Valerie Rezek

Antibodies targeting the highly conserved prehairpin intermediate (PHI) of class I viral membrane-fusion proteins are generally weakly neutralizing and are not considered viable therapeutic agents. We previously demonstrated that antibodies targeting the gp41 N-heptad repeat (NHR), which is transiently exposed in the HIV-1 PHI, exhibit enhanced broad neutralization in cells expressing the Fc receptor, FcγRI. To enhance neutralization in cells lacking FcγRI, we here develop a bispecific antibody (bsAb) by fusing an NHR-targeting antibody to an antibody against CD4, the HIV-1 receptor on T cells. The bsAb provides a 5000-fold neutralization enhancement and shows unprecedented neutralization breadth compared to existing broadly neutralizing antibodies. Importantly, the bsAb reduces viral load in HIV-1-infected humanized male mice, and viral envelope sequencing under bsAb pressure revealed an NHR mutation that potentially impairs viral fitness. These findings validate the NHR as a potential HIV-1 therapeutic target, setting the stage for a new class of broadly neutralizing antibodies.

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.

Chimeric antigen receptor (CAR)-engineered T cell therapy holds promise for treating myeloid malignancies, but challenges remain in bone marrow (BM) infiltration and targeting BM-resident malignant cells. Current autologous CAR-T therapies also face manufacturing and patient selection issues, underscoring the need for off-the-shelf products. In this study, we characterize primary patient samples and identify a unique therapeutic opportunity for CAR-engineered invariant natural killer T (CAR-NKT) cells. Using stem cell gene engineering and a clinically guided culture method, we generate allogeneic CD33-directed CAR-NKT cells with high yield, purity, and robustness. In preclinical mouse models, CAR-NKT cells exhibit strong BM homing and effectively target BM-resident malignant blast cells, including CD33-low/negative leukemia stem and progenitor cells. Furthermore, CAR-NKT cells synergize with hypomethylating agents, enhancing tumor-killing efficacy. These cells also show minimal off-tumor toxicity, reduced graft-versus-host disease and cytokine release syndrome risks, and resistance to allorejection, highlighting their substantial therapeutic potential for treating myeloid malignancies.

The processes that govern human haematopoietic stem cell (HSC) self-renewal and engraftment are poorly understood and challenging to recapitulate in culture to reliably expand functional HSCs^1–3. Here we identify MYC target 1 (MYCT1; also known as MTLC) as a crucial human HSC regulator that moderates endocytosis and environmental sensing in HSCs. MYCT1 is selectively expressed in undifferentiated human haematopoietic stem and progenitor cells (HSPCs) and endothelial cells but becomes markedly downregulated during HSC culture. Lentivirus-mediated knockdown of MYCT1 prevented human fetal liver and cord blood (CB) HSPC expansion and engraftment. By contrast, restoring MYCT1 expression improved the expansion and engraftment of cultured CB HSPCs. Single-cell RNA sequencing of human CB HSPCs in which MYCT1 was knocked down or overexpressed revealed that MYCT1 governs important regulatory programmes and cellular properties essential for HSC stemness, such as ETS factor expression and low mitochondrial activity. MYCT1 is localized in the endosomal membrane in HSPCs and interacts with vesicle trafficking regulators and signalling machinery. MYCT1 loss in HSPCs led to excessive endocytosis and hyperactive signalling responses, whereas restoring MYCT1 expression balanced culture-induced endocytosis and dysregulated signalling. Moreover, sorting cultured CB HSPCs on the basis of lowest endocytosis rate identified HSPCs with preserved MYCT1 expression and MYCT1-regulated HSC stemness programmes. Our work identifies MYCT1-moderated endocytosis and environmental sensing as essential regulatory mechanisms required to preserve human HSC stemness. Our data also pinpoint silencing of MYCT1 as a cell-culture-induced vulnerability that compromises human HSC expansion.

A hallmark of HIV-1 infection is chronic inflammation, even in patients treated with antiretroviral therapy (ART). Chronic inflammation drives HIV-1 pathogenesis, leading to loss of CD4 ⁺ T cells and exhaustion of antiviral immunity. Therefore, strategies to safely reduce systematic inflammation are needed to halt disease progression and restore defective immune responses. Autophagy is a cellular mechanism for disposal of damaged organelles and elimination of intracellular pathogens. Autophagy is pivotal for energy homeostasis and plays critical roles in regulating immunity. However, how it regulates inflammation and antiviral T cell responses during HIV infection is unclear. Here, we demonstrate that autophagy is directly linked to IFN-I signaling, which is a key driver of immune activation and T cell exhaustion during chronic HIV infection. Impairment of autophagy leads to spontaneous IFN-I signaling, and autophagy induction reduces IFN-I signaling in monocytic cells. Importantly, in HIV-1–infected humanized mice, autophagy inducer rapamycin treatment significantly reduced persistent IFN-I–mediated inflammation and improved antiviral T cell responses. Cotreatment of rapamycin with ART led to significantly reduced viral rebound after ART withdrawal. Taken together, our data suggest that therapeutically targeting autophagy is a promising approach to treat persistent inflammation and improve immune control of HIV replication.

