Mice with human immune system components as in vivo models for infections with human pathogens

Many pathogens relevant to human disease do not infect other animal species. Therefore, animal models that reconstitute or harbor human tissues are explored as hosts for these. In this review, we will summarize recent advances to utilize mice with human immune system components, reconstituted from hematopoietic progenitor cells in vivo. Such mice can be used to study human pathogens that replicate in leukocytes. In addition to studying the replication of these pathogens, the reconstituted human immune system components can also be analyzed for initiating immune responses and control against these infections. Moreover, these new animal models of human infectious disease should replicate the reactivity of the human immune system to vaccine candidates and, especially, the adjuvants contained in them, more faithfully

Adoptive transfer of EBV specific CD8+ T cell clones can transiently control EBV infection in humanized mice

Epstein Barr virus (EBV) infection expands CD8+ T cells specific for lytic antigens to high frequencies during symptomatic primary infection, and maintains these at significant numbers during persistence. Despite this, the protective function of these lytic EBV antigen-specific cytotoxic CD8+ T cells remains unclear. Here we demonstrate that lytic EBV replication does not significantly contribute to virus-induced B cell proliferation in vitro and in vivo in a mouse model with reconstituted human immune system components (huNSG mice). However, we report a trend to reduction of EBV-induced lymphoproliferation outside of lymphoid organs upon diminished lytic replication. Moreover, we could demonstrate that CD8+ T cells against the lytic EBV antigen BMLF1 can eliminate lytically replicating EBV-transformed B cells from lymphoblastoid cell lines (LCLs) and in vivo, thereby transiently controlling high viremia after adoptive transfer into EBV infected huNSG mice. These findings suggest a protective function for lytic EBV antigen-specific CD8+ T cells against EBV infection and against virus-associated tumors in extra-lymphoid organs. These specificities should be explored for EBV-specific vaccine development

Lytic replication does not significantly contribute to EBV transformation <i>in vitro</i>.

<p>Bulk PBMCs were inoculated with WT EBV, ZKO EBV or mock treated in the presence of cyclosporine A. (<b>A</b>) Cells were harvested and absolute cell numbers per well were determined on indicated days. (<b>B</b>) Outgrowth of transformed CD19⁺ B cells was monitored using flow cytometry. (<b>C</b>) Proliferation of EBV transformed B cells was demonstrated by CFSE dilution and the subsequent increase in percentage of CFSE^low B cells. The experiments were performed in duplicate and data is presented as the mean ± SEM. Similar results were obtained in samples from two donors; the average of two experiments is presented (p _{mock vs WT/ZKO} <0.0001 by two-way ANOVA).</p

Lytic EBV antigen specific CD8⁺ T cell clones can transiently control EBV infection in humanized mice.

<p>(<b>A</b>) Schematic diagram of adoptive transfer experiments. BMLF1- or LMP2-specific CD8⁺ T cell clones were transferred i.v. into humanized NSG-A2tg mice. The next day, mice were infected i.p. with WT EBV, ZKO EBV or PBS. Mice were bled weekly starting at week 2 post-infection and were euthanized at week 4 or 6 post-infection (n = 94). (<b>B</b>) Whole blood EBV loads for the second, third and fourth week post-infection of mice, which received BMLF1- or LMP2-specific CD8⁺ T cell clones prior to WT or ZKO EBV infection, is demonstrated for a representative experiment (cohort 2, n = 3–4 per group, total n = 21). Dotted line represents detection limit. (<b>C</b>) Representative figures and quantification of the ZEBRA⁺ cells in the spleen sections of WT EBV infected huNSG-A2tg mice, which received no T cells, BMLF1-specific T cell clones and LMP2-specific T cell clones for two initial experiments (n = 6–7 per group, total n = 20). Data represent the mean ± SEM (p _{no T vs BMLF1-T} <0.01, p _{BMLF1-T vs LMP2-T} = 0.07). Scale bars, 100 µm. (<b>D</b>) Whole blood EBV loads for the second, third and fourth week post-infection of mice from five independent adoptive transfer experiments (n = 94). Data are represented as the mean ± SEM. (<b>E</b>) Incidence of high blood viremia on the third week post-infection in the WT EBV infected mice, which received no T cells, BMLF1-specific T cell clone and LMP2-specific T cell clone (n.s. by one-way Chi-square). (<b>F</b>) Tumor incidence per organ is presented as the percentage of animals which developed visible tumors in the organs of the abdominal cavity (n = 92).</p

EBNA3B-deficient EBV promotes B cell lymphomagenesis in humanized mice and is found in human tumors

