Rapamycin Enhances CAR-T Control of HIV Replication and Reservoir Elimination in vivo

Chimeric Antigen Receptor (CAR) T cell therapy shows promise for various diseases. Our studies in humanized mice and non-human primates (NHPs) demonstrate that hematopoietic stem cell (HSCs) modified with anti-HIV CAR achieve lifelong engraftment, providing functional anti-viral CAR-T cells that reduce viral rebound after ART withdrawal. However, T cell exhaustion due to chronic immune activation remains a key obstacle for 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 mitochondria 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. RNAseq analysis of CAR-T cells from humanized mice showed that rapamycin downregulated multiple checkpoint inhibitors and the upregulated key survival genes. Mice treated with CAR-HSCs and rapamycin had delayed viral rebound post-ART and reduced HIV reservoir compared to CAR-HSCs alone. These findings suggest that HSCs-based anti-HIV CAR-T combined with rapamycin treatment is a promising approach for treating persistent inflammation and improving immune control of HIV replication. RNA-seq profiling of CAR T cells was performed to investigate the transcriptional effects of rapamycin treatment. CAR T cells were treated with rapamycin (0.5 mg/kg, i.p., 3 times/week) and compared to untreated controls. Additionally, the R5-tropic strain HIV-1NFNSXSL9 was generated by transfecting 293T cells with a plasmid containing the full-length HIV-1 genome. Humanized mice were infected with HIV-1NFNSXSL9 (500 ng p24/mouse) via retro-orbital injection and treated with antiretroviral therapy (TDF 80 mg/kg, FTC 120 mg/kg, ELV 160 mg/kg) for 6 weeks. Rapamycin (0.5 mg/kg) was administered i.p. 3 times/week in combination with ART to assess its effects.