PD-1 Blockade Aggravates Epstein-Barr Virus+ Post-Transplant Lymphoproliferative Disorder in Humanized Mice Resulting in Central Nervous System Involvement and CD4+ T Cell Dysregulations

Abstract

Post-transplant lymphoproliferative disorder (PTLD) is one of the most common malignancies after solid organ or allogeneic stem cell transplantation. Most PTLD cases are B cell neoplasias carrying Epstein-Barr virus (EBV). A therapeutic approach is reduction of immunosuppression to allow T cells to develop and combat EBV. If this is not effective, approaches include immunotherapies such as monoclonal antibodies targeting CD20 and adoptive T cells. Immune checkpoint inhibition (ICI) to treat EBV⁺ PTLD was not established clinically due to the risks of organ rejection and graft-versus-host disease. Previously, blockade of the programmed death receptor (PD)-1 by a monoclonal antibody (mAb) during ex vivo infection of mononuclear cells with the EBV/M81⁺ strain showed lower xenografted lymphoma development in mice. Subsequently, fully humanized mice infected with the EBV/B95-8 strain and treated in vivo with a PD-1 blocking mAb showed aggravation of PTLD and lymphoma development. Here, we evaluated vis-a-vis in fully humanized mice after EBV/B95-8 or EBV/M81 infections the effects of a clinically used PD-1 blocker. Fifteen to 17 weeks after human CD34⁺ stem cell transplantation, Nod.Rag.Gamma mice were infected with two types of EBV laboratory strains expressing firefly luciferase. Dynamic optical imaging analyses showed systemic EBV infections and this triggered vigorous human CD8⁺ T cell expansion. Pembrolizumab administered from 2 to 5 weeks post-infections significantly aggravated EBV systemic spread and, for the M81 model, significantly increased the mortality of mice. ICI promoted Ki67⁺CD30⁺CD20⁺EBER⁺PD-L1⁺ PTLD with central nervous system (CNS) involvement, mirroring EBV⁺ CNS PTLD in humans. PD-1 blockade was associated with lower frequencies of circulating T cells in blood and with a profound collapse of CD4⁺ T cells in lymphatic tissues. Mice treated with pembrolizumab showed an escalation of exhausted T cells expressing TIM-3, and LAG-3 in tissues, higher levels of several human cytokines in plasma and high densities of FoxP3⁺ regulatory CD4⁺ and CD8⁺ T cells in the tumor microenvironment. We conclude that PD-1 blockade during acute EBV infections driving strong CD8⁺ T cell priming decompensates T cell development towards immunosuppression. Given the variety of preclinical models available, our models conferred a cautionary note indicating that PD-1 blockade aggravated the progression of EBV⁺ PTLD.

Keywords: Epstein-Barr Virus (EBV); PD-1; humanized mice; immune checkpoint inhibition (ICI); immuno-oncology; lymphoma; pembrolizumab; post-transplant lymphoproliferative disease (PTLD).

