Regulatory T-cells (Tregs) play a crucial role in maintaining immune homeostasis, but their dynamics are altered in a subset of people living with Human Immunodeficiency Virus (HIV) known as immunological non-responders (INRs). INRs fail to reconstitute CD4⁺ T-cell counts despite viral suppression. This study aimed to examine Treg dysregulation in INRs, comparing them to immunological responders (IRs) and healthy controls (HCs).

The study included 40 INRs, 42 IRs, and 23 HCs. Peripheral blood mononuclear cells were isolated and analyzed by flow cytometry. Conventional CD4⁺ T-cells (Tconvs) were identified as CD25^–/loFOXP3^– cells, while Tregs were identified as CD25⁺CD127^loFOXP3⁺ CD4⁺ T-cells. Cells were further divided into naive, central memory, effector memory, and effector memory cells re-expressing CD45RA (TEMRA) subsets. Activated/cycling cells were identified as CD71⁺ and quiescent cells were CD71^–. Mitochondrial mass and transmembrane potential were measured using MitoTracker Green and MitoTracker Orange dyes, respectively. Statistical comparisons were made using the Kruskal-Wallis test with Dunn's post-hoc analysis and Mann-Whitney U-test.

INRs exhibited the highest frequencies of activated/cycling CD4⁺ T-cells. The proportion of activated/cycling cells was higher in Tregs compared to Tconvs in all groups. Cycling rates of Tregs and Tconvs were correlated, suggesting Tregs help control Tconv proliferation. Despite high overall Treg frequencies in INRs, they showed a Treg deficiency in activated/cycling CD4⁺ T-cells, specifically in naive and central memory subsets, causing an imbalance in the Tconv/Treg ratio. This deficiency was hidden by increased Treg frequencies in quiescent effector memory CD4⁺ T-cells. Activated/cycling naive and memory Tregs from INRs had normal forkhead box P3 (FOXP3) and CD25 expression, but activated/cycling memory Tregs showed decreased ability to regulate mitochondrial transmembrane potential, indicating impaired mitochondrial fitness. These mitochondrial abnormalities were similar to those observed in memory conventional T-cells.

The complex Treg dysregulation in immunological non-responders involves quantitative and functional alterations, including a Treg deficiency within activated/cycling naive and central memory CD4⁺ T-cells, impaired mitochondrial fitness of activated/cycling memory Tregs, and functional disorders of the parent conventional T-lymphocytes. These findings underscore the need for a nuanced understanding of Treg dynamics in suboptimal CD4⁺ T-cell reconstitution during HIV-infection.