Epstein-Barr virus (EBV) persistently infects more than 90% of the human population and is etiologically linked to several B cell malignancies, including Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and diffuse large B cell lymphoma (DLBCL). Despite its growth transforming properties, most immune-competent individuals control EBV infection throughout their lives. EBV encodes various oncogenes, and of the 6 latency-associated EBV-encoded nuclear antigens, only EBNA3B is completely dispensable for B cell transformation in vitro. Here, we report that infection with EBV lacking EBNA3B leads to aggressive, immune-evading monomorphic DLBCL-like tumors in NOD/SCID/γc–/– mice with reconstituted human immune system components. Infection with EBNA3B-knockout EBV (EBNA3BKO) induced expansion of EBV-specific T cells that failed to infiltrate the tumors. EBNA3BKO-infected B cells expanded more rapidly and secreted less T cell–chemoattractant CXCL10, reducing T cell recruitment in vitro and T cell–mediated killing in vivo. B cell lines from 2 EBV-positive human lymphomas encoding truncated EBNA3B exhibited gene expression profiles and phenotypic characteristics similar to those of tumor-derived lines from the humanized mice, including reduced CXCL10 secretion. Screening EBV-positive DLBCL, HL, and BL human samples identified additional EBNA3B mutations. Thus, EBNA3B is a virus-encoded tumor suppressor whose inactivation promotes immune evasion and virus-driven lymphomagenesis

Lytic replication transiently influences viremia and may account to extra-lymphoid tumorigenesis in humanized mice.

<p>(<b>A</b>) Schematic diagram of <i>in vivo</i> infection experiments. Three cohorts of huNSG-A2tg mice were infected intraperitoneally (i.p.) with WT EBV, ZKO EBV or PBS, bled weekly starting from week 2 post-infection and euthanized at week 6. (<b>B</b>) Whole blood viral load at the indicated time points and presented as the mean ± SEM. Data for the initial four weeks represent composite data from 7 independent experimental cohorts of animals (n = 21–24 per group), data for the fifth and sixth represent composite data from 3 cohorts (n = 11–12 per group) (n.s. p _{WT vs ZKO} = 0.23 by two-way ANOVA; p _{week 3: WT vs ZKO}<0.05 unpaired <i>t</i> test with Welch's correction). Dotted line represents detection limit. (<b>C</b>) Peripheral blood composition was analyzed using flow cytometry and the inversion of CD8-CD4 ratio was assessed weekly during the course of infection. Data is presented as the mean ± SEM (p _{WT vs ZKO} <0.001 by two-way ANOVA). (<b>D</b>) EBV DNA load was determined in spleen biopsies and presented as the geometric mean (n.s. p = 0.4 by unpaired <i>t</i> test). (<b>E</b>) Lymphoproliferative lesions of B cell origin were found in the organs of the abdominal cavity of some infected animals (spleen, liver and kidneys). Tumor burden is shown as percentage of tumor-bearing animals per group (n.s. p _{WT vs ZKO} = 0.09 by two-way ANOVA, n.s. p _{liver:WT vs ZKO} = 0.08 and p _{kidney:WT vs ZKO} = 0.16 by one-way Chi-square). (<b>F</b>) Immunohistochemical analysis of tumor sections confirmed the EBV-associated B cell origin of tumors by CD20 (<b>left panel</b>) and EBNA2 (<b>right panel</b>) staining. Scale bars, 200 µm. (<b>G</b>) Splenomegaly was assessed by comparing spleen weight to body weight. Data represents the mean ± SEM (p<0.05). (<b>H</b>) The absolute number of CD3⁺ T cells in the spleen was determined at the termination time point and represented as the mean ± SEM (p _{PBS vs WT} <0.05, n.s. p _{PBS vs ZKO} = 0.06, n.s. p _{WT vs ZKO} = 0.69). (<b>I</b>) The inversion of CD8:CD4 ratio in the spleen was determined by flow cytometry and represented as the mean ± SEM (n.s. p _{WT vs ZKO} = 0.98, p _{PBS vs WT, PBS vs ZKO} <0.05). Figures C–E represent composite data from three independent experiments with a total of 26 animals. Figure B represents composite data from seven independent experiments with a total of 45 animals.</p

CD8⁺ T cell clones of lytic (BMLF1) and latent (LMP2) specificity were established by single cell sorting.

