Cold exposure protects from neuroinflammation through immunologic reprogramming.

Autoimmunity is energetically costly, but the impact of a metabolically active state on immunity and immune-mediated diseases is unclear. Ly6Chi monocytes are key effectors in CNS autoimmunity with an elusive role in priming naive autoreactive T cells. Here, we provide unbiased analysis of the immune changes in various compartments during cold exposure and show that this energetically costly stimulus markedly ameliorates active experimental autoimmune encephalomyelitis (EAE). Cold exposure decreases MHCII on monocytes at steady state and in various inflammatory mouse models and suppresses T cell priming and pathogenicity through the modulation of monocytes. Genetic or antibody-mediated monocyte depletion or adoptive transfer of Th1- or Th17-polarized cells for EAE abolishes the cold-induced effects on T cells or EAE, respectively. These findings provide a mechanistic link between environmental temperature and neuroinflammation and suggest competition between cold-induced metabolic adaptations and autoimmunity as energetic trade-off beneficial for the immune-mediated diseases.

Monitoring HIV DNA and cellular activation markers in HIV-infected humanized mice under cART.

BACKGROUND: The major obstacle to cure of HIV type-1 infection is the presence of the HIV reservoir, hidden from the immune system and insensitive to combined antiretroviral therapy (cART). Eradication approaches have been hindered by the difficulty for accurately monitoring its size in vivo, especially in the lymphoid organs. Humanized mouse models are a valuable tool for systematically assess the efficacy of therapeutic interventions in reducing the HIV reservoir. Nonetheless, persistence of the HIV reservoir over time, in the presence of cART, has yet to be analyzed in this in vivo model. FINDINGS: We found that the proviral DNA as well as the total DNA were very stable in the spleen and mesenteric lymph node irrespective of the length of cART. Notably, the amount of proviral DNA was very similar in the spleen and lymph node. Furthermore, we observed a correlation between the percentage of splenic human CD4+ T-cells with total HIV DNA, between the number of human CD38 + CD8+ T-cells in the spleen with the amount of integrated HIV DNA, and eventually between the hCD4/hCD8 ratio in the spleen with integrated as well as total HIV DNA implying that the CD8+ T cells influence the size of the HIV reservoir. CONCLUSIONS: Here, we demonstrated the stability of this reservoir in humanized mice irrespective of the length of cART, confirming the relevancy of this model for HIV latency eradication investigations. Notably, we also found correlates between the frequency of CD4+ T-cells, their activation status and viral parameters, which were analogous to the ones in HIV-infected patients. Thus, hu-mice represent a very valuable HIV latency model.

Antiviral Activity of HIV gp120-Targeting Bispecific T Cell Engager Antibody Constructs.

Today's gold standard in HIV therapy is combined antiretroviral therapy (cART). It requires strict adherence by patients and lifelong medication, which can lower the viral load below detection limits and prevent HIV-associated immunodeficiency but cannot cure patients. The bispecific T cell-engaging (BiTE) antibody technology has demonstrated long-term relapse-free outcomes in patients with relapsed and refractory acute lymphocytic leukemia. Here, we generated BiTE antibody constructs that target the HIV-1 envelope protein gp120 (HIV gp120) using either the scFv B12 or VRC01, the first two extracellular domains (1 + 2) of human CD4 alone or joined to the single chain variable fragment (scFv) of the antibody 17b fused to an anti-human CD3ε scFv. These engineered human BiTE antibody constructs showed engagement of T cells for redirected lysis of HIV gp120-transfected CHO cells. Furthermore, they substantially inhibited HIV-1 replication in peripheral blood mononuclear cells (PBMCs) as well as in macrophages cocultured with autologous CD8+ T cells, the most potent being the human CD4(1 + 2) BiTE [termed CD(1 + 2) h BiTE] antibody construct and the CD4(1 + 2)L17b BiTE antibody construct. The CD4(1 + 2) h BiTE antibody construct promoted HIV infection of human CD4-/CD8+ T cells. In contrast, the neutralizing B12 and the VRC01 BiTE antibody constructs, as well as the CD4(1 + 2)L17b BiTE antibody construct, did not. Thus, BiTE antibody constructs targeting HIV gp120 are very promising for constraining HIV and warrant further development as novel antiviral therapy with curative potential.IMPORTANCE HIV is a chronic infection well controlled with the current cART. However, we lack a cure for HIV, and the HIV pandemic goes on. Here, we showed in vitro and ex vivo that a BiTE antibody construct targeting HIV gp120 resulted in substantially reduced HIV replication. In addition, these BiTE antibody constructs display efficient killing of gp120-expressing cells and inhibited replication in ex vivo HIV-infected PBMCs or macrophages. We believe that BiTE antibody constructs recognizing HIV gp120 could be a very valuable strategy for a cure of HIV in combination with cART and compounds which reverse latency.

Humanised mouse models for haematopoiesis and infectious diseases.

