Assessing the phenotypic and migratory behaviors of HIV-infected latent CD4+ T cells
Antiretroviral therapy (ART) has helped Human Immunodeficiency Virus (HIV) become a manageable chronic infection by repressing viremia to undetectable levels in People living with HIV (PLWH). However, this treatment is not curative as treatment interruption causes viremia to rebound to pre-treatment levels due to viral latency, a state of reversible non-productive infection of individual cells established early during acute infection. These latently infected cells serve as HIV reservoirs, residing in various anatomical sites and presenting a significant obstacle to achieving a complete HIV cure. Notably, central and effector memory CD4+ T cells are regarded as the major component of the long-lived HIV reservoirs. Memory CD4+ T cells play an essential role in the adaptive immune response. Guided by chemokine cues, memory CD4+ T cells continually recirculate between lymphoid and non-lymphoid tissues, enabling them to carry out immunosurveillance of the body for pathogens. These long-lived cells are sustained by tonic signalling through the T cell receptor (TCR) and cytokines within the secondary lymphoid organs (SLOs), where they can undergo clonal expansion. Therefore, the SLOs are important tissue reservoirs that allow for persistent viral replication in PLWH under suppressive ART. However, an unaddressed question is whether latently infected memory CD4+ T cells retain their ability to recirculate between lymphoid and non-lymphoid organs, allowing them to seed various tissues and access the same survival signals as uninfected memory CD4+ T cells to persist. Therefore, to address this question, I developed a fluorescent protein-based HIV reporter system that allows for the assessment of the phenotypic and migratory capacity of latently infected CD4+ T cells. The full-length, R5-tropic dual-fluorescent HIV reporter, HIV TosZy, encodes two fluorescent markers: Nef-ZsGreen fusion protein under the control of the HIV-LTR and dTomato protein driven by constitutively expressed EF1α-HTLV1 promoter. Infection of primary CD4+ T-cells with HIV TosZy allows the visualization of latently infected cells. Evaluations of receptors and molecules essential for T cell homing and migration demonstrate that viral latency does not alter their expression. Live-cell in vitro imaging studies in a 3D collagen matrix showed that latently CD4+ T-cells displayed robust migration similar to uninfected CD4+ T-cells and retained normal migratory behaviours. These in vitro observations were also recapitulated in the popliteal draining lymph node of humanized (hu-PBMC) mice using intravital imaging. The results of my research study indicate that latently infected memory CD4+ T cells maintain the expression of homing and migratory markers and retain their ability to migrate within the lymph node, potentially enabling them to continuously access survival signals, thus facilitating their persistence in ART-treated PLWH. This may also allow these cells to recirculate constantly between lymphoid and non-lymphoid organs, allowing them to continually seed various tissues. These findings provide valuable insights into how the homing and recirculation of latently infected cells may be targeted to prevent reservoir establishment and persistence in PLWH.
HIV, CD4+ T cells, HIV Latency, Cell migration, HIV reservoirs, Secondary lymphoid organs, Dual-fluorescent reporter