Adult T-cell leukemia/lymphoma (ATLL) is an aggressive malignancy of CD4+ T-cells associated with HTLV-1 infection. In this study, we used the model of immunodeficient NSG mice reconstituted with a functional human immune system (HIS) to investigate early events in HTLV-1 pathogenesis. Upon infection, human T-cells rapidly increased in the blood and lymphoid tissues, particularly CD4+CD25+ T-cells. Proliferation of CD4+ T-cells in the spleen and mesenteric lymph nodes (MLN) correlated with HTLV-1 proviral load and CD25 expression. In addition, splenomegaly, a common feature of ATLL in humans, was also observed. CD4+ and CD8+ T-cells predominantly displayed an effector memory phenotype (CD45RA−CCR7−) and expressed CXCR3 and CCR5 chemokine receptors, suggesting the polarization into a Th1 phenotype. Activated CD8+ T-cells expressed granzyme B and perforin; however, the interferon-γ response by these cells was limited, possibly due to elevated PD-1 expression and increased frequency of CD4+FoxP3+ regulatory T-cells in MLN. Thus, HTLV-1-infected HIS-NSG mice reproduced several characteristics of infection in humans, and it may be helpful to investigate ATLL-related events and to perform preclinical studies. Moreover, aspects of chronic infection were already present at early stages in this experimental model. Collectively, we suggest that HTLV-1 infection modulates host immune responses to favor viral persistence.

Recent studies have shown the potential of broadly neutralizing antibodies (bnAbs) for HIV-1 treatment. One of the candidate antibodies moving into clinical trials is the bnAb PGDM1400. Here, we studied the therapeutic potency and escape pathways of bnAb PGDM1400 during monovalent therapy in human immune system (HIS) mice using the BG505, REJO, MJ4 and AMC008 virus isolates. PGDM1400 administered during chronic infection caused a modest decrease in viral load in the first week of administration in 7 out of 10 animals, which correlated with the in vitro neutralization sensitivity of the viruses to PGDM1400. As expected for monotherapy, viral loads rebounded after about a week and different viral escape pathways were observed, involving the deletion of glycans in the envelope glycoprotein at positions 130 or 160. (Pre)clinical trials should reveal whether PGDM1400 is a useful component of an antibody combination treatment or as part of a tri-specific antibody.

NOTCH1 is a prevalent signaling pathway in T cell acute lymphoblastic leukemia (T-ALL), but crucial NOTCH1 downstream signals and target genes contributing to T-ALL pathogenesis cannot be retrospectively analyzed in patients and thus remain ill defined. This information is clinically relevant, as initiating lesions that lead to cell transformation and leukemia-initiating cell (LIC) activity are promising therapeutic targets against the major hurdle of T-ALL relapse. Here, we describe the generation in vivo of a human T cell leukemia that recapitulates T-ALL in patients, which arises de novo in immunodeficient mice reconstituted with human hematopoietic progenitors ectopically expressing active NOTCH1. This T-ALL model allowed us to identify CD44 as a direct NOTCH1 transcriptional target and to recognize CD44 overexpression as an early hallmark of preleukemic cells that engraft the BM and finally develop a clonal transplantable T-ALL that infiltrates lymphoid organs and brain. Notably, CD44 is shown to support crucial BM niche interactions necessary for LIC activity of human T-ALL xenografts and disease progression, highlighting the importance of the NOTCH1/CD44 axis in T-ALL pathogenesis. The observed therapeutic benefit of anti-CD44 antibody administration in xenotransplanted mice holds great promise for therapeutic purposes against T-ALL relapse.

Broadly neutralizing antibodies (bNAbs) such as PGDM1400 show promise as prophylactic and therapeutic agents against HIV-1. Human immune system mice were passively immunized with different doses of PGDM1400 and challenged 24h later with a high dose of HIV-1(JRCSF). We found that PGDM1400 provided protection against HIV-1 challenge in a concentration dependent manner and that the protective concentration in blood was similar to 75-fold higher than the in vitro 50% inhibitory concentration. The results demonstrate that PGDM1400 might be a promising component of strategies to prevent HIV-1 infection and provide support for the pursuit of vaccines that induce PGDM1400-like bNAbs.

