Human subjects and sample collection.

The patient cohort has been described elsewhere [17]. Briefly, all patients (1) were ART-naïve (n = 56) or had interrupted treatment for at least one year (n = 4, plus n = 2 who had previously received brief mono- or dual therapy) with a viral rebound of > 10,000 copies/ml; (2) had ≤ 200 CD4⁺ T cells/μl at baseline; (3) suppressed their HIV-1 viral load to <500 copies/ml within one year of ART; and (4) had available peripheral blood mono-nuclear cell (PBMC) samples taken pre-ART as well as after 1, 3, 6, and 12 months of ART. Seventeen patients developed episodes of immune reconstitution inflammatory syndrome (IRIS; defined according to the AIDS Clinical Trials Group criteria, <https://actgnetwork.org/IRIS_Case_Definitions>) following commencement of ART, while 39 underwent uneventful immune reconstitution. PBMC from 12 healthy donors served as controls (Table 1).

PATIENT COHORT CHARACTERISTICS

For the elucidation of T-helper subsets (Figures 5–6), PBMC of an additional 13 HIV-1⁺ individuals were sampled before, as well as one month, and 12 months after initiation of ART. Their clinical parameters were comparable to that of the main cohort, with the following medians and inter-quartile ranges pre-ART: 56 (20-77) CD4⁺ T cells/μl, 572 (469-744) CD8⁺ T cells/μl, 4.8 (4.5-5.4) log₁₀ PVL; after 1 month of ART: 129 (101-152) CD4⁺ T cells/μl, 918 (589-1105) CD8⁺ T cells/μl, 2.3 (1.9-2.7) log₁₀ PVL; and after 12 month of ART: 210 (199.8-264.5) CD4⁺ T cells/μl, 795.5 (555.5-950) CD8⁺ T cells/μl, 1.7 (1.7-1.7) log₁₀ PVL. None of these patients experienced IRIS. PBMC from an additional 16 healthy donors served as controls for this part of the study.

The gene expression profile of CCR4⁺ T_CM is different from that of Th₂-like cells. PBMC from healthy donors, as well as cells isolated before or after 1 or 12 months of ART from HIV-1-infected adults were stained with the “sorting” panel (Supplementary Table 1). Subsets of CD4⁺ T cells were sorted as indicated in Supplementary Figure 4 and their gene expression profiles determined by multi-parametric quantitative RT-PCR. (A) Th₂-associated and (B) other cytokine genes were compared in Th₂-like, CCR4⁺ T_CM, and Th₁-like cells isolated from healthy donors. CCR4⁺ T_CM from HIV-1⁺ patients after 1 month of ART were compared to their counterparts from healthy donors in respect to expression of (C) Th₂-associated or (D) other cytokine genes. (E) The expression profile of cytokine genes was investigated in nonnaïve cells. Relative expression in HIV-1-infected individuals before ART, after 1 month of ART, or 1 year of ART was compared to that in healthy donors. (F) The overall gene expression pattern of CCR4⁺ T_CM, CCR4⁺ T_CM⁻, CCR4⁻ T_CM, and CCR4⁻ T_CM⁻ cells was compared to that of Th₁- or Th₂-like cells sorted from healthy donors. Their calculated “Th-ness” is expressed as a point between those two extremes. Bar graphs show interquartile ranges, median bars, as well as individual data points. Statistically significant differences are indicated: *P < 0.01, **P < 0.001, ***P < 0.0001.

CCR4⁺ T_CM appear to be released from peripheral tissue sites upon ART initiation. PBMC from healthy donors, as well as cells isolated before or after 1 or 12 months of ART from HIV-1-infected adults were stained with the “sorting” panel (Supplementary Table 1). Subsets of CD4+ T cells were sorted as indicated in Supplementary Figure 4 and their gene expression profiles determined by multi-parametric quantitative RT-PCR. (A) CD103 expression on Th₂-like, CCR4⁺ T_CM, and Th₁-like cells isolated after 1 month of antiretroviral therapy. (B) Expression of CD103 in CCR4⁺ T_CM, Th₂-, and Th₁-like cells from healthy donor PBMC (n = 9), and longitudinal samples from HIV-1 patients (n = 12). Bar graphs show interquartile ranges, median bars, as well as individual data points. Statistically significant differences are indicated: *P < 0.01, **P < 0.001, ***P < 0.0001.