Figure 1

Humanized mice infected with EBV/fLuc and treated with pembrolizumab show high mortality associated with increased EBV spread and tumor development. (A) Scheme of the experiments. 2 × 10⁵ human CD34⁺ cord blood (CB) purified hematopoietic stem cells (HSCs) were used for hematopoietic stem cell transplantation (HCT) of sub-lethally irradiated NRG mice. Fifteen to 17 weeks after HCT, the long-term human immune reconstitution in blood (huCD45⁺) was confirmed. At this point, mice were infected i.v., with the preferentially latent EBV B95-8/fLuc strain (10⁵ GRU) or with the lytic EBV M81/fLuc strain (10⁶ GRU). Two independent experiments using HSCs from two different CB donors were performed for each EBV model. At 2 weeks post-infections (wpi), bioluminescence imaging (BLI) analyses were performed to determine the baseline of EBV infection level and to randomize the mice among three cohorts: (i) injected i.p. with phosphate-buffered saline (PBS, corresponding to the control group, CTR), (ii) injected i.p. a low dose (LD: First administration 3.33 mg/kg at 2 wpi; 1.7 mg/kg every other week applied six times), or with (iii) a high dose (HD: First administration 10 mg/kg at 2 wpi; 5 mg/kg every other week applied six times). Mice were monitored for disease severity every 2–3 days and body weights were measured weekly. Scores of disease severity or loss of 20% of body weight were used as termination criteria. Optical imaging analyses and analyses of human immune reconstitution in peripheral blood collections were performed longitudinally (2, 4, 6, 8 wpi). The experimental endpoint was eight weeks post-infections. The following data was acquired for terminal analyses: % survival, occurrence of weight loss, occurrence of tumors, EBV load, histopathology, characterization of T cells in blood and tissues, human cytokine profile in plasma. (B, C) Survival curves for humanized mice infected with the B95-8/fLuc strain (B) or with the M81/fLuc strain (C). A log-rank test (Mantel-Cox) was applied to evaluate the differences in survival. Cohorts: CTR (gray line), LD (blue line); HD (red line). (D, E) Measurements of body weight relative to the baseline weights on the day of EBV infection. Mice infected with B95-8/fLuc strain (D) or M81/fLuc strain (E). Cohorts: CTR (grea line), LD (blue line); HD (red line). (F, G) Numbers of macroscopically detectable tumors in mice infected with B95-8/fLuc (F) or with M81/fLuc (G). Cohorts: CTR (gray dots), LD (blue dots); HD (red dots). (H, I) BLI pictures generated sequentially from week 2 to 8 after EBV infection performed in duplicate experiments for each EBV model and treatment. Pictures were taken of left body side of mice infected with B95-8/fLuc (H) or with M81/fLuc (I). Bioluminescence signal intensities (photons/sec) are depicted by the color bars on the right side. Black boxes depict dead mice. (J–L) Quantification of EBV spread by BLI for the B95-8/fLuc (J) or M81/fLuc (L) strain. Total Flux corresponds to the radiance (photons/sec) in each pixel summed over the regions of interest (ROI) area containing the whole left side of the body. The dots represent quantifications at 2, 4, 6, 8 wpi for each mouse in CTR (open circles) or in pembrolizumab treatment cohorts (filled triangles, LD and HD were combined). (K–M) Quantification of EBV spread by RT-qPCR. EBV copies per µg DNA were quantified in spleen (SPL) and bone marrow (BM) of mice infected with B95-8/fLuc (K) or with M81/fLuc (M). The dots represent quantifications for each mouse in CTR (open circles, PBS and KIOVIG controls combined) or in pembrolizumab treatment cohort (filled triangles, LD and HD were combined). Results of measurements were log-transformed and statistical analyses were performed using unpaired t test with Welch’s correction. Standard deviation is indicated. Statistical significances are indicated with *P < 0.05, ****P < 0.0001.

Figure 2

Analyses of explanted spleen and liver showed increased EBV/fLuc spread, inflammation, and neoplasia after pembrolizumab treatment. (A, B) Bioluminescence imaging analyses performed with explanted spleen and liver from representative B95-8/fLuc (A) or from representative M81/fLuc-infected mice (B). Tissues from representative non-treated control (CTR) mice are shown. Bioluminescence signal intensities (photons/sec) are depicted by the color bars on the right side. (C–J) Histopathological analyses of spleen (C–I) and liver (D–J) of representative mice infected with EBV/B95-8/fLuc and non-treated (CTR, left side) or treated with pembrolizumab (Pembro, right side). (C, D) Giemsa, EBER: In both organs, the perivascular spread of EBV/B95-8/fLuc infected neoplastic cells is observed. More disseminated infection and larger tumors were observed in pembrolizumab-treated than in control mice. Stars indicate perivascular angiocentric neoplastic growth. (E, F) CD30, CD20/CD3 duplex: The CD30- and CD20-expressing tumors and perivascular infiltrates resemble human B-cell neoplasia, corresponding to diffuse large B-cell lymphoma, and are surrounded or invaded by variable amounts of tumor-infiltrating CD3⁺ lymphocytes (TILs). (G, H) CD8, CD4: Early perivascular lesions display a largely balanced CD4/CD8 ratio. In the center of tumor bulk mass, the balance is shifted towards CD8⁺ cytotoxic T lymphocytes (CTLs). (I, J) Ki67, PD-L1: proliferating cells observed at a higher density in tumor areas, which also correlates with PD-L1 expression. Digital whole slides scanned at 40×; black bars correspond to 600 µm (full image) and 100 µm (inserts).

