braunschweig, Germany). Cover-slipping was done using Aquatex® aqueous mounting medium (Merck KGaA, Darmstadt, Germany). Sample permeabilization, antibody concentrations, antibody reactions, and staining procedures were previously optimized for each antibody (29) to get clear and specific immunohistochemical signals. Slides were elevated using a bright-field microscope. Whole slides were digitized using the Mirax scanner (Zeiss, Oberkochen, Germany) equipped with a 20x plan-apochromat lens (Zeiss) and a camera (Hitachi_HV_F22CL, Hitachi Kokusai Electric Europe, Neu-Isenburg, Germany). Pictures for the figures were taken with the Panoramic Viewer (3dHistech, Budapest, Hungary) as viewing software.

BM samples were thawed from individual mice (Exp. 4, Table 1, CTR = 3, INF = 4, REAC = 4), and mononuclear cells were used directly for IFN-γ ELISpot assay using the T-track human Kit according to the vendor's recommendations (Lophius Bioscience, Regensburg, Germany) (31). PBMCs obtained from HCMV sero-positive and sero-negative donors were used as controls. Cell suspensions (2 × 10⁵ cells/ 100 μl) were mixed in duplicates with IE1 or pp65 recombinant proteins (following the vendor's recommendations, Lophius bioscience) or with gB recombinant protein (250 μg/ml, Sino Biologicals, Beijing, China). Subsequently, the mixtures of cells plus antigens were transferred onto ELISpot plates pre-coated with anti-IFN-γ antibodies and incubated for 20 h at 37°C. Immune detection of IFN-γ spots was performed following the vendor's recommendations (Lophius Bioscience). Spots on the wells were photographed and counted using a microscope and the results depicted as spot forming cells (SFC) within 2 × 10⁵ PBMC.

LN and mLN samples were thawed and pooled (Exp. 4, Table 1, CTR = 3, INF = 4, REAC = 4). The corresponding CB CD34 negative fraction was used to isolate CD14+ monocytes. For the generation of Conventional DCs (conDC), cells were cultured in X-vivo media (Lonza, Basel, Switzerland) with recombinant human IL-4 and GM-CSF (both 50 ng/ml; Cellgenix, Freiburg, Germany) for 7 days. For generation of conDC infected with HCMV-GLuc (conDC/HCMV), monocytes cultured with cytokines for 4 days were infected with virus at M.O.I. = 1 and maintained in culture for additional 3 days. Prior to ELISpot, DCs were analyzed by flow cytometry to confirm expression of relevant markers (CD80, CD86; HLA-DR, gB see Table S1). Then, DCs and mononuclear cells (1 × 10⁵ cells each in 100 μl) were co-cultured on the ELISpot plate as described before for 20 h. Spots were counted automatically using an ELISpot reader (AID, Straßberg, Germany) and the results depicted as SFC/1 × 10⁵ PBMC.

293T-wt and 293T-gB cells were re-suspended in PBS and maintained on ice. Cell lysates were produced by sonication and the protein concentration was determined with Bicinchoninic acid assay (BCA). One microliter corresponding to 1 μg of cell lysate was spotted onto strips of nitrocellulose membranes (GE Healthcare, Little Chalfont, UK) and air-dried for 10 min. Five percent milk/PBS/0.05% Tween (Merck Millipore) was used to block the membrane for 30 min at room temperature. Plasma from mice was added to the membrane strips which were incubated overnight at 4°C and washed 3x with PBS/0.05% Tween for 10 min at RT. Anti-human IgG-HRP (Roth, Karlsruhe, Germany) (1:500) was added for 1 h at RT. Next, the membrane was washed 4x with PBS/Tween for 10 min at room temperature (RT). After incubation with HRP chemiluminescent substrate (ThermoFisher), signals were measured with a Chemidoc station (Bio-Rad, Hercules, CA). ImageJ software (NIH, freeware) was used for signal quantifications of dot-blots.