Purine nucleoside phosphorylase (PNP) enables the breakdown and recycling of guanine nucleosides. PNP insufficiency in humans is paradoxically associated with both immunodeficiency and autoimmunity, but the mechanistic basis for these outcomes is incompletely understood. Here, we identify two immune lineage-dependent consequences of PNP inactivation dictated by distinct gene interactions. During T cell development, PNP inactivation is synthetically lethal with downregulation of the dNTP triphosphohydrolase SAMHD1. This interaction requires deoxycytidine kinase activity and is antagonized by microenvironmental deoxycytidine. In B lymphocytes and macrophages, PNP regulates Toll-like receptor 7 signaling by controlling the levels of its (deoxy)guanosine nucleoside ligands. Overriding this regulatory mechanism promotes germinal center formation in the absence of exogenous antigen and accelerates disease in a mouse model of autoimmunity. This work reveals that one purine metabolism gene protects against immunodeficiency and autoimmunity via independent mechanisms operating in distinct immune lineages and identifies PNP as a potentially novel metabolic immune checkpoint.

Chimeric Antigen Receptor (CAR) T-cells have emerged as a powerful immunotherapy for various forms of cancer and show promise in treating HIV-1 infection. However, significant limitations are persistence and whether peripheral T cell-based products can respond to malignant or infected cells that may reappear months or years after treatment remains unclear. Hematopoietic Stem/Progenitor Cells (HSPCs) are capable of long-term engraftment and have the potential to overcome these limitations. Here, we report the use of a protective CD4 chimeric antigen receptor (C46CD4CAR) to redirect HSPC-derived T-cells against simian/human immunodeficiency virus (SHIV) infection in pigtail macaques. CAR-containing cells persisted for more than 2 years without any measurable toxicity and were capable of multilineage engraftment. Combination antiretroviral therapy (cART) treatment followed by cART withdrawal resulted in lower viral rebound in CAR animals relative to controls, and demonstrated an immune memory-like response. We found CAR-expressing cells in multiple lymphoid tissues, decreased tissue-associated SHIV RNA levels, and substantially higher CD4/CD8 ratios in the gut as compared to controls. These results show that HSPC-derived CAR T-cells are capable of long-term engraftment and immune surveillance. This study demonstrates for the first time the safety and feasibility of HSPC-based CAR therapy in a large animal preclinical model. Hematopoietic Stem/Progenitor Cell (HSPC) based gene therapy can be used to treat many infectious and genetic diseases. Here, we used an HSPC-based approach to redirect and enhance host immunity against HIV-1. We engineered HSPCs to carry chimeric antigen receptor (CAR) genes that detect and destroy HIV-infected cells. CAR therapy has shown huge potential in the treatment of cancer, but has only been applied in peripheral blood T-cells. HSPC-based CAR therapy has several benefits over T cell gene therapy, as it allows for normal T cell development, selection, and persistence of the engineered cells for the lifetime of the patient. We used a CAR molecule that hijacks the essential interaction between the virus and the cell surface molecule CD4 to redirect HSPC-derived T-cells against infected cells. We observed >2 years of stable production of CAR-expressing cells without any adverse events, and wide distribution of these cells in lymphoid tissues and gastrointestinal tract, which are major anatomic sites for HIV replication and persistence in suppressed patients. Most importantly, HSPC-derived CAR T-cells functionally responded to infected cells. This study demonstrates for the first time the safety and feasibility of HSPC based therapy utilizing an HIV-specific CAR for suppressed HIV infection.

Chronic immune activation, immunosuppression, and T cell exhaustion are hallmarks of HIV infection, yet the mechanisms driving these processes are unclear. Chronic activation can be a driving force in immune exhaustion, and type I interferons (IFN-I) are emerging as critical components underlying ongoing activation in HIV infection. Here, we have tested the effect of blocking IFN-I signaling on T cell responses and virus replication in a murine model of chronic HIV infection. Using HIV-infected humanized mice, we demonstrated that in vivo blockade of IFN-I signaling during chronic HIV infection diminished HIV-driven immune activation, decreased T cell exhaustion marker expression, restored HIV-specific CD8 T cell function, and led to decreased viral replication. Antiretroviral therapy (ART) in combination with IFN-I blockade accelerated viral suppression, further decreased viral loads, and reduced the persistently infected HIV reservoir compared with ART treatment alone. Our data suggest that blocking IFN-I signaling in conjunction with ART treatment can restore immune function and may reduce viral reservoirs during chronic HIV infection, providing validation for IFN-I blockade as a potential therapy for HIV infection.