Figure 3

Pembrolizumab treatment is associated with spread of EBV/fLuc to the central nervous system. (A, B) Bioluminescence images showing representative EBV signal patterns in brains from mice infected with B95-8/fLuc (A) or with M81/fLuc (B) and treated with pembrolizumab (Pembro; most severely affected animals shown in each group), compared with non-treated controls (CTR). The bioluminescence signal intensities (photons/sec) are depicted by the color bar on the right side. (C, D) In-situ hybridization for detection of Epstein-Barr encoded RNA (EBER)-1 in brains of mice infected with B95-8/fLuc and treated with pembrolizumab. Observed patterns include invasion of tumor cells into the plexus choroideus (white inserts), as well as into periventricular brain parenchyma (red insert) and meningeal tumor spread (blue inserts). The green arrows indicate single tumor cells associated with blood vessels. (E) Comparison with three representative examples of human EBV-positive monomorphic transplantation-associated B-PTLD, with clinical manifestation as primary CNS lymphoma (PCNSL) revealed similar patterns of intraparenchymal (Case 01), perivascular (Case 02), and massive angiocentric/diffuse growth with meningeal spread (Case 03). (F) Control animals did not show any involvement of the plexus choroideus or periventricular brain areas. Only single EBER-positive cells in intravascular localization were detected (green arrows). (G–J) The immunophenotype of the neoplastic cells invading the brain of ICI-treated mice corresponded to the phenotype of the peripheral neoplastic population with variable CD30⁺, and constant CD20⁺ expression. The majority of neoplastic cells expressed EBNA2 but not LMP1 and were actively proliferating (CD20⁺/Ki67⁺). (G–H) and (I–J) Analyses of duplicate mice infected with B95-8/fLuc and M81/fLuc and treated with pembrolizumab, respectively. (K) Multiplexed image of two mouse brains from B95-8/fLuc infection model after pembrolizumab treatment confirmed co-localization of neoplastic cells (CD20⁺, white; CD30⁺, magenta) and T lymphocytes (CD8⁺ yellow; CD4⁺, green). Lymphocytes were actively proliferating (Ki67⁺, red), particularly CD8 (blue arrows). DAPI (blue) is used to mark cell nucleus. Bars correspond to 200 µm (full image) and 50 µm (inserts).