Cell lysates were produced as described above from 293T-wt, 293T-gB, MRC-5, and MRC-5 cells infected with HCMV-GLuc (3 days post-infection, MOI = 1). After determination of the protein concentrations, 96 well plates (Coring, Corning, NY) were coated with 640 ng/50 μl of each cell lysate. The plates were incubated overnight at 4°C and washed 5x with PBS/0.1%Tween. The wells were pre-blocked by replacing the lysates with 100 μl of PBS/5% FBS in a wet chamber for 2 h at 37°C and then washed 3X with PBS/0.1% Tween. As a reference to validate the quantification obtained with the ELISA measurements we used 50 μl a human purified SM5-1 monoclonal antibody that was serially diluted up to 1 × 10⁻⁴ ng/ml and applied to wells coated with the 293T-gB lysate. The plasma samples from individual mice were diluted at 1:10 (in PBS/2% FBS) and applied to the wells coated with the different cell lysates. The plasma samples from each mouse were analyzed in duplicates for each cell lysate. The plates were incubated for 2 h at 37°C, washed 5x with PBS/0.1% Tween and then incubated with anti-human IgG-HRP (1:1,000) for 45 min at 37°C. After washing 3x with PBS/0.1% Tween, 100 μl of the TMB solution (ThermoFisher) were added to each well and reaction was stopped with 100 μl H₂SO₄ (Roth). The OD450 was measured in an ELISA plate reader.

Data consisting of the percentage of human lymphocyte subsets in blood and tissues and the absolute counts of different human lymphocyte subsets in tissues (# of positive cells and % positive) were organized in a PivotTable using Excel software 2010 (Microsoft, Redmond, WA). Each sample was coded according to the reconstitutions (R1 or R2), cohorts (CTR, INF, and REAC), mice IDs, time-point of analyses, tissues, and lymphocyte subtypes. Negative binomial regression was used to compare the cohorts in terms of total cell counts of lymphocyte subtypes in tissues using rate ratio, whereas beta regression was applied to compare the cohorts regarding the percentage of human lymphocyte subsets in blood and tissues using odds ratio. One-way analysis of variance (ANOVA) was performed for statistical analyses to differentiate the cohorts regarding the measurement of bio-luminescence signals after optical imaging analyses, human cytokines in plasma, human immune globulins in plasma, frequencies, and absolute counts of Tregs and Tfh cells and the determination of viral neutralization in vitro. Parameter estimation was performed by least square means. P-values were calculated at significance levels 0.05 using two-sided t-test statistical analyses (estimated p-values lower than 0.01 have been indicated). All analyses were implemented using the SAS 9.3 software (SAS, Cary, NC). PROC GENMOD and PROC GLIMMIX procedures were used for the negative binomial and beta regression analysis, respectively. GraphPad Prism version 5 software (GraphPad Software, Inc., La Jolla, CA) was used to create all figures.

Principal component analysis (PCA) is a multivariate statistical technique used to simplify high-dimensional data by emphasizing the data variations. PCA expresses the underlying information in data with a new set of variables called principal components (PC). PCs specify directions where the data is widely distributed and a significant proportion of data variability can be explained. By calculating the correlation of original data with the PCs, one can identify the markers with the highest correlation/anti-correlation value. We employed PCA in our experimental data to identify a subset of markers accounted for data variations. The relationship between the identified markers was obtained by calculating their correlations. Our analysis consisted of the following steps: (1) variables (markers) were standardized, i.e., normalized with 0 mean value and unit variance; (2) PCA was performed on different subsets of experimental data according to the unit [absolute (#)vs. frequency (%)], cell-type (T-cell vs. B-cell associated markers), and organ (LN, SPL, BM, single-organ/multi-organs); (3) most important T cell and B cell associated markers with highest maximum correlation with the first 3 PCs were selected; (4) correlations between markers were calculated and presented in the form of heat-maps. In order to find the PCs that could distinguish individuals according to their group (control, after HCMV infections, or reactivations), we searched for PCs (among top 5 PCs) that could separate the distribution of samples of distinct groups. Then, the markers with highest contribution to the composition of the separating PCs were obtained as the potential predictors of the group.

Linear Discriminant analysis (LDA) is a multivariate statistical technique for data profiling and clustering which aims to find a linear combination of variables that best separates samples of distinct groups. LDA creates discriminant functions maximizing the “between-class” separability while minimizing the “within-class” variability. We employed LDA on the experimental data to identify top markers that are contributing in the separability of groups. The most important markers for group discrimination were identified according to the contribution of markers in the composition of discriminant functions (the absolute value of associated coefficient). PCA and LDA were performed with 23 observations (7 CTR, 8 INF, 8 REAC) with 130 phenotypic markers (65 absolute numbers and 65 frequencies) using FactoMineR (32) and MASS (33) libraries of the statistical computing software R (version 3.4.3) (34).

