MHC class II-deficient mice allow functional human CD4+^{+} T-cell development

Humanized mouse models have been developed to study cell-mediated immune responses to human pathogens in vivo. How immunocompetent human T cells are selected in a murine thymus in such humanized mice remains poorly explored. To gain insights into this mechanism, we investigated the differentiation of human immune compartments in mouse MHC class II-deficient immune-compromised mice (humanized Ab0 mice). We observed a strong reduction in human CD4$^{+}$ T-cell development but despite this reduction Ab0 mice had no disadvantage during Epstein-Barr virus (EBV) infection. Viral loads were equally well controlled in humanized Ab0 mice compared to humanized NSG mice, and improved T-cell recognition of autologous EBV-transformed B cells was observed, especially with respect to cytotoxicity. MHC class II blocking experiments with CD4$^{+}$ T cells from humanized Ab0 mice demonstrated MHC class II restriction of lymphoblastoid cell line recognition. These findings suggest that a small number of CD4$^{+}$ T cells in humanized mice can be solely selected on human MHC class II molecules, presumably expressed by reconstituted human immune cells, leading to improved effector functions

KSHV infection drives poorly cytotoxic CD56-negative natural killer cell differentiation in vivo upon KSHV/EBV dual infection

Funding Information: This research was supported in part by Cancer Research Switzerland , Switzerland ( KFS-4091-02-2017 ); KFSP-PrecisionMS and HMZ ImmunoTargET of the University of Zurich , Switzerland; the Cancer Research Center Zurich , Switzerland; the Vontobel Foundation , Switzerland; the Baugarten Foundation , Switzerland; the Sobek Foundation , Germany; the Swiss Vaccine Research Institute , Switzerland; Roche , Switzerland; Novartis , Switzerland; and the Swiss National Science Foundation , Switzerland ( 310030B_182827 and CRSII5_180323 ). A.M.M. was funded by a National Institutes of Health , United States, grant ( R01 CA189806 ). N.C. was supported by a career advancement grant from the University of Zurich , Switzerland ( FK-18-026 ). D.M. and M.B. were supported by MD-PhD fellowships from the Swiss National Science Foundation , Switzerland, and the Swiss Academy of Medical Sciences , Switzerland ( 323530_145247 and 323630_19938 ).Peer reviewedPublisher PD

Gammaherpesvirus-Induced T Cell-Mediated Immune Control and Tumorigenesis in a Model of Dual-Infection

Epstein-Barr virus (EBV) and Kaposi sarcoma-associated herpesvirus (KSHV) are two humanotropic g-herpesviruses that infect a large part of the human population. They can both persist lifelong in human B cells and contribute to a number of B cell lymphoproliferative diseases, that mostly arise in immunocompromised individuals. Primary effusion lymphoma (PEL) is associated with KSHV, but about 90% of tumors are also positive for EBV. The first part of this PhD thesis is a review that sums up the knowledge on EBV and KSHV in PEL and innate and adaptive immune responses that can control EBV and KSHV but are also counteracted by both viruses. A recently established humanized mouse model of KSHV infection showed that KSHV can persist in EBV co-infected mice, and arising tumors share characteristics of PEL. The second part of this thesis utilized KSHV and EBV co-infection to investigate KSHV-specific adaptive immune responses in vivo. While EBV-directed immune responses are well understood, knowledge on KSHV-directed immune responses is limited to epidemiological and patient-based studies. We showed that cytotoxic CD8+ T cells expanded in KSHV coinfected animals, and KSHV-specific IgM antibodies with specificities similar to those observed in KSHV+ individuals were generated in these mice. Depletion of T cells increased both EBV and KSHV viral titers and tumor burden in KSHV co-infected animals. Additionally, T cells isolated from KSHV co-infected humanized animals could specifically kill KSHV and EBV dual infected B cell lines and were specific to KSHV ORF6. The third part of this thesis utilized KSHV lytic gene mutant viruses deficient for complete KSHV lytic gene expression through a stop mutation in the replication and transcription activator (RTA) or the KSHV lytic gene K2 that encodes for viral IL-6 through gene deletion. We observed decreased tumor formation in animals co-infected with EBV and KSHV rta-stop mutant or KSHV delta K2 mutant compared to KSHV wildtype co-infection independent of infectivity or viral titers. In vitro generated B cell lines revealed that cells coinfected with EBV and KSHV rta-stop or KSHV delta K2 were deficient in cell growth, and viability of KSHV rta-stop co-infected cells was impaired. In vivo transfer of these cells revealed that expansion capacity of KSHV+ cells was increased in KSHV wildtype co-infected cells compared KSHV mutant virus co-infected cells, which revealed a role of KSHV lytic genes in cell survival and expansion. Over all, humanized mice are a suitable platform to investigate basic functions of the immune system in human g-herpesvirus infections and enable studies on the role of individual KSHV genes through the use of KSHV mutant viruses in EBV co-infections. They support the investigation of immune responses, and thereby make the investigation of immune-based therapeutics and vaccine development targeting KSHV possible