Figure 4

Reduced frequencies of human T lymphocytes in peripheral blood and increased concentrations of human cytokines in plasma after PD-1 blockade during active EBV infections. Peripheral blood was collected on the day prior to EBV infection (time-point 0) and 2 weeks post-infections (wpi), as baseline values for randomization (shaded graphs). Non-treated control mice (CTR, open circles) and pembrolizumab treated cohorts are shown (filled triangles; low-dose and high-dose treatments were merged for the analyses, each dot represents a mouse analyzed at the corresponding time point). After initiation of pembrolizumab treatment, peripheral blood was collected at 3–4, 5–6, and 8 wpi. The different EBV infection models (B95-8/fLuc; left panels and M81/fLuc; right panels) were analyzed separately regarding lymphocytes and cytokines and then the data was merged for power analyses and identification of markers associated with response. (A–J) Flow cytometry analyses of peripheral blood lymphocytes. (A, B) Frequencies (%) of human CD45⁺/CD3⁺ lymphocytes were reduced after pembrolizumab treatment. (C, D) Frequencies (%) of human CD45⁺/CD8⁺ lymphocytes were reduced after pembrolizumab treatment. (E, F) Mean fluorescence intensities (MFI) of PD-1 detected on the surface of CD8⁺ T cells were reduced after pembrolizumab treatment. (G, H) Frequencies (%) of human CD45⁺/CD4⁺ lymphocytes were reduced after pembrolizumab treatment. (I, J) MFI of PD-1 detected on the surface of CD4⁺ T cells were significantly reduced after pembrolizumab treatment. Statistical analyses for flow cytometry data was performed using unpaired t test with Welch’s correction. Standard deviation is indicated. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (K, L) Concentration human cytokines (pg/ml) at 8 wpi showing elevation for several cytokines in plasma of mice infected B95-8/fLuc (K) or M81/fLuc (L) treated with pembrolizumab. Mann-Whitney t test was used to compare the control and treatment groups. Standard deviation is indicated. *P < 0.05, **P < 0.01. # indicates parameters with some values being beyond the lower limit of detection (LOD), LOD was used for these samples to analyze the differences. (M) Global analysis for power analyses and identification of T cell and cytokine patterns at 8 wpi. The datasets from the B95-8/fLuc and M81/fLuc infection models were merged. Kolmogorov-Smirnov-Test (KS test; D > 0.3, P < 0.05) is depicted for flow cytometry and cytokine datasets showing markers with significant difference between control (CTR, red) and pembrolizumab treatment (Pembro, blue). P-values determined for each biomarker are indicated below the box plots. The top biomarkers associated with pembrolizumab treatment were: Reduced %CD3/CD45 (P = 0.0003), reduced %CD4/CD45 (P = 0.0182), reduced MFI of PD-1 expression on CD4⁺ cells (P = 0.0001), increased IL-10 (P = 0.0086) and IFN-α (P = 0.0090) levels in plasma.

Figure 5

The absolute numbers of human T lymphocytes in lymphatic tissues increased for the B95-8/fLuc model and decreased for the M81/fLuc model after PD-1 blockade. The absolute numbers of viable lymphocytes recovered from spleen (SPL), lymph nodes (LN), bone marrow (BM), and thymus (Thy) were counted and, after flow cytometry analyses, the absolute numbers of each lineage was calculated. Non-treated control mice (CTR, open circles) and pembrolizumab-treated cohorts are shown (filled triangles; low-dose and high-dose treatments were merged for the analyses; each dot represents a mouse analyzed at the terminal time point). Only mice surviving until the terminal analyses at 8 wpi were included in the terminal flow cytometry analyses. (A, B, E, F). Quantified total lymphocyte numbers (# cells) calculated for CD8⁺ T cells (A, B) and CD4⁺ T cells (E, F) for each organ. Note the increased numbers of lymphocytes for the B95-8/fLuc models and the decreased numbers for the M81/fLuc models for the pembrolizumab-treated mice. (C, D, G, H) Mean fluorescence intensity (MFI) for PD-1 detection for CD8⁺ T cells (C, D) and CD4⁺ T cells (G, H) showing overall significantly reduced PD-1 detection after pembrolizumab treatment for both models. Unpaired t test with Welch’s correction was used to compare treatment groups. *P < 0.05, **P < 0.01, ****P < 0.0001. Standard deviation is indicated. (I) Global power analyses and biomarker identification. The flow cytometry datasets of spleen of B95-8/fLuc and M81/fLuc models were merged. Kolmogorov-Smirnov Test (KS test; D > 0.65, P < 0.05) is depicted. The biomarkers identified with significant differences between control (CTR, open circles) and pembrolizumab treatment (Pembro, filled triangles) were: Lower SPL % CD4/CD45 (P = 0.0008); lower BM #CD4/CD45 (P = 0.0319); lower MFI for PD-1 in CD4⁺ cells (P < 0.0001); lower MFI for PD-1 in CD8⁺ cells (P = 0.0003). P-values determined for each marker are indicated below the graphs.