We explored the use of the novel HCMV/GLuc strain expressing the Gaussia luciferase reporter gene downstream of the IE1 promoter (30). This promoter is strongly active immediately after HCMV infection, and thus IE1 expression is suitable for determining HCMV infection in several types of cells, including peripheral blood mononuclear cells (PBMCs) (Figure S1A). A high correlation between inoculation dose and bio-luminescence signal was measured 1 day post-infection of MRC-5 fibroblasts with the HCMV/GLuc at MOIs 10⁻⁴ to 10² (Figure S1B). Cryopreserved batches of HCMV/GLuc-infected MRC-5 cells were produced and, after thawing, flow cytometry analyses showed that 70–90% of the viable cells expressed the HCMV glycoprotein B (gB) on the cell surface (Figures S1C,D). G-CSF is known to mobilize and activate stem cells and myeloid precursors, thereby promoting HCMV infection. In pilot experiments we evaluated the immune modulatory effects of hG-CSF on mice transplanted with human CD34⁺ cells (Figure 1). Importantly, we utilized only banked and cryopreserved CD34⁺ purified cells pre-tested in couple of transplanted NRG mice showing robust long-term (>15 weeks) human reconstitutions. In our laboratory, a sufficient “humanization” is arbitrarily defined by the presence of >20% human CD45⁺ cells within all PBMCs measured at 15 weeks after CB-HSCT or prior to the experimental start. Seventeen weeks after HSCTs, human cell frequencies analyzed in PBL for the four different experiments described below varied: 28–52% huCD45⁺; 11–47% huCD3⁺ in huCD45⁺; 6–21% huCD4⁺ in huCD45⁺, and 4–14% huCD8⁺ in huCD45⁺ (Table 1). Despite this variability among the different experiments, these values were within our expected ranges. At 17 weeks after HSCT, humanized mice were administered subcutaneously and daily for 5 days with a low dose of hG-CSF (150 ng). Twenty-four weeks after HSCT, prior to experimental termination, mice were administered with a high dose for 7 days (2.5 μg/mouse/day) (Figure S1E). Flow cytometry analyses of CD45⁺, CD19⁺, CD3⁺, CD4⁺, CD8⁺ cells performed at 25 weeks post-HSCT showed that the hG-CSF treatment schedule on its own did not affect the development or expansion of human lymphocytes in SPL or BM when compared to transplantation only controls (CTR) (Figure S1F).

Next, we used the same hG-CSF treatment schedule but combined it with HCMV infections (reconstitution data analyzed for PBL shown in Table 1, Exp 2). We established a cryopreserved stock of MRC-5 cells infected with a lentiviral vector (LV) expressing GLuc (MRC5-LV) and a cryopreserved stock of MRC-5 cells infected with HCMV/GLuc. After thawing and serially diluting the cell stocks in a 96 well plate, we confirmed equivalent levels of bio-luminescence by optical imaging analyses (Figure 1A). Humanized NRG mice at 17 weeks after CD34⁺ HSCT were administered with 1x10⁶ MRC-5-LV cells (n = 4) and then treated with hG-CSF daily from weeks 24 to 25. These mice showed only background bio-luminescence signals detectable on weeks 21 and 25, similar to the transplantation only controls (CTR, n = 2) (Figure 1B). This indicated that the MRC-5 cell did not engraft in the mice and basically disappeared. In contrast, all mice injected with 1 × 10⁶ HCMV/GLuc-infected MRC-5 cells (HCMV-REAC, n = 4) showed conspicuous bio-luminescence signals in the ventral anatomical regions, indicating ongoing viral reactivations (Figure 1B). Compared to the MRC-5-LV cohort, mice with HCMV reactivations exhibited higher absolute numbers of CD3⁺, CD4⁺, and CD8⁺ T cells in SPL and BM (Figure 1C, see gating strategy in Figure S2A). We further defined the CD3⁺ T cell maturation subtypes based on the expression of CD62L and CD45RA as previously described (35): naïve (N, 62L⁺, and RA⁺), central memory (CM, 62L⁺, and RA⁻), effector memory (EM, 62L⁻, and RA⁻), and terminal effector (TE, 62L⁻, and RA⁺) (see gating strategy in Figure S2B). Upon HCMV reactivations, the immune phenotype profiles of the CD4⁺ T cells from SPL and BM showed a pronounced elevation in the absolute numbers of central memory (CM) and effector memory (EM) cells (Figure 1D, S1G). Additional examination of T cells in these tissues through expression analyses of PD-1 revealed that HCMV reactivations stimulated both CD4⁺ and CD8⁺ T cell activations Figure 1E, S1G. Remarkably, analyses of B cell markers indicative of class switch showed that HCMV reactivations were associated with major rises in the numbers of mature IgG⁺ memory B cells (IgGmBC), plasma blasts (PB, CD138^neg), and plasma cells (PC, CD138^pos) (Figure 1F, Figure S1H) (see gating strategy in Figures S2C,D). Thus, collectively, this data established the set-up and feasibility of the model recapitulating HCMV/GLuc reactivations, introduced the usage of spatial optical imaging analyses to track viral bio-distribution and showed associations between the HCMV reactivations with T and B cell activations.

