Ferroptosis and Dysfunction of CD3+CD4-CD8- T Cells are Associated with Poor Immune Reconstitution in HIV Patients.

Introduction: Some HIV patients stay in an immune unresponsive state after antiretroviral therapy (ART), with a notably higher risk of AIDS-related and non-AIDS-related complications. Double-negative T cells (DNT) can compensate for immunity and prevent immune overactivation in HIV patients. Also, immune non-responders (INRs) have fewer DNT cells than immune responders (IRs). HIV infection and ART can change the dynamic function of cell mitochondria, which are crucial in ferroptosis. Ferroptosis is a form of cell death marked by the accumulation of reactive oxygen species (ROS) and iron-dependent lipid peroxidation. Yet, the changes in DNT cell function in INRs and the impact of ferroptosis on immune reconstitution remain unclear.

Aims: Our study focused on the expression level of DNT cells in HIV immune non-responders. Then, we detected markers of ferroptosis, cell activation, proliferation, killing function, and inflammatory states of DNT cells in INRs.

Methods: The study involved 88 PLHIVs who had received antiretroviral therapy for over 4 years and tested virus-negative. These patients were classified into two groups: 28 INRs (CD4 < 350/μl) and 60 IRs (CD4 ≥350/μl). Additionally, 25 sex- and age-matched HCs were included. Flow cytometry was used to detect ferroptosis markers (JC-1, Lipid ROS, lipid peroxidation), cell proliferation, and cell activation. Transmission electron microscopy (TEM) was applied to observe mitochondrial morphology. Finally, statistical analysis was performed on the detection results.

Results: After long-term antiretroviral therapy, we found that INRs had a lower DNT cell count than IRs. Regarding proliferation and activation, our results showed higher CD38/HLA-DR co-expression and Ki67 expression in INRs' DNT cells than in IRs', indicating over-activation of DNT cells in INRs. In terms of killing function, the perforin and granzyme B levels in INRs' DNT cells were lower than those in IRs', suggesting impaired killing function of DNT cells in INRs. For ferroptosis, the proportion of DNT cells with decreased MMP in INRs was higher than in IRs and HCs. INRs' DNT cells also had higher levels of lipid ROS and lipid peroxidation compared to those in IRs and HCs. TEM revealed that the mitochondria of INRs' DNT cells had typical morphological features. Moreover, INRs' DNT cells had a greater degree of inflammation.

Conclusion: Our study centered on the proliferation, activation, ferroptosis, killing function, and inflammatory status of DNT cells in INRs. We found that DNT cells in INRs had more active proliferation and activation, weakened killing function, mitochondrial function with typical ferroptosis features, and increased TNF-αlevels. Correlation analysis indicated that DNT cell overactivation (Ki-67+, CD38+HLA-DR+), MMP reduction ratio, and TNF-αexpression were negatively related to immune reconstitution in PLHIVs. In contrast, the killing function (perforin+) of DNT cells was positively related to it. These findings provide a theoretical basis for targeting the functional remodeling of DNT cells. In the future, therapeutic strategies can be explored, such as regulating the mitochondrial metabolic pathway or enhancing the immunoregulatory activity of DNT cells. These strategies can thus offer innovative solutions to the dilemma of immune reconstitution in HIV-infected individuals.