Mesenchymal stromal cells (MSCs) are applied as novel therapeutics for their regenerative and immune-suppressive capacities. Clinical applications, however, require extensive expansion of MSCs. Fetal bone marrow-derived MSCs (FBMSCs) proliferate faster than adult bone marrow-derived MSC (ABMSCs). To optimize expansion and function of MSC in general, we explored the differences between ABMSC and FBMSC. Gene expression profiling implicated differential expression of genes encoding proteins in the Wnt signaling pathway, including excreted inhibitors of Wnt signaling, particularly by ABMSC. Both MSC types had a similar basal level of canonical Wnt signaling. Abrogation of autocrine Wnt production by inhibitor of Wnt production-2 (IWP2) reduced canonical Wnt signaling and cell proliferation of FBMSCs, but hardly affected ABMSC. Addition of exogenous Wnt3a, however, induced expression of the target genes lymphocyte enhancer-binding factor (LEF) and T-cell factor (TCF) faster and at lower Wnt3a levels in ABMSC compared to FBMSC. Medium replacement experiments indicated that ABMSC produce an inhibitor of Wnt signaling that is effective on ABMSC itself but not on FBMSC, whereas FBMSC excrete (Wnt) factors that stimulate proliferation of ABMSC. In contrast, FBMSC were not able to support hematopoiesis, whereas ABMSC displayed hematopoietic support sensitive to IWP2, the inhibitor of Wnt factor excretion. In conclusion, ABMSC and FBMSC differ in their Wnt signature. While FBMSC produced factors, including Wnt signals, that enhanced MSC proliferation, ABMSC produced Wnt factors in a setting that enhanced hematopoietic support. Thus, further unraveling the molecular basis of this phenomenon may lead to improvement of clinical expansion protocols of ABMSCs.

Human adult bone marrow-derived mesenchymal stromal cells MSC (ABMSC) are frequently applied cells in the clinic, but fetal bone marrow-derived MSC (FBMSC) share most of immune-modulatory and regenerative capacities with ABMSCs. The proliferation capacity of FBMSC, is much larger than that of ABMSC. Unraveling the underlying molecular mechanism governing this process in FBMSCs may enable us to improve clinical expansion protocols for ABMSCs. Differential gene expression comparing ABMSCs and FBMSCs identified 687 differentially expressed genes from which 16 were Wnt-related, mainly Wnt-inhibitors and Frizzled receptors. Therefore we investigated the role of Wnt signaling in adult and fetal bone marrow-derived MSC. Wnt3a induced a concentration dependent increase of the canonical Wnt-target genes TCF and LEF in both ABMSC and FBMSC. However, ABMSC responded faster, and at a lower concentration of Wnt3a compared to FBMSC. In addition, Wnt3a increased the proliferation of ABMSC, but not of FBMSC. Interestingly, a complete medium change was sufficient to increase TCF/LEF expression in ABMSC, but not in FBMSC, suggesting that ABMSC produced a soluble Wnt-inhibitor. Moreover, switching MSC conditioned medium (CM) between FBMSC and ABMSC indicated that FBMSC-CM significantly stimulated the expansion of ABMSC while the reverse experiments did not show an inhibiting effect of ABMSC-CM on the expansion of FBMSC. Mass Spec analysis was performed on CM from both ABMSCs and FBMSCs. Preliminary data indicate that both MSC sources produce similar amounts of proteins, but 400 proteins exclusively produced by FBMSCs were detected. With GEO term analysis we linked those proteins back to the Wnt- and notch pathway. Two pathways involved in cell proliferation and differentiation.Thus pursuing proteomics on vesicle/exosome enriched CMs may enable us to narrow down the search for FBMSC derived factor(s) that lead to improvement of clinical expansion protocols of ABMSCs.