For the subsequent experiments with larger mice cohorts for in-depth immunological analyses, we compared the effects of HCMV reactivations with primary latent infections. Thus, in addition to the G-CSF double treatment schedule (REAC, n = 8), we included one latent infection cohort which did not receive the second hG-CSF daily administrations between weeks 24 and 25 (INF, n = 8) (Figure 2A). Mice transplanted with HSCs but not treated were used as baseline control (CTR, n = 7). Experiments were performed in duplicate with mice reconstituted with CD34⁺ cells obtained from two different CB donors (Table 1, Exp 3/R1 and Exp 3/R2). Optical imaging analysis performed prior to euthanasia demonstrated high bio-luminescence signals after HCMV infections, which increased after reactivations (Figure 2B). The bio-distribution of the bio-luminescence ranged from speckled signals in the abdominal region to intense spots in the anatomical regions of SG, LI, and LN (Figure 2B). No major changes in body weight were observed after primary HCMV infections or after reactivations (Figure 2C), and no signs of xeno-GVHD were observed macroscopically. HCMV viral DNA was detected in LI of all infected mice (average 10⁴ viral copies/μg DNA), which increased substantially after reactivations (average 10⁶ viral copies/μg DNA) (Figure 2D). This increase was also observed in SPL and BM, but was less pronounced. Further confirming the optical imaging bio-distribution analyses, HCMV DNA copies were detectable in LN and SG of some of the mice and at higher levels after HCMV reactivations (Figure 2D). Remarkably, although all mice were infected, HCMV DNA was inconsistently detected in PBMCs of blood (PBL), i.e., in only about half of the mice after infections and reactivations (Figure 2D). We were able to detect HCMV DNA only in monocytes (CD45⁺/CD14⁺) sorted from spleen after primary infections (INF) but not after reactivations (Figure 2E). On the other hand, we detected HCMV DNA in sorted macrophages (CD45⁺/CD169⁺) after reactivations (REAC), but not after primary infections (INF) only (Figure 2E). CD34⁺ cells, known to become latently infected with HCMV (36), also showed HCMV DNA copies after infections, which increased upon reactivations (Figure 2F). Thus, whereas analyses of BL were not accurate (i.e., showing frequently false negatives values), viral copy numbers in LI, BM, SPL, LN, and SG and in sorted CD34⁺ cells and monocytes were consistently higher after reactivations. Histology analyses revealed that the spleens of infected and reactivated mice were enlarged with clear follicle formations containing human T (CD3⁺) and B (CD79a⁺) cells (Figure 2G). Immune histochemical analyses of LI for detection of human nuclei (HN) showed higher rates of human cells in infected and reactivated mice than in controls, but the occurrence of HCMV/IE1⁺ cells was rare (Figure 2H). All together, these results demonstrated robust qualitative and quantitative differences between HCMV infections and reactivations in this humanized mouse model, providing in addition comprehensible bio-distribution information by non-invasive imaging analyses.

We next tried to detect immune effects of HCMV infections and reactivations in BL samples that were obtained longitudinally. Whereas, there was only a slight decline in hCD45⁺ frequencies in the CTR and INF groups in the period between 17 and 25 weeks (to 30%), a prominent drop was detectable after reactivations (to 15%) (data did not reach statistical significance) (Figure S3A). But other than that, the relative frequency of B cells, CD4⁺ T, and CD8⁺ T within huCD45⁺ PBL were not much different among the experimental groups (Figures S3B–D). Regarding the T cell maturation subtypes in blood, only the relative frequencies of CD4⁺ and CD8⁺ CM T cells were moderately increased after primary infections, but this effect disappeared after subsequent reactivations (Figures S3E,F). Analyses of human cytokines in plasma revealed that, compared with the other groups, infected mice contained lower concentrations of two important Th1-type cytokines, namely IFN-γ (INF Vs. CTR p = 0.07 and INF Vs. REAC p = 0.11) and IL-12 (n.s.). On the other hand, analyses of IL-10, which is a typical Th2-type cytokine, showed a moderate increase after HCMV infections, but more so after reactivations (n.s.) (Figure 3A, Table S2). Thus, overall, the HCMV immune modulatory effects observed in circulating lymphocytes and plasma of humanized mice were mild.

