Longitudinal clonal tracking in humanized mice reveals sustained polyclonal repopulation of gene-modified human-HSPC despite vector integration bias

Background Current understanding of hematopoiesis is largely derived from mouse models that are physiologically distant from humans. Humanized mice provide the most physiologically relevant small animal model to study human diseases, most notably preclinical gene therapy studies. However, the clonal repopulation dynamics of human hematopoietic stem and progenitor cells (HSPC) in these animal models is only partially understood. Using a new clonal tracking methodology designed for small sample volumes, we aim to reveal the underlying clonal dynamics of human cell repopulation in a mouse environment. Methods Humanized bone marrow-liver-thymus (hu-BLT) mice were generated by transplanting lentiviral vector-transduced human fetal liver HSPC (FL-HSPC) in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice implanted with a piece of human fetal thymus. We developed a methodology to track vector integration sites (VIS) in a mere 25 µl of mouse blood for longitudinal and quantitative clonal analysis of human HSPC repopulation in mouse environment. We explored transcriptional and epigenetic features of human HSPC for possible VIS bias. Results A total of 897 HSPC clones were longitudinally tracked in hu-BLT mice—providing a first-ever demonstration of clonal dynamics and coordinated expansion of therapeutic and control vector-modified human cell populations simultaneously repopulating in the same humanized mice. The polyclonal repopulation stabilized at 19 weeks post-transplant and the contribution of the largest clone doubled within 4 weeks. Moreover, 550 (~ 60%) clones persisted over 6 weeks and were highly shared between different organs. The normal clonal profiles confirmed the safety of our gene therapy vectors. Multi-omics analysis of human FL-HSPC revealed that 54% of vector integrations in repopulating clones occurred within ± 1 kb of H3K36me3-enriched regions. Conclusions Human repopulation in mice is polyclonal and stabilizes more rapidly than that previously observed in humans. VIS preference for H3K36me3 has no apparent negative effects on HSPC repopulation. Our study provides a methodology to longitudinally track clonal repopulation in small animal models extensively used for stem cell and gene therapy research and with lentiviral vectors designed for clinical applications. Results of this study provide a framework for understanding the clonal behavior of human HPSC repopulating in a mouse environment, critical for translating results from humanized mice models to the human settings.

TAILORED DC INDUCE PROTECTIVE HIV-1 SPECIFIC POLYFUNCTIONAL CD8+ T CELLS IN THE LYMPHOID TISSUE FROM HUMANIZED BLT MICE

Effective function of CD8+ T cells and enhanced innate activation of dendritic cells (DC) in response to HIV-1 is linked to protective antiviral immunity in controllers. Manipulation of DC targeting the master regulator TANK-binding Kinase 1 (TBK1) might be useful to acquire controller-like properties. Here, we evaluated the impact of TBK1-primed DC inducing protective CD8+ T cell responses in lymphoid tissue and peripheral blood and their association with reduced HIV-1 disease progression in vivo in the humanized bone marrow, liver and thymus (hBLT) mouse model. A higher proportion of hBLT-mice vaccinated with TBK1-primed DC exhibited less severe CD4+ T cell depletion following HIV-1 infection compared to control groups. This was associated with infiltration of CD8+ T cells in the white pulp from the spleen, reduced spread of infected p24+ cells to secondary lymphoid organs and with preserved abilities of CD8+ T cells from the spleen and blood of vaccinated animals to induce specific polyfunctional responses upon antigen stimulation. Therefore, TBK1-primed DC might be an useful tool for subsequent vaccine studies.

Human Microglia Extensively Reconstitute in Humanized-BLT Mice With Human Interleukin-34 Transgene and Support HIV-1 Brain Infection

Humanized bone marrow-liver-thymic (hu-BLT) mice develop a functional immune system in periphery, nevertheless, have a limited reconstitution of human myeloid cells, especially microglia, in CNS. Further, whether bone marrow derived hematopoietic stem and progenitor cells (HSPCs) can enter the brain and differentiate into microglia in adults remains controversial. To close these gaps, in this study we unambiguously demonstrated that human microglia in CNS were extensively reconstituted in adult NOG mice with human interleukin-34 transgene (hIL34 Tg) from circulating CD34+ HSPCs, nonetheless not in hu-BLT NOG mice, providing strong evidence that human CD34+ HSPCs can enter adult brain and differentiate into microglia in CNS in the presence of hIL34. Further, the human microglia in the CNS of hu-BLT-hIL34 NOG mice robustly supported HIV-1 infection reenforcing the notion that microglia are the most important target cells of HIV-1 in CNS and demonstrating its great potential as an in vivo model for studying HIV-1 pathogenesis and evaluating curative therapeutics in both periphery and CNS compartments.