<p>(<b>A</b>) A dextramer positive population of live CD3⁺/CD8⁺ cells was sorted by flow cytometry from the blood of an HLA-A*0201 positive healthy EBV carrier. Flow cytometry plots show the frequency of starting populations within CD8⁺ T cells. (<b>B</b>) After two rounds of PHA re-expansions of sorted single cells, the purity of the T cell clones was confirmed by staining for T cell receptor specificity (MHC-dextramers), CD3, CD4, and CD8. Representative dextramer staining plots for three clones of LMP2-specificity (<b>dark grey</b>) and three clones of BMLF1-specificity (<b>black</b>) are shown, together with an overlay of control staining with an irrelevant dextramer (<b>light grey</b>). (<b>C</b>) Epitope avidity was determined in peptide titration assays for BMLF1-specifc clones (<b>top</b>) and LMP2-specific clones (<b>bottom</b>). The presence of IFNγ in supernatants upon stimulation with various concentrations of cognate peptide was assessed by ELISA and is presented as the mean ± SEM. Assays were performed in duplicate. (<b>D</b>) The ability to recognize HLA-A*02 negative WT LCLs, autologous WT LCLs and ZKO LCLs as well as peptide-pulsed WT LCLs was assessed for BMLF1-specific clones (<b>top panels</b>) and LMP2-specific clones (<b>bottom panel</b>) by IFNγ ELISA of supernatants after co-culture (n.d. – not detected). (<b>E</b>) FACS histograms of intracellular perforin and granzyme B staining for selected clones after stimulation with LMP2 (<b>grey</b>) and BMLF1 (<b>black</b>) peptides. An isotype control for intracellular staining is represented as a shaded grey histogram.</p

CD141+ dendritic cells produce prominent amounts of IFN-α after dsRNA recognition and can be targeted via DEC-205 in humanized mice

Functional differences between human dendritic cell (DC) subsets and the potential benefits of targeting them with vaccines remain poorly defined. Here we describe that mice with reconstituted human immune system components (huNSG mice) develop all human conventional and plasmacytoid DC compartments in lymphoid organs. Testing different Toll-like receptor agonists for DC maturation in vivo, we found that IL-12p70 and interferon (IFN)-α production correlated with the maturation of CD141+ (BDCA3+) conventional DCs in huNSG mice. Furthermore, depletion of CD141+ DCs before stimulation significantly reduced IFN-α levels in vivo. This DC subset produced similar total amounts but different subtypes of IFN-α in response to synthetic double-stranded RNA compared with plasmacytoid DCs in response to a single-stranded RNA equivalent. Moreover, synthetic double-stranded RNA as adjuvant and antigen targeting to the endocytic receptor DEC-205, a combination that focuses antigen presentation for T-cell priming on CD141+ DCs, stimulated antigen-specific human CD4+ T-cell responses. Thus, the human CD141+ DC subset is a prominent source of IFN-α and interleukin-12 production and should be further evaluated for vaccine development

Persistent KSHV Infection Increases EBV-Associated Tumor Formation In Vivo via Enhanced EBV Lytic Gene Expression

The human tumor viruses Epstein-Barr virus (EBV) and Kaposi sarcoma-associated herpesvirus (KSHV) establish persistent infections in B cells. KSHV is linked to primary effusion lymphoma (PEL), and 90% of PELs also contain EBV. Studies on persistent KSHV infection in vivo and the role of EBV co-infection in PEL development have been hampered by the absence of small animal models. We developed mice reconstituted with human immune system components as a model for KSHV infection and find that EBV/KSHV dual infection enhanced KSHV persistence and tumorigenesis. Dual-infected cells displayed a plasma cell-like gene expression pattern similar to PELs. KSHV persisted in EBV-transformed B cells and was associated with lytic EBV gene expression, resulting in increased tumor formation. Evidence of elevated lytic EBV replication was also found in EBV/KSHV dually infected lymphoproliferative disorders in humans. Our data suggest that KSHV augments EBV-associated tumorigenesis via stimulation of lytic EBV replication

Human NK cells of mice with reconstituted human immune system components require preactivation to acquire functional competence

To investigate human natural killer (NK)–cell reactivity in vivo we have reconstituted human immune system components by transplantation of human hematopoietic progenitor cells into NOD-scid IL2Rγnull mice. We demonstrate here that this model allows the development of all NK-cell subsets that are also found in human adult peripheral and cord blood, including NKp46+CD56− NK cells. Similar to human cord blood, NK cells from these reconstituted mice require preactivation by interleukin-15 to reach the functional competence of human adult NK cells. Mainly the terminally differentiated CD16+ NK cells demonstrate lower reactivity without this stimulation. After preactivation, both CD16+ and CD16− NK cells efficiently produce interferon-γ and degranulate in response to stimulation with NK cell–susceptible targets, including K562 erythroleukemia cells. NK-cell lines, established from reconstituted mice, demonstrate cytotoxicity against this tumor cell line. Importantly, preactivation can as well be achieved by bystander cell maturation via poly I:C stimulation in vitro and injection of this maturation stimulus in vivo. Preactivation in vivo enhances killing of human leukocyte antigen class I negative tumor cells after their adoptive transfer. These data suggest that a functional, but resting, NK-cell compartment can be established in immune-compromised mice after human hematopoietic progenitor cell transfer