We next examined the effects of HCMV infections and reactivations on the T and B cell lineages in primary and secondary lymphatic tissues and several organs for comparative analysis. For comparative analyses, we mostly adopted the absolute human lymphocytes counts, since we showed previously that they were more suitable than relative cell frequencies to reliably characterize immune modulations (26). For thymus, HCMV infections and reactivations stimulated higher hCD45 cellularity and higher absolute numbers of CD4/CD8 double negative (DN, REAC vs. CTR p = 0.02), double positive (DP, n.s.), and CD4 single positive (CD4SP, INF vs. CTR p = 0.01, REAC vs. CTR p = 0.01) T cells (Figure 3B, Table S3). For LN, significantly increased numbers of hCD45⁺ cells (INF Vs. CTR p = 0.04, INF Vs. REAC p = 0.03), CD3⁺ (INF Vs. CTR p = 0.03, INF Vs. REAC p = 0.02), CD4⁺ (INF Vs. CTR p = 0.03, INF Vs. REAC p = 0.03), and CD8⁺ T cells (INF Vs. CTR p = 0.04, INF Vs. REAC p = 0.02) were observed after HCMV infections compared with the other groups. Similar trends were observed for SPL, BM, and SG (Figure 3C, Table S4). After HCMV infections, and more markedly after reactivations, the numbers of PD-1⁺ T cells were consistently increased for several tissues (Figure 3D: LN, BM, SG; Figure S3: LI and mLN; Table S5). Noteworthy, HCMV infections and reactivations also promoted significantly higher frequencies of CD4⁺/CD25⁺/FoxP3⁺/CD45RA⁻ regulatory T cells (Tregs) (INF Vs. CTR p < 0.05, REAC Vs. CTR p < 0.05) and CD4⁺/PD-1⁺/CXCR5⁺ T follicular helper cells (Tfh) (INF Vs. CTR p < 0.05, REAC Vs. CTR p < 0.05) in SPL (Figure 3E). In order to check for functional T cell responses against HCMV antigens, lymphocytes recovered from bone marrow were assayed by IFN-γ ELISpot using pp65, IE1, and gB recombinant proteins as antigens (Figure 3F). Blood cells of healthy donors known to be HCMV seropositive (HD⁺) or seronegative (HD⁻) were used as controls to validate the assay. No reactive spots could be detected with cells retrieved from CTR mice (n = 3), whereas T cells recovered from mice after HCMV infections (n = 3) and reactivations (n = 3) showed detectable spots. Since the detectable number of SFCs was very low using this antigen-specific assay (in the range of 3–12 SFCs per 2 × 10⁵ PBMCs) and we could not differentiate if the responses in mice undergoing HCMV infection or reactivation were distinguishable, we developed another more robust ELISpot assay. We had previously shown that conventional dendritic cells (conDCs) obtained upon culture of monocytes with GM-CSF and IL-4 could be efficiently infected with HCMV laboratory strains (37). Therefore, we generated conDCs matched with the CB CD34⁺ cells used to humanize the mice and infected them with HCMV/GLuc (Figure 3G; as control for the assay, we used non-infected conDCs). Mononuclear cells present in pooled LN and mLN samples (CTR = 3, INF = 4, REAC = 4) were co-cultured with the HCMV-infected or non-infected conDCs on the ELISpot plate for 20 h. Background reactivity due to homeostatic stimulation was detectable for all co-cultures with conDCs. Nevertheless, comparing the number of spots observed when the assay was performed with conDC/HCMV and using effector cells from mice undergoing HCMV infections, we obtained almost 300 SFCs per 1 × 10⁵ PBMCs, and these numbers were half as high for mice undergoing reactivations. Thus, the results obtained with this ELISpot assay seem to provide an inverse correlation between the elevated numbers of PD-1⁺ T cells after HCMV reactivations with lower T cell responses against the HCMV-gB antigen. Since the T cells that develop in these humanize mice are not expected to be restricted to the human HLA, the use of tetramer analyses to verify the PD-1 upregulation on HCMV-specific T cells cannot be explored.