Human microglia extensively reconstitute in humanized BLT mice with human interleukin-34 transgene and support HIV-1 brain infection

Humanized bone marrow-liver-thymic (hu-BLT) mice develop a functional immune system in periphery but have a limited reconstitution of human myeloid cells, especially microglia, in CNS. Further, whether bone marrow derived hematopoietic stem and progenitor cells (HSPCs) can enter the brain and differentiate into microglia in adults remains controversial. To close these gaps, in this study we unambiguously demonstrated that human microglia in CNS were extensively reconstituted in adult NOG mice with human interleukin-34 transgene (hIL34 Tg) from circulating CD34+ HSPCs but no in hu-BLT NOG mice, providing strong evidence that human CD34+ HSPCs can enter adult brain and differentiate into microglia in CNS in the presence of hIL34. Further, the human microglia in the CNS of hu-BLT-hIL34 NOG mice robustly supported HIV-1 infection reenforcing the notion that microglia are the most important target cells of HIV-1 in CNS and demonstrating its great potential as an in vivo model for studying HIV-1 pathogenesis and evaluating curative therapeutics in both periphery and CNS compartments.

Osteoclast-expanded super-charged NK-cells preferentially select and expand CD8+ T cells

Osteoclasts (OCs) and much less dendritic cells (DCs) induce significant expansion and functional activation of NK cells, and furthermore, the OC-expanded NK cells preferentially increase the expansion and activation of CD8+ T cells by targeting CD4+ T cells. When autologous OCs were used to expand patient NK cells much lower percentages of expanded CD8+ T cells, decreased numbers of expanded NK cells and decreased functions of NK cells could be observed, and the addition of allogeneic healthy OCs increased the patients’ NK function. Mechanistically, OC-expanded NK cells were found to lyse CD4+ T cells but not CD8+ T cells suggesting potential selection of CD8+ T cells before their expansion by OC activated NK cells. In agreement, Increased IFN-γ secretion, and NK cell-mediated cytotoxicity and higher percentages of CD8+ T cells, in various tissue compartments of oral tumor-bearing hu-BLT mice in response to immunotherapy by OC-expanded NK cells were observed. Thus, our results indicate an important relationship between NK and CD8+ T cells.

Probiotic-Treated Super-Charged NK Cells Efficiently Clear Poorly Differentiated Pancreatic Tumors in Hu-BLT Mice

Background and Aims: We have previously demonstrated that the stage of differentiation of tumors has profound effect on the function of NK cells, and that stem-like/poorly differentiated tumors were preferentially targeted by the NK cells. Therefore, in this study we determined the role of super-charged NK cells in immune mobilization, lysis, and differentiation of stem-like/undifferentiated tumors implanted in the pancreas of humanized-BLT (hu-BLT) mice fed with or without AJ2 probiotics. The phenotype, growth rate and metastatic potential of pancreatic tumors differentiated by the NK cells (NK-differentiated) or patient derived differentiated or stem-like/undifferentiated pancreatic tumors were investigated. Methods: Pancreatic tumor implantation was performed in NSG and hu-BLT mice. Stage of differentiation of tumors was determined using our published criteria for well-differentiated tumors exhibiting higher surface expression of MHC- class I, CD54, and PD-L1 (B7H1) and lower expression of CD44 receptors. The inverse was seen for poorly-differentiated tumors. Results: Stem-like/undifferentiated pancreatic tumors grew rapidly and formed large tumors and exhibited lower expression of above-mentioned differentiation antigens in the pancreas of NSG and hu-BLT mice. Unlike stem-like/undifferentiated tumors, NK-differentiated MP2 (MiaPaCa-2) tumors or patient-derived differentiated tumors were not able to grow or grew smaller tumors, and were unable to metastasize in NSG or hu-BLT mice, and they were susceptible to chemotherapeutic drugs. Stem-like/undifferentiated pancreatic tumors implanted in the pancreas of hu-BLT mice and injected with super-charged NK cells formed much smaller tumors, proliferated less, and exhibited differentiated phenotype. When differentiation of stem-like tumors by the NK cells was prevented by the addition of antibodies to IFN-γ and TNF-α, tumors grew rapidly and metastasized, and they remained resistant to chemotherapeutic drugs. Greater numbers of immune cells infiltrated the tumors of NK-injected and AJ2-probiotic bacteria-fed mice. Moreover, increased IFN-γ secretion in the presence of decreased IL-6 was seen in tumors resected and cultured from NK-injected and AJ2 fed mice. Tumor-induced decreases in NK cytotoxicity and IFN-γ secretion were restored/increased within PBMCs, spleen, and bone marrow when mice received NK cells and were fed with AJ2. Conclusion: NK cells prevent growth of pancreatic tumors through lysis and differentiation, thereby curtailing the growth and metastatic potential of stem-like/undifferentiated-tumors.

