HIV Infection and Persistence in Pulmonary Mucosal Double Negative T Cells In Vivo

Abstract

The lungs are relatively unexplored anatomical HIV reservoirs in the antiretroviral therapy (ART) era. Double negative (DN) T-cells are a subset of T-cells which lack expression of CD4 and CD8 (CD4-CD8-) and may play both regulatory and effector functions during HIV infection. Notably, circulating DN T-cells were previously described as cellular HIV reservoirs. Here, we undertook a thorough analysis of pulmonary versus blood DN T-cells of people living with HIV (PLWH) under ART. Bronchoalveolar lavage (BAL) fluid and matched peripheral blood were collected from n=35 PLWH on ART and n=16 uninfected volunteers without respiratory symptoms. Both PLWH and HIV- adults displayed higher frequencies of DN T-cells in BAL versus blood, and these cells mostly exhibited an effector memory phenotype. In PLWH, pulmonary mucosal DN T-cells expressed higher levels of HLA-DR and several cellular markers associated with HIV persistence (CCR6, CXCR3 and PD-1) compared to blood. We also observed that DN T-cells were less senescent (CD28-CD57+) and expressed less immunosuppressive ectonucleotidase (CD73/CD39), granzyme B and perforin in the BAL compared to the blood of PLWH. Importantly, FACS-sorted DN T-cells from the BAL of PLWH under suppressive ART harbored HIV-DNA. Using the humanized bone marrow-liver-thymus (hu-BLT) mouse model of HIV infection, we observed higher infection frequencies of lung DN T-cells compared to blood and spleen in both early and late HIV infection. Overall, our findings show that HIV is seeded in pulmonary mucosal DN T-cells early following infection and persists in these potential cellular HIV reservoirs even during long-term ART. Importance Reservoirs of HIV during ART are the primary reasons why HIV/AIDS remains an incurable disease. Indeed, HIV remains latent and unreachable by antiretrovirals in cellular and anatomical sanctuaries, preventing its eradication. The lungs have received very little attention compared to other anatomical reservoirs, despite being immunological effector sites exhibiting characteristics ideal for HIV persistence. Furthermore, PLWH suffer from a high burden of pulmonary non-opportunistic infections suggesting impaired pulmonary immunity despite ART. Meanwhile, various immune cell populations have been proposed to be cellular reservoirs in blood, including CD4-CD8- DN T-cells, a subset that may originate from CD4 downregulation by HIV proteins. The present study aims to describe DN T-cells in human and humanized mice lungs in relation to intrapulmonary HIV burden. The characterization of DN T-cells as cellular HIV reservoirs and the lungs as an anatomical HIV reservoir will contribute towards the development of targeted HIV eradication strategies.

Pulmonary Immune Dysregulation and Viral Persistence During HIV Infection

“…Moreover, the lungs were enriched in activated memory CD4+ T-cells subsets that can further promote HIV replication and persistence (173). We also observed that pulmonary mucosal DN T cells of PLWH on ART expressed higher levels of HLA-DR and several cellular markers associated with HIV persistence (CCR6, CXCR3, and PD-1) compared to the blood (58). Importantly, CD3+CD4-CD8-DN T-cells from the BAL fluid of these participants harbored HIV DNA.…”

“…Importantly, CD3+CD4-CD8- DN T-cells from the BAL fluid of these participants harbored HIV DNA. Using the humanized bone marrow-liver-thymus mouse model, our group also observed higher infection frequencies of lung DN T-cells than those of the blood and spleen in both early and late HIV infection stages, meaning that apart from AMs and CD4 T-cells, HIV is also seeded in pulmonary mucosal DN T-cells early following infection and persists in these potential cellular HIV reservoirs even during long-term ART ( 58 ).…”

“…Human ex vivo AM analysis shows that HIV preferentially infects monocyte-like small AMs, which show higher pro-inflammatory gene expression and greater phagocytic capacity compared to large AMs ( 56 , 57 ). In addition to infecting AMs, we have also shown in a humanized mouse model of early HIV infection that the virus is preferentially seeded within lung DN T-cells, a rather novel lung HIV reservoir cell subset, which is enriched in the lungs of both HIV+ and seronegative individuals compared to other tissues ( 58 ).…”

“…Although most of these studies are typically done in the blood, the lungs of PLWH show a similar phenomenon. All lung T-cells show high frequencies of activation (HLA-DR) ( 17 , 58 , 193 ). Lung CD4 T-cells from treated PLWH also show higher frequencies of senescent cells (CD57), while CD8+ and double negative T-cells show high expression of PD-1 ( 17 , 58 , 193 ).…”

“…All lung T-cells show high frequencies of activation (HLA-DR) ( 17 , 58 , 193 ). Lung CD4 T-cells from treated PLWH also show higher frequencies of senescent cells (CD57), while CD8+ and double negative T-cells show high expression of PD-1 ( 17 , 58 , 193 ). These high levels of immune activation likely contribute to lung HIV reservoir persistence under ART, which our team has demonstrated to be larger in BAL CD4 T-cells compared to the blood ( 17 ).…”