Figure 6

The frequencies of T cells expressing TIM-3 or LAG-3 exhaustion markers or a regulatory T cell immunophenotype increased after pembrolizumab treatment. Frequencies of marker-positive lymphocytes recovered from spleen (SPL) comparing non-treated control mice (CTR, open circles) and pembrolizumab-treated cohorts are shown (filled triangles; low-dose and high-dose treatments were merged for the analyses). Only mice surviving until the terminal analyses at 8 wpi were included in the terminal flow cytometry analyses. (A–D) Frequencies (%) of TIM-3⁺ in CD45⁺/CD8⁺ (A, B) and of TIM-3⁺ in CD45⁺/CD4⁺ (C, D) T cells are shown. PD-1⁺ (left side) or PD-1⁻ (right side) CD8 or CD4 subpopulations were analyzed separately. Higher proportion of TIM-3⁺ cells was observed more clearly for PD1⁺ T cells for both models. (E–H) Frequencies (%) of LAG-3⁺ in CD45⁺/CD8⁺ (E, F) and LAG-3⁺ in CD45⁺/CD4⁺ (G, H) T cells are shown. CD69⁺ (left side) or CD69^- (right side) CD8 or CD4 subpopulations were analyzed separately. Significantly higher proportion of LAG-3⁺ T cells were observed for activated CD69⁺ T cells for mice infected with M81/EBV/fLuc and treated with pembrolizumab. (I–L) Frequencies (%) of CD25⁺/FoxP3⁺/CD45⁺ regulatory (I, K) and CD45RA^-/CD25⁺/FoxP3⁺/CD45⁺ activated regulatory T cells (J, L) detected within the human CD4⁺ cells for B95-8 (I, J) and M81 model (K, L). Pembrolizumab treatment was associated with higher proportion of regulatory T cells for both models. Unpaired Welch’s t test was used to compare treatment groups. *P < 0.05. Standard deviation is indicated.

Figure 7

Lower overall infiltration of CD4⁺ T cells but higher density and chance of intercellular interactions of regulatory T cells in tumors of mice infected with EBV/B95-8/fLuc and treated with pembrolizumab. (A) Representative example of a multiplexed immunohistochemical (mIHC) staining of liver tissue of a B95-8/fLuc-infected and pembrolizumab-treated humanized mouse followed by multispectral image (MSI) analysis/color deconvolution into six channels representing CD20⁺ (white), Ki67⁺ (magenta), CD4⁺ (green), CD8⁺ (yellow), FoxP3⁺ (red), and DAPI (blue) markers. (B) Tissue segmentation between the perivascular tumor mass (red) and the non-neoplastic parenchyma (green). Expectedly, the tumor area corresponds with high density of proliferating CD20⁺ blast-like cells (A, B). (C) Comprehensive distance mapping between FoxP3⁺ (red dots) and FoxP3^- T-cells (cyan dots). (D) The tumor areas of treated animals (black triangles, n = 11) showed fewer CD4⁺ T cells than controls (open circles, n = 3). (E) The parenchyma area is characterized by a prominent population of proliferating CD8⁺ cells, with a trend towards higher densities in treated animals (not significant). (F) Densities of proliferating Ki67⁺CD4⁺ Treg cells in the parenchyma or in the tumor tissue, with an increase of tumor-infiltrating FoxP3⁺/CD4⁺ Treg cells in the pembrolizumab-treated group (not significant), (G) that was also observed for parenchyma FoxP3⁺/CD8⁺ Treg cells (not significant). (H) Distance metrics between FoxP3⁺/CD4⁺ Tregs or (I) FoxP3⁺/CD8⁺ Tregs revealing significantly increased average number of FoxP3⁺/CD4⁺ within a radius of 200 µm around any single CD4⁺ or CD8⁺ T cells residing in the tumor area for pembrolizumab-treated (black triangles) mice compared with control (open circles) mice. Unpaired t test with Welch’s correction was applied to calculate statistical significance. Standard deviation is indicated. *P < 0.05.