Biomechanical and Epigenetic Determinants of CTL Resistance in HIV Reservoirs

About This Grant

SUMMARY Anti-retroviral therapy suppresses HIV replication but fails to completely eradicate the virus, which persists in a reservoir of infected CD4+ T cells that resist immune-mediated clearance. Immune evasion by reservoir cells has canonically been attributed to viral latency, which silences HIV gene expression and thereby precludes the recognition of infected cells by HIV-specific cytotoxic T lymphocytes (CTLs). It is now clear, however, that some reservoir cells continue to express HIV antigens, raising the question of how they resist killing by CTLs. To identify these latency-independent mechanisms of resistance, we have employed molecular, cellular, and biophysical approaches to analyze HIV-infected T cells that survive extended coculture with cognate CTLs. Our results indicate that survivor cells express lower levels of cytoskeletal proteins and cell surface adhesion molecules, accompanied by reduced cortical stiffness and plasma membrane tension. These architectural and mechanical phenotypes are intriguing because CTLs kill their targets using an adhesive and physically active immune synapse, and recent studies indicate that perturbing the biophysical properties of target cells can protect them from CTL-mediated destruction. Accordingly, we hypothesize that a subset of HIV-infected cells elude the cytotoxic immune system by becoming soft and slippery. Our proposed studies, which are divided into two Specific Aims, will investigate the molecular mechanisms underlying these resistance pathways with the end goal of identifying novel strategies for targeting the HIV reservoir. Aim 1 will combine biophysical and immunological assays with in vivo experiments to determine the basis for cell softening in resistant cells. We are particularly interested in the interplay between HIV Nef, a viral protein known to induce actin remodeling, and the host cell cytoskeleton. Aim 2 will focus on the molecular foundation(s) of the adhesion phenotype. Our preliminary studies suggest a role for the histone methyltransferase EZH2, which has been shown to mediate the downregulation of adhesion molecules in the context of B cell lymphoma. This proposal is anchored by the conceptually innovative idea that HIV exploits biophysical vulnerabilities to achieve immune evasion, and it will be carried out using technically innovative approaches and reagents, including single cell biophysical measurements as well as first-of-their-kind authentic reservoir CD4+ T cells clones isolated directly from people with HIV. We will also establish avenues for clinical translation based on small molecule modulators of the cytoskeleton and Tazemetostat, a clinically approved EZH2 inhibitor. These efforts will incorporate samples and data from an ongoing Tazemetostat clinical trial, enabling us to evaluate the effects of this drug in actual patients. The successful realization of our Specific Aims has the potential to substantially broaden the of study HIV resistance mechanisms, paving the way for a different kind of cure research. As such, this work is highly relevant to the NIH mission in that it will contribute to the advancement of knowledge that could improve human health.