"Humanised" mouse models have emerged over past years as powerful tools for investigating human haematopoiesis and immunity. They allowed the identification of key factors for the maintenance and function of normal and leukaemic human haematopoietic stem cells. These findings have been widely used to dissect the pathogenesis of multiple myeloid and lymphoid neoplasms, such as acute myeloid leukaemia and acute lymphoblastic leukaemia. Furthermore, these models can serve as a stepping-stone to clinical trials by testing novel drugs that target leukaemic stem cells. The investigation of human immunity in vivo is also of great interest in both the context of understanding the innate and adaptive immune system and responses to viral infections with exclusive human tropism, such as Epstein-Barr virus and human immunodeficiency virus. This review focuses on recent advances in the study of human haematopoiesis and immunity in humanised mouse models, underlining their relevance and limitations.

Long-term leukocyte reconstitution in NSG mice transplanted with human cord blood hematopoietic stem and progenitor cells.

BACKGROUND: Humanized mice (hu mice) are based on the transplantation of hematopoietic stem and progenitor cells into immunodeficient mice and have become important pre-clinical models for biomedical research. However, data about their hematopoiesis over time are scarce. We therefore characterized leukocyte reconstitution in NSG mice, which were sublethally irradiated and transplanted with human cord blood-derived CD34+ cells at newborn age, longitudinally in peripheral blood and, for more detailed analyses, cross-sectionally in peripheral blood, spleen and bone marrow at different time points. RESULTS: Human cell chimerism and absolute human cell count decreased between week 16 and 24 in the peripheral blood of hu mice, but were stable thereafter as assessed up to 32 weeks. Human cell chimerism in spleen and bone marrow was maintained over time. Notably, human cell chimerism in peripheral blood and spleen as well as bone marrow positively correlated with each other. Percentage of B cells decreased between week 16 and 24, whereas percentage of T cells increased; subsequently, they levelled off with T cells clearly predominating at week 32. Natural killer cells, monocytes and plasmacytoid dendritic cells (DCs) as well as CD1c + and CD141+ myeloid DCs were all present in hu mice. Proliferative responses of splenic T cells to stimulation were preserved over time. Importantly, the percentage of more primitive hematopoietic stem cells (HSCs) in bone marrow was maintained over time. CONCLUSIONS: Overall, leukocyte reconstitution was maintained up to 32 weeks post-transplantation in our hu NSG model, possibly explained by the maintenance of HSCs in the bone marrow. Notably, we observed great variation in multi-lineage hematopoietic reconstitution in hu mice that needs to be taken into account for the experimental design with hu mice.

Vpx mediated degradation of SAMHD1 has only a very limited effect on lentiviral transduction rate in ex vivo cultured HSPCs.

Understanding how to achieve efficient transduction of hematopoietic stem and progenitor cells (HSPCs), while preserving their long-term ability to self-reproduce, is key for applying lentiviral-based gene engineering methods. SAMHD1 is an HIV-1 restriction factor in myeloid and resting CD4+ T cells that interferes with reverse transcription by decreasing the nucleotide pools or by its RNase activity. Here we show that SAMHD1 is expressed at high levels in HSPCs cultured in a medium enriched with cytokines. Thus, we hypothesized that degrading SAMHD1 in HSPCs would result in more efficient lentiviral transduction rates. We used viral like particles (VLPs) containing Vpx, shRNA against SAMHD1, or provided an excess of dNTPs or dNs to study this question. Regardless of the method applied, we saw no increase in the lentiviral transduction rate. The result was different when we used viruses (HR-GFP-Vpx+) which carry Vpx and encode GFP. These viruses allow assessment of the effects of Vpx specifically in the transduced cells. Using HR-GFP-Vpx+ viruses, we observed a modest but significant increase in the transduction efficiency. These data suggest that SAMHD1 has some limited efficacy in blocking reverse transcription but the major barrier for efficient lentiviral transduction occurs before reverse transcription.

Lentivector Knockdown of CCR5 in Hematopoietic Stem and Progenitor Cells Confers Functional and Persistent HIV-1 Resistance in Humanized Mice.