Moving toward a more in-depth and complex analyses of the T cell signatures in tissues, we assessed the correlations between T cell immune phenotypic patterns with PD-1 expression (gating strategy provided in Figure S2). LN contained the highest numbers of CM and EM CD4⁺ and CD8⁺ T cells after HCMV infections (Figure 4A, Tables S6A–D, Figure S4A). The PD-1 expression pattern within the T cell subtypes in LN was between INF and REAC, but for T effector T cells of the INF cohort, PD-1 expression was lower. After HCMV reactivations, the numbers of TE CD4⁺ cells were significantly increased in SPL, BM, and SG, and this was associated with high numbers of CD4⁺ PD-1⁺ cells (Figure 4A, Tables S6A–D, Figures S3, S4A). Therefore, LN seemed to represent a distinct tissue regarding immunomodulation of T cells by HCMV. In general terms, these analyses emphasized the relevance of specific niches such as LN for central T cell memory homeostasis vs. SPL, BM, mLN, and LI for T cell activations toward PD-1⁺ activated effector T cells. Thus, the allocation of T cells with different maturation and activation status within different tissues showed a very complex pattern, likely defined by multifactorial variables. To dissect structural relationships (trends) between phenotypic markers and to uncover their changes according to the HCMV infection status, we employed PCA and correlation analyses. This approach evaluates any possible relationship between variables and its alteration (lost, formed or inversed) between the groups (CTR, INF, REAC). Due to a large set of measured quantities, we first employed PCA to identify the markers that best explain the data variations. This was achieved by considering all the groups simultaneously (INF, REAC, CTR). We tested all combinations of data subsets according to the organ and the type of measurements (frequencies, absolute numbers, B cell, or T cell associated markers), and only showed the data subsets that could enlighten the different groups.

Using the absolute (#) or frequency (%) of T cell associated markers in LN, we found two PCs that moderately separated REAC from INF and CTR groups. Terminal effector (#) and effector memory (%) CD4⁺ T cells in LN were important markers in these separating PCs (Figure 4B). The markers with absolute numbers gave a better separation of REAC group from INF and CTR compared to the frequencies, yet, the variation of the whole data was much smaller (compare the range of y-axis in Figure 4B). A separation of REAC from CTR and INF group could also be obtained using the SPL data (Figure 4D). Several important markers that contributed to these PCs contained PD-1. Then, the correlation analyses were performed between markers (absolute numbers #) within each group and the comparisons were depicted in the form of heat-maps (Figures 4C,E). The LN data of the INF and REAC groups were almost undistinguishable (Figure 4C), whereas the BM and SPL data showed clearly different interrelations of phenotypic markers between REAC vs. INF (Figure 4E). In sum, the PCA combined with the correlation analyses applied for immune modulation of T cells in SPL and BM distinguished reactivations from infections through a signature of memory and terminal effector T cells expressing the activation marker PD-1. This signature was less evident for LN data, where other markers such as CD19 were overrepresented for the REAC cohort.

