Location
Philadelphia, PA
Start Date
17-4-2026 1:30 PM
End Date
17-4-2026 2:30 PM
Description
Introduction: Even in antiretroviral therapy-treated people with HIV (PWH) exhibiting controlled viremia, infection is characterized by a sustained activation of type I interferon (IFN) pathways. Indeed, IFNβ is persistently expressed in the tissues of PWH, and the anti-proliferative effect of IFNβ represents an unappreciated hurdle to immunotherapies aimed at achieving an HIV functional cure.
Objectives: The primary objectives are to i) characterize the anti-proliferative effects of IFNβ on HIV-specific CAR T cells, ii) describe the mechanism(s) underlying IFNβ-mediated attenuation of CAR T cell function, and iii) devise base-editing strategies to ameliorate the negative effects of IFNβ on HIV-specific CAR T cell function in vivo.
Methods: Second Generation 4-1BBz CAR T-cells targeting HIV with a CD4 ectodomain based CAR were generated through lentiviral transduction of healthy human donors. CAR T cells were exposed to IFNβ in vitro and in the context of antigen stimulation and markers of apoptosis as well as expansion kinetics were measured via flow cytometry. STAT1-6 activation after IFNβ exposure was characterized through phospho-flow.
Results:
IFNβ exposure increased markers of apoptosis and cell death in CAR T cells as soon as 30 minutes following exposure both in the presence and absence of CAR-antigen sensing. Moreover, we studied the effects of chronic IFNβ exposure on CAR T cell proliferation over a 7-week period of antigen exposure. At nanomolar concentrations, IFNβ was sufficient to cause logarithmic deficits in CAR T cell proliferation, as well perturb mitochondria phenotypes and inhibitory receptor expression profiles.
To interrogate the mechanism of IFNβ-mediated apoptosis, IFNβ-exposed CAR T cells were screened for activating phosphorylation events of the Signal Transducer and Activator of Transcription (STAT) family proteins. IFNβ caused an increase of pSTAT1 and pSTAT2 but demonstrated a negligible effect on phosphorylation of STATs 3, 4, and 6. Intriguingly, IFNβ downregulated pSTAT5, known as a cell survival signal, providing a putative mechanism of increased net apoptosis.
To confirm the clinical relevance of IFNβ exposure on HIV-specific CAR T cells, we adoptively transferred HIV-specific CAR T cells into HIV-infected, ART-suppressed humanized mice and treated with an IFNβ-neutralizing antibody. We observed a significant advantage in engraftment and persistence of CAR T cells in mice treated with IFNβ-neutralizing antibody over IgG control. These data support a negative regulatory role for IFNβ in ART-suppressed HIV infection, a hurdle to the persistence of adoptive T cell therapies.
Conclusion: The putative mechanism for IFNβ induced proliferation deficit is largely driven by apoptosis rather than quiescent effects. We propose that the mechanism of increased apoptosis is driven by an upregulation of the pSTAT1 and pSTAT2 axis, inducing pro-apoptotic machinery, while suppressing pSTAT5, destabilizing tonic cellular survival signals in T cells. Current work is centered on devising a base-editing strategy targeting the IFNAR1/2 receptor complex to tune CAR T cell sensitivity to type I interferon signals, with the ultimate goal of retaining the stimulatory properties of type-I IFN signaling while blocking the anti-proliferative effects of potent and sustained IFN-I signaling which is often driven by IFNβ in the context of chronic and treated HIV infection.
Embargo Period
6-4-2026
Included in
Neutralization of Interferon-beta Promotes HIVspecific CAR T cell Engraftment and Proliferation
Philadelphia, PA
Introduction: Even in antiretroviral therapy-treated people with HIV (PWH) exhibiting controlled viremia, infection is characterized by a sustained activation of type I interferon (IFN) pathways. Indeed, IFNβ is persistently expressed in the tissues of PWH, and the anti-proliferative effect of IFNβ represents an unappreciated hurdle to immunotherapies aimed at achieving an HIV functional cure.
Objectives: The primary objectives are to i) characterize the anti-proliferative effects of IFNβ on HIV-specific CAR T cells, ii) describe the mechanism(s) underlying IFNβ-mediated attenuation of CAR T cell function, and iii) devise base-editing strategies to ameliorate the negative effects of IFNβ on HIV-specific CAR T cell function in vivo.
Methods: Second Generation 4-1BBz CAR T-cells targeting HIV with a CD4 ectodomain based CAR were generated through lentiviral transduction of healthy human donors. CAR T cells were exposed to IFNβ in vitro and in the context of antigen stimulation and markers of apoptosis as well as expansion kinetics were measured via flow cytometry. STAT1-6 activation after IFNβ exposure was characterized through phospho-flow.
Results:
IFNβ exposure increased markers of apoptosis and cell death in CAR T cells as soon as 30 minutes following exposure both in the presence and absence of CAR-antigen sensing. Moreover, we studied the effects of chronic IFNβ exposure on CAR T cell proliferation over a 7-week period of antigen exposure. At nanomolar concentrations, IFNβ was sufficient to cause logarithmic deficits in CAR T cell proliferation, as well perturb mitochondria phenotypes and inhibitory receptor expression profiles.
To interrogate the mechanism of IFNβ-mediated apoptosis, IFNβ-exposed CAR T cells were screened for activating phosphorylation events of the Signal Transducer and Activator of Transcription (STAT) family proteins. IFNβ caused an increase of pSTAT1 and pSTAT2 but demonstrated a negligible effect on phosphorylation of STATs 3, 4, and 6. Intriguingly, IFNβ downregulated pSTAT5, known as a cell survival signal, providing a putative mechanism of increased net apoptosis.
To confirm the clinical relevance of IFNβ exposure on HIV-specific CAR T cells, we adoptively transferred HIV-specific CAR T cells into HIV-infected, ART-suppressed humanized mice and treated with an IFNβ-neutralizing antibody. We observed a significant advantage in engraftment and persistence of CAR T cells in mice treated with IFNβ-neutralizing antibody over IgG control. These data support a negative regulatory role for IFNβ in ART-suppressed HIV infection, a hurdle to the persistence of adoptive T cell therapies.
Conclusion: The putative mechanism for IFNβ induced proliferation deficit is largely driven by apoptosis rather than quiescent effects. We propose that the mechanism of increased apoptosis is driven by an upregulation of the pSTAT1 and pSTAT2 axis, inducing pro-apoptotic machinery, while suppressing pSTAT5, destabilizing tonic cellular survival signals in T cells. Current work is centered on devising a base-editing strategy targeting the IFNAR1/2 receptor complex to tune CAR T cell sensitivity to type I interferon signals, with the ultimate goal of retaining the stimulatory properties of type-I IFN signaling while blocking the anti-proliferative effects of potent and sustained IFN-I signaling which is often driven by IFNβ in the context of chronic and treated HIV infection.