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Mechanistic insight into the CD32-driven enhancement of HIV-1 susceptibility of resting CD4 T cells
Mechanistic insight into the CD32-driven enhancement of HIV-1 susceptibility of resting CD4 T cells
The current treatment of HIV-1-infected patients relies on life-long antiretroviral therapy (ART) that can suppress virus replication, but not eradicate the pathogen. The persistence of latent and ART-resistant viral reservoirs, particularly in resting CD4 T cells, remains the major bar-rier to HIV cure. The current limitation of strategies aiming at reversal of HIV latency or killing of infected cells in a clinical setting, in part results from a lack of biomarkers that are selec-tively exposed on latently HIV-1-infected, resting CD4 T cells. This knowledge could facilitate the selective elimination of the viral reservoir in infected individuals. Several candidate bi-omarkers were recently proposed, including the Fcγ receptor (FcγR)-IIa (CD32a), but its role has remained controversial. Here we show, that the FcγR CD32 (FcγRII) is not expressed de novo by HIV-1-infected CD4 T cells, but acquired from CD32-positive cells such as macro-phages under conditions of close cell-to-cell contact. This cell communication process is re-ferred to as FcγR-mediated trogocytosis. To study it, we established a cell line-based FcγR-mediated trogocytosis model. Here, we tested the functionality and auto-transfer of three isoforms of CD32, i.e. CD32A, B and C, as well as a panel of CD32A and B mutants. This re-vealed that CD32 drives the transient, cell contact-dependent transfer of itself, but also the co-transfer of other cell surface receptors (CD32B>CD32C≥CD32A) including chemokine core-ceptors for HIV-1. Additionally, we explored the role of antibodies in modulating CD32-mediated trogocytosis. We found that the HIV-1 broadly neutralizing antibody (bNAb) PGT151 is autoreactive to CD4 T cells and thereby enhances trogocytosis. Intriguingly, also a subset of patients with chronic HIV-1 infection harbors T cell-reactive IgG autoantibodies with this capacity in their blood. Moreover, in co-cultures of primary macrophages with primary CD4 T cells we detected the transfer of CD32 as well as the co-transfer of a number of other recep-tors from the plasma membrane of macrophages to that of T cells. Transferred receptors were able to confer cell migration and adhesion properties to recipient cells. By confocal microsco-py we were able to visualize transferred receptors on T cells within distinct membrane patch-es, which originated from the donor cells. Importantly, these macrophage-derived membrane patches served as hotspots for binding and fusion of HIV-1 particles and rendered resting CD4 T cells susceptible to infection. In order to further elucidate mechanisms that may be directly involved in preferential HIV-1 binding to membrane patches transferred by trogocytosis we investigated a set of receptors, previously implicated in HIV-1 binding to macrophages. While being able to detect the co-transfer of such HIV-1 binding receptors, including DC-SIGN, CD206 and CD11a/b/c, we ex-cluded their functional contribution in this process using highly efficient knockout approaches in donor macrophages. Unexpectedly, we observed that CD4 endogenously expressed by T cells aggregated within transferred CD32+ ganglioside GM1+ membrane patches. Based on gene perturbation and antibody inhibition studies, we show that the recruited CD4 is an im-portant factor for preferential virion binding and fusion at these hotspots, resulting in increased infection of these viral reservoir cells. Taken together, the antibody-enhanced, CD32-driven trogocytotic transfer of membrane patches containing a number of surface receptors can change the proteomic and functional plasticity of primary CD4 T cells. On a more general level, FcγR-mediated trogocytosis should be taken into account when investigating primary immune cells in co-culture and analyzing the expression and functionality of cell surface markers untypical for the respective cell lineage. Even though we can exclude CD32 as a bona fide biomarker for latently HIV-1-infected cells, the discovery of FcγR-driven trogocytosis which enhances the HIV-1 infection rate of resting CD4 T cells is important as this process may contribute to seeding and expansion of the latent reservoir in patients.