Hepatic infections by hepatitis B virus (HBV), hepatitis C virus (HCV) and Plasmodium parasites leading to acute or chronic diseases constitute a global health challenge. The species tropism of these hepatotropic pathogens is restricted to chimpanzees and humans, thus model systems to study their pathological mechanisms are severely limited. Although these pathogens infect hepatocytes, disease pathology is intimately related to the degree and quality of the immune response. As a first step to decipher the immune response to infected hepatocytes, we developed an animal model harboring both a human immune system (HIS) and human hepatocytes (HUHEP) in BALB/c Rag2-/- IL-2Rγc-/- NOD.sirpa uPAtg/tg mice. The extent and kinetics of human hepatocyte engraftment were similar between HUHEP and HIS-HUHEP mice. Transplanted human hepatocytes were polarized and mature in vivo, resulting in 20-50% liver chimerism in these models. Human myeloid and lymphoid cell lineages developed at similar frequencies in HIS and HIS-HUHEP mice, and splenic and hepatic compartments were humanized with mature B cells, NK cells and naïve T cells, as well as monocytes and dendritic cells. Taken together, these results demonstrate that HIS-HUHEP mice can be stably (> 5 months) and robustly engrafted with a humanized immune system and chimeric human liver. This novel HIS-HUHEP model provides a platform to investigate human immune responses against hepatotropic pathogens and to test novel drug strategies or vaccine candidates.

Human adult bone marrow-derived mesenchymal stromal cells MSC (ABMSC) are increasingly applied in the clinic to decrease graft versus host disease and to enhance hematopoietic recovery. Fetal bone marrow-derived MSC (FBMSC) display similar immune suppressive and regenerative capacities as adult MSC, and have been transplanted into patients. The proliferation capacity of FBMSC, however, is much larger than that of ABMSC. The aim of our studies is to understand the molecular mechanism of proliferation and hematopoietic support by MSC to optimize the expansion of functional MSC for clinical use. Comparison of gene expression between ABMSCs and FBMSCs identified 687 differentially expressed genes. Of these, 16 were Wnt-related, mainly Wnt-inhibitors and Frizzled receptors. Expression of SFRP4, WISP1, WISP2, WISP3, FZD1, FZD5, FZD8 and MYCBP2 was upregulated in ABMSC, whereas DKK1, DKK2, CCND2, WNT5a, MYC, FZD2, FZD6 and FZD7 are expressed at a higher level in FBMSC. Although the expression of few genes (e.g. DKK1) was culture density dependent, other genes such as Wnt5a, DKK2 and SFRP4 were consistently differentially expressed independent of culture conditions. Therefore we investigated the role of Wnt signaling in adult and fetal bone marrow-derived MSC. Wnt3a induced a concentration dependent increase of the canonical Wnt-target genes TCF and LEF in both ABMSC and FBMSC. However, ABMSC responded faster, and at a lower concentration of Wnt3a compared to FBMSC. In addition, Wnt3a increased the proliferation of ABMSC, but not of FBMSC. Interestingly, a complete medium change was already sufficient to increase TCF/LEF expression in ABMSC, but not in FBMSC, suggesting that ABMSC produced a soluble Wnt-inhibitor. Moreover, switching MSC conditioned medium between FBMSC and ABMSC indicated that FBMSC conditioned medium significantly stimulated the expansion of ABMSC while the reverse experiments did not show an inhibiting effect of ABMSC conditioned medium on the expansion of FBMSC. Thus, ABMSC produce a factor that only affects ABMSC, but not (the factors produced by) FBMSC. To block autocrine Wnt production, MSC were exposed for 48 h to the Inhibitor of Wnt Production 2 (IWP2). Abrogation of Wnt-production in FBMSC modestly decreased beta-catenin expression, and strongly decreased TCF/LEF expression, but did not affect ABMSC. Addition of IWP-2 to long-term cultures strongly inhibited proliferation of FBMSCs compared to ABMSCs. To unravel the role of MSC-produced Wnt factors in hematopoiesis, we co-cultured adult or fetal MSCs together with cord blood derived CD34+ cells in the presence or absence of IWP2 inhibitor. Addition of IWP2 to ABMSC decreased the short term support of hematopoietic stem and progenitors (HSPC), while IWP2 did not affect the support of HSPCs by FBMSC. Overall, ABMSCs provided a significant better short term hematopoietic support than FBMSCs. In conclusion, our data demonstrate that ABMSC produce both Wnt factors and inhibitors. FBMSC, in contrast, produce Wnt-related factors that seem to contribute more to the expansion capacity of FBMSC than to their hematopoietic support. To identify factors we current use mass spectroscopy of supernatant to determine the secretome. Modulation of (parts) of the Wnt pathway may improve clinical expansion protocols of ABMSC. Disclosures No relevant conflicts of interest to declare.