Next, we evaluated the effects of HCMV infections and reactivations on the B cell development, maturation and class switch (Figure 5, Tables S7A,B). The B cell antigen CD19 assembles with the B cell receptor (BCR) and is continuously expressed along the different stages of early B cell development but it is lost upon maturation to plasma cells. On the other hand, CD27 is a later B cell marker, which is the ligand for CD70, and plays a key role in regulating B-cell activation and immunoglobulin synthesis. Since B cells initially develop in BM, this was the tissue initially analyzed. As expected, most of the B cells there were immature (CD19^pos/CD27^neg), and could be further sub-grouped in immature/ transitional type 1 (T1, CD24^hi/CD38^hi) (Figure S5B) cells, transitional type 2 (T2, CD24^int/CD38^int), mature naïve (MNBC, CD24^low/CD38^low), and pre-memory B cells (pre-mBC, CD24^pos/CD38^neg), whereas very few plasma blasts (PB, CD138^neg), plasma cells (PC, CD138^pos), and IgG⁺ cells could be detected (see gating strategy in Figure S2). Most of the detected CD19^pos/ CD27^pos B cells in BM were IgM⁺ but IgG⁺ cells were also detectable. Thus, HCMV infections and reactivations did not alter much the pattern of B cell development in BM. In contrast, reactivations pushed the development and maturation of B cells in SPL, resulting in higher numbers of T1 (REAC vs. CTR p = 0.01, Table S7A), T2 (REAC vs. CTR p = 0.03, Table S7A), PB (REAC vs. CTR p = 0.03, Table S7B), and IgG⁺ B cells (REAC vs. CTR p = 0.01, Figure 5A, Table S7B, Figure S6A). The effects of HCMV reactivations obtained for SPL were in line with the results obtained for mLN and LI (Table S7B, Figures 5B). In contrast, LN showed a deviant pattern, as primary infections suppressed the development of IgG⁺ memory B cells (REAC vs. INF p < 0.01, Table S7B, Figure 5A) and reactivations pushed significantly the development of mature PB, PC, and IgG⁺ B cells (Figure 5A, Table S7B, Figure S6A). A PCA performed with B cell associated markers using absolute cell numbers (#) in SPL resulted in two separating PCs, which clearly distinguished REAC group from the other groups (Figure 5B). The phenotypic markers in PC1 (pre-MBC, PB, IgM⁺, PC, and CD19⁺CD27⁺) and PC4 (IgG⁺, T1 BC, and IgM⁺) were among the markers overrepresented in the REAC group. Therefore, the heat map based on the SPL data clearly showed different interrelations of phenotypic B cell markers in REAC vs. CTR (Figure 5C).

We further evaluated whether the distinctive B cell-related immune phenotypic patterns associated with HCMV reactivations correlated with functional antibody generation. Levels of human IgM and IgG in plasma were both higher after HCMV reactivations (figure 5D, Table S8). The levels of human IgG (determined by least square mean estimation, LSM) were considerably higher after reactivations (27.89 μg/ml) relative to controls (15.37 μg/ml) or primary infections (6.44 μg/ml). In order to evaluate reactivity of human IgGs recovered from plasma against HCMV-gB, dot-blots using protein extracts obtained from wild type (w.t.) 293T cells and 293T cells expressing gB were used for immune detection (Figure 5E). No signs of IgG reactivity against gB could be found in plasma of control mice. Plasma obtained from mice after HCMV infections showed detectable reactivity, which was further enhanced with plasma from mice after HCMV reactivations. Further, an ELISA assay was developed in order to provide additional evidence by other methods that mice infected with HCMV and undergoing reactivations could mount IgG responses against HCMV (Figure 5F). ELISA plates were coated with protein lysates generated with 293Twt, 293TgB, MRC-5 cells, or MRC-5 cells infected with HCMV. As a reference for the assay we used a high affinity purified human monoclonal antibody recognizing gB (SM5-1). Plasma samples from mice obtained in experiment #2 (receiving either MRC-5/LV-GLuc or MRC-5/HCMV-GLuc) were diluted 10-fold and applied to the wells of the plates. The results showed clearly that plasma obtained from several mice undergoing HCMV reactivation reacted against proteins present in 293TgB and in MRC-5 cells infected with HCMV. Only background signals were obtained for plasma obtained from mice administered with MRC-5 cells infected with LV-GLuc. These results confirmed the data shown by Crawford et al using other ELISA methods (25) that humanized mice infected with HCMV can mount detectable IgG responses.

As another strategy to reduce the complexity of our data and find phenotypic markers that might be essential for defining HCMV infections vs. reactivations status, we employed LDA on our experimental data in similar fashion to PCA (see Methods). LDA performed with a subset of measurements (frequencies and/or absolute values) obtained from the T and B cell panels (individually or combined) resulted into distinct separations of CTR, INF, and REAC groups (Figure 6A). LDA analyses using only the frequencies of T lymphocyte types in blood identified clusters for each experimental arm, but with some overlap. When the frequencies and absolute numbers of T lymphocyte types in spleen were used in combination, the experimental arms could be better divided. Nevertheless, LDA performed with frequencies and absolute numbers of the B cell panel clearly showed the best distinction among the clustered groups. However, when the data obtained with B and T cell panels were combined, the clustering was less prominent, indicating that data obtained from T and B panels were not additive or synergistic. In summary, the LDA analyses advocated that using different subsets of immune phenotypic markers, and predominantly the B cell panel, mice with HCMV infections and reactivations could be distinctively distinguished from the control group.

RS received honoraria as a lecturer in conferences from The Jackson Laboratory, which commercially distributes the NRG mice. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a past co-authorship with one of the authors MMe.