CD32, HIV reservoir, autoantibodies, immune cell communication, trogocytosis
Gapp, Madeleine
2024
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Gapp, Madeleine (2024): Mechanistic insight into the CD32-driven enhancement of HIV-1 susceptibility of resting CD4 T cells. Dissertation, LMU München: Faculty of Medicine
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Abstract

The current treatment of HIV-1-infected patients relies on life-long antiretroviral therapy (ART) that can suppress virus replication, but not eradicate the pathogen. The persistence of latent and ART-resistant viral reservoirs, particularly in resting CD4 T cells, remains the major bar-rier to HIV cure. The current limitation of strategies aiming at reversal of HIV latency or killing of infected cells in a clinical setting, in part results from a lack of biomarkers that are selec-tively exposed on latently HIV-1-infected, resting CD4 T cells. This knowledge could facilitate the selective elimination of the viral reservoir in infected individuals. Several candidate bi-omarkers were recently proposed, including the Fcγ receptor (FcγR)-IIa (CD32a), but its role has remained controversial. Here we show, that the FcγR CD32 (FcγRII) is not expressed de novo by HIV-1-infected CD4 T cells, but acquired from CD32-positive cells such as macro-phages under conditions of close cell-to-cell contact. This cell communication process is re-ferred to as FcγR-mediated trogocytosis. To study it, we established a cell line-based FcγR-mediated trogocytosis model. Here, we tested the functionality and auto-transfer of three isoforms of CD32, i.e. CD32A, B and C, as well as a panel of CD32A and B mutants. This re-vealed that CD32 drives the transient, cell contact-dependent transfer of itself, but also the co-transfer of other cell surface receptors (CD32B>CD32C≥CD32A) including chemokine core-ceptors for HIV-1. Additionally, we explored the role of antibodies in modulating CD32-mediated trogocytosis. We found that the HIV-1 broadly neutralizing antibody (bNAb) PGT151 is autoreactive to CD4 T cells and thereby enhances trogocytosis. Intriguingly, also a subset of patients with chronic HIV-1 infection harbors T cell-reactive IgG autoantibodies with this capacity in their blood. Moreover, in co-cultures of primary macrophages with primary CD4 T cells we detected the transfer of CD32 as well as the co-transfer of a number of other recep-tors from the plasma membrane of macrophages to that of T cells. Transferred receptors were able to confer cell migration and adhesion properties to recipient cells. By confocal microsco-py we were able to visualize transferred receptors on T cells within distinct membrane patch-es, which originated from the donor cells. Importantly, these macrophage-derived membrane patches served as hotspots for binding and fusion of HIV-1 particles and rendered resting CD4 T cells susceptible to infection. In order to further elucidate mechanisms that may be directly involved in preferential HIV-1 binding to membrane patches transferred by trogocytosis we investigated a set of receptors, previously implicated in HIV-1 binding to macrophages. While being able to detect the co-transfer of such HIV-1 binding receptors, including DC-SIGN, CD206 and CD11a/b/c, we ex-cluded their functional contribution in this process using highly efficient knockout approaches in donor macrophages. Unexpectedly, we observed that CD4 endogenously expressed by T cells aggregated within transferred CD32+ ganglioside GM1+ membrane patches. Based on gene perturbation and antibody inhibition studies, we show that the recruited CD4 is an im-portant factor for preferential virion binding and fusion at these hotspots, resulting in increased infection of these viral reservoir cells. Taken together, the antibody-enhanced, CD32-driven trogocytotic transfer of membrane patches containing a number of surface receptors can change the proteomic and functional plasticity of primary CD4 T cells. On a more general level, FcγR-mediated trogocytosis should be taken into account when investigating primary immune cells in co-culture and analyzing the expression and functionality of cell surface markers untypical for the respective cell lineage. Even though we can exclude CD32 as a bona fide biomarker for latently HIV-1-infected cells, the discovery of FcγR-driven trogocytosis which enhances the HIV-1 infection rate of resting CD4 T cells is important as this process may contribute to seeding and expansion of the latent reservoir in patients.