Highly active antiretroviral therapy (HAART) has significantly improved the quality of life and the life expectancy of HIV-infected individuals. Still, drug-induced side effects and emergence of drug-resistant viral variants remain important issues that justify the exploration of alternative therapeutic options. One strategy consists of a gene therapy based on RNA interference to induce the sequence-specific degradation of the HIV-1 RNA genome. We have selected four potent short hairpin RNA (shRNA) candidates targeting the viral capside, integrase, protease and tat/rev open-reading frames and screened the safety of them during human hematopoietic cell development, both in vitro and in vivo. Although the four shRNA candidates appeared to be safe in vitro, one shRNA candidate impaired the in vivo development of the human immune system in Balb/c Rag2(-/-)IL-2R gamma(-/-)(BRG) mice. The three remaining shRNA candidates were combined into one single lentiviral vector (LV), and safety of the shRNA combination during human hematopoietic cell development was confirmed. Overall, we demonstrate here the preclinical in vivo safety of a LV expressing three shRNAs against HIV-1, which is proposed for a future Phase I clinical trial.

Innate lymphoid cells are cytokine-producing cells that contribute to tissue homeostasis. Spits and colleagues identify a human innate cell population that expresses T-bet and IFN-γ and is prevalent in Crohn's disease.

Sézary syndrome ( SS ) is an aggressive cutaneous T ‐cell lymphoma with CD 4+ tumor cells localized in the skin, lymph nodes and peripheral blood. Characteristic molecular aberrancies in SS have been identified; however, paucity of functional models severely hampered the translation of these observations into pathogenic mechanisms, and subsequent validation of novel therapeutic targets. We therefore developed a mouse model for SS using intrahepatic injection of SS cells in newborn immunodeficient RAG 2 −/− γc −/− mice that are completely devoid of T‐, B‐ and NK ‐cell activity. Injection of the SS cell line SeAx led to long‐term and reproducible systemic repopulation of the mice. Injection of mice with the SS cell line HuT‐78 led to the death of the mice owing to massive growth of internal tumors. Four weeks after injection of primary SS cells, human CD 3+ T cells could be tracked back in the liver, peripheral blood, lymph nodes, spleen and skin of the mice, although the engraftment rate varied when using cells from different patients. In conclusion, we demonstrate that injection of SS cell lines or primary cells in newborn RAG 2 −/− γc −/− mice results in long‐term systemic repopulation of the mice, thereby providing a novel mouse model for S ézary syndrome.

A novel genetic approach for the control of virus replication was used for the design of a conditionally replicating human immunodeficiency virus (HIV) variant, HIV-rtTA. HIV-rtTA gene expression and virus replication are strictly dependent on the presence of a non-toxic effector molecule, doxycycline (dox), and thus can be turned on and off at will in a graded and reversible manner. The in vivo replication capacity, pathogenicity and genetic stability of this HIV-rtTA variant were evaluated in a humanized mouse model of haematopoiesis that harbours lymphoid and myeloid components of the human immune system (HIS). Infection of dox-fed BALB Rag/γc HIS (BRG-HIS) mice with HIV-rtTA led to the establishment of a productive infection without CD4(+) T-cell depletion. The virus did not show any sign of escape from dox control for up to 10 weeks after the onset of infection. No reversion towards a functional Tat-transactivating responsive (TAR) RNA element axis was observed, confirming the genetic stability of the HIV-rtTA variant in vivo. These results demonstrate the proof of concept that HIV-rtTA replicates efficiently in vivo. HIV-rtTA is a promising tool for fundamental research to study virus-host interactions in vivo in a controlled fashion.

