Characterization of HIV variants from paired Cerebrospinal fluid and Plasma samples in primary microglia and CD4+ T-cells.

Despite antiretroviral therapy (ART), HIV persistence in the central nervous system (CNS) continues to cause a range of cognitive impairments in people living with HIV (PLWH). Upon disease progression, transmigrating CCR5-using T-cell tropic viruses are hypothesized to evolve into macrophage-tropic viruses in the CNS that can efficiently infect low CD4-expressing cells, such as microglia. We examined HIV-1 RNA concentration, co-receptor usage, and CSF compartmentalization in paired CSF and blood samples from 19 adults not on treatment. Full-length envelope CSF- and plasma-derived reporter viruses were generated from 3 subjects and phenotypically characterized in human primary CD4+ T-cells and primary microglia. Median HIV RNA levels were higher in plasma than in CSF (5.01 vs. 4.12 log10 cp/mL; p = 0.004), and coreceptor usage was mostly concordant for CCR5 across the paired samples (n = 17). Genetically compartmentalized CSF viral populations were detected in 2 subjects, one with and one without neurological symptoms. All viral clones could replicate in T-cells (R5 T cell-tropic). In addition, 3 CSF and 1 plasma patient-derived viral clones also had the capacity to replicate in microglia/macrophages and, therefore have an intermediate macrophage tropic phenotype. Overall, with this study, we demonstrate that in a subset of PLWH, plasma-derived viruses undergo genetic and phenotypic evolution within the CNS, indicating viral infection and replication in CNS cells. It remains to be studied whether the intermediate macrophage-tropic phenotype observed in primary microglia represents a midpoint in the evolution towards a macrophage-tropic phenotype that can efficiently replicate in microglial cells and propagate viral infection in the CNS.

Human microglial models to study HIV infection and neuropathogenesis: a literature overview and comparative analyses.

HIV persistence in the CNS despite antiretroviral therapy may cause neurological disorders and poses a critical challenge for HIV cure. Understanding the pathobiology of HIV-infected microglia, the main viral CNS reservoir, is imperative. Here, we provide a comprehensive comparison of human microglial culture models: cultured primary microglia (pMG), microglial cell lines, monocyte-derived microglia (MDMi), stem cell-derived microglia (iPSC-MG), and microglia grown in 3D cerebral organoids (oMG) as potential model systems to advance HIV research on microglia. Functional characterization revealed phagocytic capabilities and responsiveness to LPS across all models. Microglial transcriptome profiles of uncultured pMG showed the highest similarity to cultured pMG and oMG, followed by iPSC-MG and then MDMi. Direct comparison of HIV infection showed a striking difference, with high levels of viral replication in cultured pMG and MDMi and relatively low levels in oMG resembling HIV infection observed in post-mortem biopsies, while the SV40 and HMC3 cell lines did not support HIV infection. Altogether, based on transcriptional similarities to uncultured pMG and susceptibility to HIV infection, MDMi may serve as a first screening tool, whereas oMG, cultured pMG, and iPSC-MG provide more representative microglial culture models for HIV research. The use of current human microglial cell lines (SV40, HMC3) is not recommended.

Integrative analysis of neuroblastoma by single-cell RNA sequencing identifies the NECTIN2-TIGIT axis as a target for immunotherapy.

Pediatric patients with high-risk neuroblastoma have poor survival rates and urgently need more effective treatment options with less side effects. Since novel and improved immunotherapies may fill this need, we dissect the immunoregulatory interactions in neuroblastoma by single-cell RNA-sequencing of 24 tumors (10 pre- and 14 post-chemotherapy, including 5 pairs) to identify strategies for optimizing immunotherapy efficacy. Neuroblastomas are infiltrated by natural killer (NK), T and B cells, and immunosuppressive myeloid populations. NK cells show reduced cytotoxicity and T cells have a dysfunctional profile. Interaction analysis reveals a vast immunoregulatory network and identifies NECTIN2-TIGIT as a crucial immune checkpoint. Combined blockade of TIGIT and PD-L1 significantly reduces neuroblastoma growth, with complete responses (CR) in vivo. Moreover, addition of TIGIT+PD-L1 blockade to standard relapse treatment in a chemotherapy-resistant Th-ALKF1174L/MYCN 129/SvJ syngeneic model induces CR. In conclusion, our integrative analysis provides promising targets and a rationale for immunotherapeutic combination strategies.