UNLABELLED: Gene-engineered CD34(+) hematopoietic stem and progenitor cells (HSPCs) can be used to generate an HIV-1-resistant immune system. However, a certain threshold of transduced HSPCs might be required for transplantation into mice for creating an HIV-resistant immune system. In this study, we combined CCR5 knockdown by a highly efficient microRNA (miRNA) lentivector with pretransplantation selection of transduced HSPCs to obtain a rather pure population of gene engineered CD34(+) cells. Low-level transduction of HSPCs and subsequent sorting by flow cytometry yielded >70% transduced cells. Mice transplanted with these cells showed functional and persistent resistance to a CCR5-tropic HIV strain: viral load was significantly decreased over months, and human CD4(+) T cells were preserved. In one mouse, viral mutations, resulting presumably in a CXCR4-tropic strain, overcame HIV resistance. Our results suggest that HSPC-based CCR5 knockdown may lead to efficient control of HIV in vivo. We overcame a major limitation of previous HIV gene therapy in humanized mice in which only a proportion of the cells in chimeric mice in vivo are anti-HIV engineered. Our strategy underlines the promising future of gene engineering HIV-resistant CD34(+) cells that produce a constant supply of HIV-resistant progeny. IMPORTANCE: Major issues in experimental long-term in vivo HIV gene therapy have been (i) low efficacy of cell transduction at the time of transplantation and (ii) transduction resulting in multiple copies of heterologous DNA in target cells. In this study, we demonstrated the efficacy of a transplantation approach with a selection step for transduced cells that allows transplantation of an enriched population of HSPCs expressing a single (low) copy of a CCR5 miRNA. Efficient maintenance of CD4(+) T cells and a low viral titer resulted only when at least 70% of the HIV target cells were genetically modified. These findings imply that clinical protocols of HIV gene therapy require a selective enrichment of genetically targeted cells because positive selection of modified cells is likely to be insufficient below this threshold. This selection approach may be beneficial not only for HIV patients but also for other patients requiring transplantation of genetically modified cells.

Triggering TLR2, -3, -4, -5, and -8 reinforces the restrictive nature of M1- and M2-polarized macrophages to HIV.

UNLABELLED: Macrophages must react to a large number of pathogens and their effects. In chronic HIV infection, the microenvironment changes with an influx of microbial products that trigger Toll-like receptors (TLRs). That dynamic nature can be replicated ex vivo by the proinflammatory (M1-polarized) and alternatively activated (M2-polarized) macrophages. Thus, we determined how polarized macrophages primed by various TLR agonists support HIV replication. Triggering of TLR2, -3, -4, -5, and -8 reinforced the low level of permissiveness in polarized macrophages. HIV was inhibited even more in M1-polarized macrophages than in macrophages activated only by TLR agonists. HIV was inhibited before its integration into the host chromosome. Polarization and triggering by various TLR agonists resulted in distinct cytokine profiles, endocytic activity, and distinct upregulation of restriction factors of HIV. Thus, different mechanisms likely contribute to the HIV-inhibitory effects. In chronic HIV infection, macrophages might become less permissive to HIV due to changes in the microenvironment. The high level of reactivity of polarized macrophages to TLR triggering may be exploited for immunotherapeutic strategies. IMPORTANCE: Macrophages are a major target of HIV-1 infection. Different cell types in this very heterogeneous cell population respond differently to stimuli. In vitro, the heterogeneity is mimicked by their polarization into proinflammatory and alternatively activated macrophages. Here we explored the extent to which agonists triggering the TLR family affect HIV replication in polarized macrophages. We found that a number of TLR agonists blocked HIV replication substantially when given before infection. We also report the mechanisms of how TLR agonists exert their inhibitory action. Our findings may advance our understanding of which and how TLR agonists block HIV infection in polarized macrophages and may facilitate the design of novel immunotherapeutic approaches.

Humanized mice recapitulate key features of HIV-1 infection: a novel concept using long-acting anti-retroviral drugs for treating HIV-1.

BACKGROUND: Humanized mice generate a lymphoid system of human origin subsequent to transplantation of human CD34+ cells and thus are highly susceptible to HIV infection. Here we examined the efficacy of antiretroviral treatment (ART) when added to food pellets, and of long-acting (LA) antiretroviral compounds, either as monotherapy or in combination. These studies shall be inspiring for establishing a gold standard of ART, which is easy to administer and well supported by the mice, and for subsequent studies such as latency. Furthermore, they should disclose whether viral breakthrough and emergence of resistance occurs similar as in HIV-infected patients when ART is insufficient. METHODS/PRINCIPAL FINDINGS: NOD/shi-scid/γ(c)null (NOG) mice were used in all experimentations. We first performed pharmacokinetic studies of the drugs used, either added to food pellets (AZT, TDF, 3TC, RTV) or in a LA formulation that permitted once weekly subcutaneous administration (TMC278: non-nucleoside reverse transcriptase inhibitor, TMC181: protease inhibitor). A combination of 3TC, TDF and TMC278-LA or 3TC, TDF, TMC278-LA and TMC181-LA suppressed the viral load to undetectable levels in 15/19 (79%) and 14/14 (100%) mice, respectively. In successfully treated mice, subsequent monotherapy with TMC278-LA resulted in viral breakthrough; in contrast, the two LA compounds together prevented viral breakthrough. Resistance mutations matched the mutations most commonly observed in HIV patients failing therapy. Importantly, viral rebound after interruption of ART, presence of HIV DNA in successfully treated mice and in vitro reactivation of early HIV transcripts point to an existing latent HIV reservoir. CONCLUSIONS/SIGNIFICANCE: This report is a unique description of multiple aspects of HIV infection in humanized mice that comprised efficacy testing of various treatment regimens, including LA compounds, resistance mutation analysis as well as viral rebound after treatment interruption. Humanized mice will be highly valuable for exploring the antiviral potency of new compounds or compounds targeting the latent HIV reservoir.