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Front Immunol. 2018 Nov 22;9:2734. doi: 10.3389/fimmu.2018.02734. eCollection 2018.

Signatures of T and B Cell Development, Functional Responses and PD-1 Upregulation After HCMV Latent Infections and Reactivations in Nod.Rag.Gamma Mice Humanized With Cord Blood CD34+ Cells.

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Clinic of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany.
Excellence Cluster REBIRTH, Laboratory of Regenerative Immune Therapies Applied, Hannover Medical School, Hannover, Germany.
Partner Site Hannover-Braunschweig, German Center for Infection Research (DZIF), Braunschweig, Germany.
Department of Systems Immunology and Braunschweig Integrated Centre of Systems Biology (BRICS), Helmholtz Centre for Infection Research, Braunschweig, Germany.
Institute for Biochemistry, Biotechnology and Bioinformatics, Technical University Braunschweig, Braunschweig, Germany.
Institute of Virology, University of Ulm, Ulm, Germany.
Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM), Hannover, Germany.
Institute for Laboratory Animal Science, Hannover Medical School, Hannover, Germany.
Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research Braunschweig, Braunschweig, Germany.
Department of Transfusion Medicine, Hannover Medical School, Hannover, Germany.
Clinic of Gynecology and Obstetrics, Hannover Medical School, Hannover, Germany.
Institute for Biostatistics, Hannover Medical School, Hannover, Germany.
Department of Pediatrics, University Medicine Greifswald, Greifswald, Germany.
Department of Hematology, Oncology and Rheumatology, GMP Core Facility, Heidelberg University Hospital, Heidelberg, Germany.
Institute of Virology, University Erlangen-Nürnberg, Erlangen, Germany.
Institute of Virology, Hannover Medical School, Hannover, Germany.


Human cytomegalovirus (HCMV) latency is typically harmless but reactivation can be largely detrimental to immune compromised hosts. We modeled latency and reactivation using a traceable HCMV laboratory strain expressing the Gaussia luciferase reporter gene (HCMV/GLuc) in order to interrogate the viral modulatory effects on the human adaptive immunity. Humanized mice with long-term (more than 17 weeks) steady human T and B cell immune reconstitutions were infected with HCMV/GLuc and 7 weeks later were further treated with granulocyte-colony stimulating factor (G-CSF) to induce viral reactivations. Whole body bio-luminescence imaging analyses clearly differentiated mice with latent viral infections vs. reactivations. Foci of vigorous viral reactivations were detectable in liver, lymph nodes and salivary glands. The number of viral genome copies in various tissues increased upon reactivations and were detectable in sorted human CD14+, CD169+, and CD34+ cells. Compared with non-infected controls, mice after infections and reactivations showed higher thymopoiesis, systemic expansion of Th, CTL, Treg, and Tfh cells and functional antiviral T cell responses. Latent infections promoted vast development of memory CD4+ T cells while reactivations triggered a shift toward effector T cells expressing PD-1. Further, reactivations prompted a marked development of B cells, maturation of IgG+ plasma cells, and HCMV-specific antibody responses. Multivariate statistical methods were employed using T and B cell immune phenotypic profiles obtained with cells from several tissues of individual mice. The data was used to identify combinations of markers that could predict an HCMV infection vs. reactivation status. In spleen, but not in lymph nodes, higher frequencies of effector CD4+ T cells expressing PD-1 were among the factors most suited to distinguish HCMV reactivations from infections. These results suggest a shift from a T cell dominated immune response during latent infections toward an exhausted T cell phenotype and active humoral immune response upon reactivations. In sum, this novel in vivo humanized model combined with advanced analyses highlights a dynamic system clearly specifying the immunological spatial signatures of HCMV latency and reactivations. These signatures can be merged as predictive biomarker clusters that can be applied in the clinical translation of new therapies for the control of HCMV reactivation.


B cell class switch; HCMV; T cell maturation; humanized mice; linear discriminant analyses; optical imaging analyses; principal component analyses; reactivation

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