Background HIV-1 is still a major public health problem and one of the priorities of the World Health Organization. The development of HAART against HIV was a considerable advance for infected individuals, but this life-long treatment does only block virus replication, and no viral eradication is obtained. Furthermore, HAART may exhibit long-term toxicity and may eventually lead to the emergence of drug-resistant viral variants. We explore a new durable therapeutic intervention based on a gene therapy that induces RNA interference (RNAi) against HIV1. In this pre-clinical research setting, “humanized” experimental mouse models are of interest considering the relative ease of handling and relatively low cost as compared to non-human primates. Methods

A girl presented during childhood with a single course of extensive chickenpox and moderate albeit recurrent pneumonia in the presence of idiopathic CD4+ T lymphocytopenia (ICL). Her clinical condition remained stable over the past 10 years without infections, any granulomatous disease, or autoimmunity. Immunophenotyping demonstrated strongly reduced naive T and B cells with intact proliferative capacity. Antibody reactivity on in vivo immunizations was normal. T-cell receptor-Vβ repertoire was polyclonal with a very low content of T-cell receptor excision circles (TRECs). Kappa-deleting recombination excision circles (KRECs) were also abnormal in the B cells. Both reflect extensive in vivo proliferation. Patient-derived CD34+ hematopoietic stem cells could not repopulate RAG2(-/-)IL2Rγc(-/-) mice, indicating the lymphoid origin of the defect. We identified 2 novel missense mutations in RAG1 (p.Arg474Cys and p.Leu506Phe) resulting in reduced RAG activity. This report gives the first genetic clue for ICL and extends the clinical spectrum of RAG mutations from severe immune defects to an almost normal condition.

Human Immune System (HIS) mice represent a novel biotechnology platform to dissect human haematopoiesis and immune responses. However, the limited human T‐cell development that is observed in HIS mice restricts its utility for these applications. Here, we address whether reduced thymopoiesis in HIS mice reflects an autonomous defect in T‐cell precursors and/or a defect in the murine thymic niche. Human thymocyte precursors seed the mouse thymus and their reciprocal interactions with murine thymic epithelial cells (TECs) led to both T‐cell and TEC maturation. The human thymocyte subsets observed in HIS mice demonstrated survival, proliferative and phenotypic characteristics of their normal human counterparts, suggesting that the intrinsic developmental program of human thymocytes unfolds normally in this xenograft setting. We observed that exogenous administration of human IL‐15/IL‐15Rα agonistic complexes induced the survival, proliferation and absolute numbers of immature human thymocyte subsets, without any obvious effect on cell‐surface phenotype or TCR Vβ usage amongst the newly selected mature single‐positive (SP) thymocytes. Finally, when IL‐15 was administered early after stem cell transplantation, we noted accelerated thymopoiesis resulting in the more rapid appearance of peripheral naïve T cells. Our results highlight the functional capacity of murine thymic stroma cells in promoting human thymopoiesis in HIS mice but suggest that the “cross‐talk” between murine thymic stroma and human haematopoietic precursors may be suboptimal. As IL‐15 immunotherapy promotes early thymopoiesis, this novel approach could be used to reduce the period of T‐cell immunodeficiency in the post‐transplant clinical setting.

The development and antigen-dependent differentiation of B lymphocytes are orchestrated by an array of growth factors, cytokines, and chemokines that require tight spatiotemporal regulation. Heparan sulfate proteoglycans specifically bind and regulate the bioavailability of soluble protein ligands, but their role in the immune system has remained largely unexplored. Modification of heparan sulfate by glucuronyl C5-epimerase (Glce) controls heparan sulfate-chain flexibility and thereby affects ligand binding. Here we show that Glce deficiency impairs B-cell maturation, resulting in decreased plasma cell numbers and immunoglobulin levels. We demonstrate that C5-epimerase modification of heparan sulfate is critical for binding of a proliferation inducing ligand (APRIL) and that Glce-deficient plasma cells fail to respond to APRIL-mediated survival signals. Our results identify heparan sulfate proteoglycans as novel players in B-cell maturation and differentiation and suggest that heparan sulfate conformation is crucial for recruitment of factors that control plasma cell survival.