Co-Infection of the Epstein–Barr Virus and the Kaposi Sarcoma-Associated Herpesvirus

The two human tumor viruses, Epstein–Barr virus (EBV) and Kaposi sarcoma-associated herpesvirus (KSHV), have been mostly studied in isolation. Recent studies suggest that co-infection with both viruses as observed in one of their associated malignancies, namely primary effusion lymphoma (PEL), might also be required for KSHV persistence. In this review, we discuss how EBV and KSHV might support each other for persistence and lymphomagenesis. Moreover, we summarize what is known about their innate and adaptive immune control which both seem to be required to ensure asymptomatic persistent co-infection with these two human tumor viruses. A better understanding of this immune control might allow us to prepare for vaccination against EBV and KSHV in the future

Immunosuppressive FK506 treatment leads to more frequent EBV-associated lymphoproliferative disease in humanized mice

Post-transplant lymphoproliferative disorder (PTLD) is a potentially fatal complication after organ transplantation frequently associated with the Epstein-Barr virus (EBV). Immunosuppressive treatment is thought to allow the expansion of EBV-infected B cells, which often express all eight oncogenic EBV latent proteins. Here, we assessed whether HLA-A2 transgenic humanized NSG mice treated with the immunosuppressant FK506 could be used to model EBV-PTLD. We found that FK506 treatment of EBV-infected mice led to an elevated viral burden, more frequent tumor formation and diminished EBV-induced T cell responses, indicative of reduced EBV-specific immune control. EBV latency III and lymphoproliferation-associated cellular transcripts were up-regulated in B cells from immunosuppressed animals, akin to the viral and host gene expression pattern found in EBV-PTLD. Utilizing an unbiased gene expression profiling approach, we identified genes differentially expressed in B cells of EBV-infected animals with and without FK506 treatment. Upon investigating the most promising candidates, we validated sCD30 as a marker of uncontrolled EBV proliferation in both humanized mice and in pediatric patients with EBV-PTLD. High levels of sCD30 have been previously associated with EBV-PTLD in patients. As such, we believe that humanized mice can indeed model aspects of EBV-PTLD development and may prove useful for the safety assessment of immunomodulatory therapies

Correction: Immunosuppressive FK506 treatment leads to more frequent EBV-associated lymphoproliferative disease in humanized mice

[This corrects the article DOI: 10.1371/journal.ppat.1008477.]

Immunosuppressive FK506 treatment leads to more frequent EBV-associated lymphoproliferative disease in humanized mice

Post-transplant lymphoproliferative disorder (PTLD) is a potentially fatal complication after organ transplantation frequently associated with the Epstein-Barr virus (EBV). Immunosuppressive treatment is thought to allow the expansion of EBV-infected B cells, which often express all eight oncogenic EBV latent proteins. Here, we assessed whether HLA-A2 transgenic humanized NSG mice treated with the immunosuppressant FK506 could be used to model EBV-PTLD. We found that FK506 treatment of EBV-infected mice led to an elevated viral burden, more frequent tumor formation and diminished EBV-induced T cell responses, indicative of reduced EBV-specific immune control. EBV latency III and lymphoproliferation-associated cellular transcripts were up-regulated in B cells from immunosuppressed animals, akin to the viral and host gene expression pattern found in EBV-PTLD. Utilizing an unbiased gene expression profiling approach, we identified genes differentially expressed in B cells of EBV-infected animals with and without FK506 treatment. Upon investigating the most promising candidates, we validated sCD30 as a marker of uncontrolled EBV proliferation in both humanized mice and in pediatric patients with EBV-PTLD. High levels of sCD30 have been previously associated with EBV-PTLD in patients. As such, we believe that humanized mice can indeed model aspects of EBV-PTLD development and may prove useful for the safety assessment of immunomodulatory therapies