Mechanisms of virus dissemination in bone marrow of HIV-1–infected humanized BLT mice

Immune progenitor cells differentiate in bone marrow (BM) and then migrate to tissues. HIV-1 infects multiple BM cell types, but virus dissemination within BM has been poorly understood. We used light microscopy and electron tomography to elucidate mechanisms of HIV-1 dissemination within BM of HIV-1–infected BM/liver/thymus (BLT) mice. Tissue clearing combined with confocal and light sheet fluorescence microscopy revealed distinct populations of HIV-1 p24-producing cells in BM early after infection, and quantification of these populations identified macrophages as the principal subset of virus-producing cells in BM over time. Electron tomography demonstrated three modes of HIV-1 dissemination in BM: (i) semi-synchronous budding from T-cell and macrophage membranes, (ii) mature virus association with virus-producing T-cell uropods contacting putative target cells, and (iii) macrophages engulfing HIV-1–producing T-cells and producing virus within enclosed intracellular compartments that fused to invaginations with access to the extracellular space. These results illustrate mechanisms by which the specialized environment of the BM can promote virus spread locally and to distant lymphoid tissues.

Immunization of BLT Humanized Mice Redirects T Cell Responses to Gag and Reduces Acute HIV-1 Viremia

ABSTRACT BLT (bone marrow-liver-thymus) humanized mice, which reconstitute a functional human immune system, develop prototypic human virus-specific CD8+ T cell responses following infection with human immunodeficiency virus type 1 (HIV-1). We explored the utility of the BLT model for HIV-1 vaccine development by immunizing BLT mice against the conserved viral Gag protein, utilizing a rapid prime-boost protocol of poly(lactic-co-glycolic) acid microparticles and a replication-defective herpes simplex virus (HSV) recombinant vector. After HIV-1 challenge, the mice developed broad, proteome-wide gamma interferon-positive (IFN-γ+) T cell responses against HIV-1 that reached magnitudes equivalent to what is observed in HIV-1-infected individuals. The functionality of these responses was underscored by the consistent emergence of escape mutations in multiple CD8+ T cell epitopes during the course of infection. Although prechallenge vaccine-induced responses were largely undetectable, the Gag immunization increased both the magnitude and the kinetics of anamnestic Gag-specific T cell responses following HIV-1 infection, and the magnitude of these postchallenge Gag-specific responses was inversely correlated with acute HIV-1 viremia. Indeed, Gag immunization was associated with a modest but significant 0.5-log reduction in HIV-1 viral load when analyzed across four experimental groups of BLT mice. Notably, the HSV vector induced elevated plasma concentrations of polarizing cytokines and chemotactic factors, including interleukin-12p70 (IL-12p70) and MIP-1α, which were positively correlated with the magnitude of Gag-specific responses. Overall, these results support the ability of BLT mice to recapitulate human pathogen-specific T cell responses and to respond to immunization; however, additional improvements to the model are required to develop a robust system for testing HIV-1 vaccine efficacy. IMPORTANCE Advances in the development of humanized mice have raised the possibility of a small-animal model for preclinical testing of an HIV-1 vaccine. Here, we describe the capacity of BLT humanized mice to mount broadly directed HIV-1-specific human T cell responses that are functionally active, as indicated by the rapid emergence of viral escape mutations. Although immunization of BLT mice with the conserved viral Gag protein did not result in detectable prechallenge responses, it did increase the magnitude and kinetics of postchallenge Gag-specific T cell responses, which was associated with a modest but significant reduction in acute HIV-1 viremia. Additionally, the BLT model revealed immunization-associated increases in the plasma concentrations of immunomodulatory cytokines and chemokines that correlated with more robust T cell responses. These data support the potential utility of the BLT humanized mouse for HIV-1 vaccine development but suggest that additional improvements to the model are warranted.