Rapamycin enhances CAR-T control of HIV replication and reservoir elimination in vivo
Wenli Mu, Shallu Tomer, Jeffrey Harding, Nandita Kedia, Valerie Rezek, Ethan Cook, Vaibahavi Patankar, Mayra A. Carrillo, Heather Martin, Hwee Ng, Li Wang, Matthew D. Marsden, Scott G. Kitchen, Anjie Zhen
Wenli Mu, Shallu Tomer, Jeffrey Harding, Nandita Kedia, Valerie Rezek, Ethan Cook, Vaibahavi Patankar, Mayra A. Carrillo, Heather Martin, Hwee Ng, Li Wang, Matthew D. Marsden, Scott G. Kitchen, Anjie Zhen
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Research Article AIDS/HIV Immunology

Rapamycin enhances CAR-T control of HIV replication and reservoir elimination in vivo

Abstract

Chimeric antigen receptor (CAR) T cell therapy shows promise for various diseases. Our studies in humanized mice and nonhuman primates demonstrate that hematopoietic stem cells (HSCs) modified with anti-HIV CAR achieve lifelong engraftment, providing functional antiviral CAR-T cells that reduce viral rebound after antiretroviral therapy (ART) withdrawal. However, T cell exhaustion due to chronic immune activation remains a key obstacle to sustained CAR-T efficacy, necessitating additional measures to achieve functional cure. We recently showed that low-dose rapamycin treatment reduced inflammation and improved anti-HIV T cell function in HIV-infected humanized mice. Here, we report that rapamycin improved CAR-T cell function both in vitro and in vivo. In vitro treatment with rapamycin enhanced CAR-T cell mitochondrial respiration and cytotoxicity. In vivo treatment with low-dose rapamycin in HIV-infected, CAR-HSC mice decreased chronic inflammation, prevented exhaustion of CAR-T cells, and improved CAR-T control of viral replication. RNA-sequencing analysis of CAR-T cells from humanized mice showed that rapamycin downregulated multiple checkpoint inhibitors and upregulated key survival genes. Mice treated with CAR-HSCs and rapamycin had delayed viral rebound after ART and reduced HIV reservoir compared with those treated with CAR-HSCs alone. These findings suggest that HSC-based anti-HIV CAR-T cells combined with rapamycin treatment are a promising approach for treating persistent inflammation and improving immune control of HIV replication.

Authors

Wenli Mu, Shallu Tomer, Jeffrey Harding, Nandita Kedia, Valerie Rezek, Ethan Cook, Vaibahavi Patankar, Mayra A. Carrillo, Heather Martin, Hwee Ng, Li Wang, Matthew D. Marsden, Scott G. Kitchen, Anjie Zhen

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Figure 1

Treatment of anti-HIV CAR-T cells with rapamycin modifies cellular metabolism in vitro.

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Treatment of anti-HIV CAR-T cells with rapamycin modifies cellular metab...
Anti-HIV CD4CAR-T cells were produced by transduction of activated primary PBMCs from healthy donors. Cells were then sorted to greater than 90% CAR+ purity and expanded using 100 IU/mL IL-2 for 2 weeks to promote exhaustion, followed by treatment with either DMSO or 50 pM of rapamycin for 2 days. Afterward, Seahorse assay was performed on treated CAR-T cells. (A) The oxygen consumption rate (OCR) over time changes under basal metabolic conditions and in response to metabolic inhibitors. (B) Basal OCR levels. (C) Maximal respiratory levels. (D) ROS were analyzed in CD4CAR-T cells labeled with MitoSOX after treatment as shown by flow cytometry and MFI summary of MitoSOX. (E and F) Cytokine assay. Vehicle- or rapamycin-treated CAR-T cells were cocultured with HIV Env–expressing (stimulated ACH2) cells overnight, followed by GolgiPlug (BD Biosciences) for 6 hours. Percentages of IFN-γ and IL-2 expression were measured by flow cytometry in CD4CAR-T cells. Representative flow plot and summary are shown. (G) Killing assay. Vehicle- or rapamycin-treated CAR-T cells were cocultured with either HIV Env+ (stimulated ACH2) or HIV Env– (unstimulated ACH2) cells overnight at 2.5:1, 5:1, and 10:1 ratio. Specific killing activity is shown for vehicle-treated and rapamycin-treated CAR-T cells. All values are means ± SD of at least 3 independent experiments. Mann-Whitney test (unpaired); *P < 0.05, **P < 0.01, ***P < 0.001.