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Objective Resting CD4+ T cells are major reservoirs of latent HIV-1 infection, and may be formed during the early phase of the infection. Although CCR5-tropic (R5) HIV-1 is highly transmissible during the early phase, newly infected individuals have usually been exposed to a mixture of R5 and CXCR4-tropic (X4) viruses, and X4 viral DNA is also detectable in the host. Our aim was to identify which subsets of resting CD4+ T cells contribute to forming the latent reservoir in the presence of both X4 and R5 viruses. Results Primary resting CD4+ naïve T (TN) cells, CCR5− memory T (TM) cells, and CCR5+ TM cells isolated by flow cytometry were infected simultaneously with X4 and R5 HIV-1, which harbored different reporter genes, and were cultured in the resting condition. Flow cytometry at 3 days post-infection demonstrated that X4 HIV-1+ cells were present in all three subsets of cells, whereas R5 HIV-1+ cells were present preferentially in CCR5+ TM cells, but not in TN cells. Following CD3/CD28-mediated activation at 3 days post-infection, numbers of R5 HIV-1+ cells and X4 HIV-1+ cells increased significantly only in the CCR5+ TM subset, suggesting that it provides a major reservoir of replication-competent, latently infected viruses.

A variety of humanized mice, which are reconstituted only with human hematopoietic stem cells (HSC) or with fetal thymus and HSCs, have been developed and widely utilized as in vivo animal models of HIV-1 infection. The models represent some aspects of HIV-mediated pathogenesis in humans and are useful for the evaluation of therapeutic regimens. However, there are several limitations in these models, including their incomplete immune responses and poor distribution of human cells to the secondary lymphoid tissues. These limitations are common in many humanized mouse models and are critical issues that need to be addressed. As distinct defects exist in each model, we need to be cautious about the experimental design and interpretation of the outcomes obtained using humanized mice. Considering this point, we mainly characterize the current conventional humanized mouse reconstituted only with HSCs and describe past achievements in this area, as well as the potential contributions of the humanized mouse models for the study of HIV pathogenesis and therapy. We also discuss the use of various technologies to solve the current problems. Humanized mice will contribute not only to the pre-clinical evaluation of anti-HIV regimens, but also to a deeper understanding of basic aspects of HIV biology.

Several mechanisms underline induction of CD4 T-cell death by human immunodeficiency virus (HIV) infection. For a long time, apoptosis was considered central to cell death involved in the depletion of CD4 T cells during HIV infection. However, which types of cell death are induced during the early phase of HIV infection in vivo remains unclear. In this study, CD4 T-cell death induced in early HIV infection was characterized using humanized mice challenged with CCR5-tropic (R5) or CXCR4-tropic (X4) HIV-1. Results showed that CD4 T-cell death was induced in the spleen 3 days post-challenge with both R5 and X4 HIV-1. Although cell death without caspase-1 and caspase-3/7 activation was preferentially observed, caspase-1+ pyroptosis was also significantly induced within the memory subpopulation by R5 or X4 HIV-1 and the naive subpopulation by X4 HIV-1. In contrast, caspase-3/7+ apoptosis was not enhanced by either R5 or X4 HIV-1. Furthermore, phosphorylated mixed lineage kinase domain-like protein+ necroptosis was induced by only X4 HIV-1. These findings indicate that various types of non-apoptotic CD4 T-cell death, such as pyroptosis and necroptosis, are induced during the early phase of HIV infection in vivo.

Two cytokines, fms-related tyrosine kinase 3 ligand (Flt3-L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are considered to be the essential regulators of dendritic cell (DC) development in vivo. However, the combined effect of Flt3-L and GM-CSF on human DCs has not been evaluated in vivo. In this study, we, therefore, aimed at evaluating this using a humanized mouse model. Humanized non-obese diabetic/SCID/Jak3null (hNOJ) mice were constructed by transplanting hematopoietic stem cells from human umbilical cord blood into newborn NOJ mice, and in vivo transfection (IVT) was performed by hydrodynamic injection-mediated gene delivery using plasmids encoding human Flt3-L and GM-CSF. Following IVT, Flt3-L and GM-CSF were successfully induced in hNOJ mice. At 10 days post-IVT, we found, in the spleen, that treatment with both Flt3-L and GM-CSF enhanced the reconstitution of two myeloid DC subsets, CD14−CD1c+ conventional DCs (cDCs) and CD14−CD141+ cDCs, in addition to CD14+ monocyte-like cells expressing CD1c and/or CD141. GM-CSF alone had less effect on the reconstitution of these myeloid cell populations. By contrast, none of the cytokine treatments enhanced CD123+ plasmacytoid DC (pDC) reconstitution. Regardless of the reconstitution levels, three cell populations (CD1c+ myeloid cells, CD141+ myeloid cells, and pDCs) could be matured by treatment with cytokines, in terms of upregulation of CD40, CD80, CD86, and CD184/CXCR4 and downregulation of CD195/CCR5. In particular, GM-CSF contributed to upregulation of CD80 in all these cell populations. Interestingly, we further observed that Foxp3+ cells within splenic CD4+ T cells were significantly increased in the presence of GM-CSF. Foxp3+ T cells could be subdivided into two subpopulations, CD45RA−Foxp3hi and CD45RA−Foxp3lo T cells. Whereas CD45RA−Foxp3hi T cells were increased only after treatment with GM-CSF alone, CD45RA−Foxp3lo T cells were increased only after treatment with both Flt3-L and GM-CSF. Treatment with Flt3-L alone had no effect on the number of Foxp3+ T cells. The correlation analysis demonstrated that the development of these Foxp3+ subpopulations was associated with the maturation status of DC(-like) cells. Taken together, this study provides a platform for studying the in vivo effect of Flt3-L and GM-CSF on human DCs and regulatory T cells.

Latently infected T lymphocytes are an important barrier toward eliminating a persistent HIV infection. Here we describe an HIV-based recombinant fluorescent-lentivirus referred to as “rfl-HIV” that enables to analyze sense and antisense transcription by means of fluorescence reporter genes. This model virus exhibited similar transcriptional and functional properties of the antisense transcript as observed with a wild type HIV, and largely facilitated the generation of latently-infected T cells clones. We show that latently-infected cells can be divided into two types, those with and those without antisense transcription. Upon addition of latency reversal agents, only the cells that lack antisense transcripts are readily reactivated to transcribe HIV. Thus, antisense transcripts may exhibit a dominant suppressor activity and can lock an integrated provirus into a non-reactivatable state. These findings could have important implications for the development of strategies to eradicate HIV from infected individuals. This work was supported by grants from Japan Society for the Promotion of Science (JSPS KAKENHI #15H06877 for MK-I, #JP17K08800 for KT), ViiV Healthcare Japan Research Grant 2015 (MK-I), Grants-in-Aid from the Ministry of Health, Labour and Welfare (H24-AIDS-008 to YT-Y) and Japan Agency for Medical Research and Development (AMED #JP17fk0410305h0103 to YT-Y and #JP18fk0410003 to KT). MK-I received Fellowships from Japan Foundation for AIDS Prevention and JSPS Oversea Research Fellow Program. AM and JM were supported by a grant from the Spanish Ministry of Economy, Industry and Competitiveness and FEDER grant no. SAF2016-75505-R (AEI/MINEICO/FEDER, UE) and through the “María de Maeztu” Program for Units of Excellence in R&D (MDM-2014-0370).