Design of the study.

<p>Footnote: (a) Eligible individuals were patients treated within 90 days of HIV exposure on stable ART for at least 12 months. Patients had to show good virological and immunological responses (at least 2 viral load <20 copies/mL and CD4 T cells higher than 500 cells/mm3 with a CD4/CD8 ratio >1). All individuals started four structured treatment interruption cycles (b) of 8 week each (<i>off-ART</i>), followed by four cycles (c) of treatment (<i>on-ART</i>). After the last treatment interruption (week 0) 6 patients started self-administrated IL-2 at a dose of 750,000 UI/m2 daily for 6 months (d). Treatment was interrupted in both arms when patients reached viral load <20 copies/mL (e). Analyses were performed at 24 and 48 weeks and after a long term follow-up period (9 years). During this period (f) treatment was restarted in patients whose CD4 cell count dropped below 350 cell/mm3.</p

Structured Treatment Interruptions and Low Doses of IL-2 in Patients with Primary HIV Infection. Inflammatory, Virological and Immunological Outcomes

<div><p>Background</p><p>Interventions during primary HIV infection (PHI) can modify the clinical course during the chronic phase. The long-term effect of structured treatment interruptions (STI) followed by low doses of interleukin-2 (IL-2) in treated PHI patients is unknown.</p><p>Methods</p><p>Twelve PHI patients with viral load (VL) <20 copies/mL, CD4 cells >500 cells/mm3, and CD4/CD8 ratio >1, on antiretroviral therapy (ART) initiated within the first 90 days of infection and continued for at least 12 months were included. They underwent four STI and were then allocated (week 0 of the study) to ART alone or ART plus low doses of IL-2. ART was stopped once VL <20 copies/mL ('final stop'). Primary endpoints were VL<3000 copies/mL and CD4 cells >500 cells/mm3 at 48 weeks; secondary endpoints were immune activation, inflammatory markers until 48 weeks and the time before resuming ART (CD4 <350 cells/mm3 or AIDS) after ‘final stop’, compared between groups.</p><p>Results</p><p>Ten out of 12 patients were males, median age was 35 years and the main risk was men-who-have-sex-with-men. Only one out of 12 patients (in the STI group) maintained VL<3000 copies/mL and CD4 cells >500 cells/mm3 without ART at 48 weeks. All other virological and immunological parameters were comparable between groups at week 0, 'final stop' and week 48. However, the proportion of CD8-CD38+ cells, tumor necrosis factor and srIL-2 were higher in the IL-2 group at 'final stop' and week 24. All these differences vanished during follow-up. At 5 years after the final stop 3 out of 6 patients in the IL-2 group and 6 out of 6 patients in the STI group have resumed ART (P = 0.19).</p><p>Conclusions</p><p>STI and IL-2 failed to achieve virological control after ART interruption. STI were not deleterious in long-term follow-up, an important issue for eradication and functional cure trials.</p><p>Trial Registration</p><p>ClinicalTrials.gov <a href="http://www.clinicaltrials.gov/ct2/show/NCT02300623" target="_blank">NCT02300623</a></p></div

Proportion of patients not receiving ART after the final stop.

<p>Proportion of patients not receiving ART after the final stop.</p

Inflammation, immune activation, immunological and virological evolution following the end of 4^th STI (week 0) until week 48 of follow-up.

<p>Values are median and IQR</p><p>*p<0.05</p><p>ART: Antiretroviral treatment</p><p>STI: structured treatment interruptions</p><p>srIL-2: seric receptor for IL-2</p><p>SFC: Spot Forming Cells</p><p>PBMC: Peripheral blood mononuclear cells</p><p>Inflammation, immune activation, immunological and virological evolution following the end of 4^th STI (week 0) until week 48 of follow-up.</p

The frequency of PD-1-expressing Treg cells correlates with markers of disease progression.

<p>(A) Representative flow cytometry dot plots showing PD-1 gating on Treg cells (including eTreg and rTreg). One HIV-infected individual from each study group is displayed. Numbers indicate the percentage of PD-1+ Treg cells in PD-1 stained samples (down) compared with isotype control antibodies (up). (B) PD-1 expression on Treg cells from different HIV-infected study groups (black circles) and healthy controls (empty triangles) are shown as indicated. Each dot represents the result from one individual. The mean ± SEM (standard error of the mean) is shown. Significant differences were determined by a Mann-Whitney U test, corrected for multiple comparisons using Bonferroni method, and indicated by asterisks (*p <0.05; **p <0.01; ***p <0.001). (C) Correlation of PD-1 expression on Treg cells from HIV-infected individuals with viral loads (up) and CD4 T cell counts (down), respectively. Spearman’s rank correlation coefficients (r) and p values (P) are indicated.</p

A) Plasma HIV Viral Load evolution after 4^th STI cycle. B) CD4+ T cell evolution after 4^th STI cycle. Footnote: The period of IL-2 administration is shown in grey.

<p>The filled points represent periods on ART. Empty points are determinations without ART.</p

Flow diagram of the trial.

<p>Flow diagram of the trial.</p

Treg cells from viremic individuals show impaired proliferative capacity that correlates with PD-1 expression.

<p>CFSE-labelled PBMC from HIV-infected individuals were stimulated with Gag peptides for 6 days. (A) Flow cytometry dot plots showing CFSE dilution of Treg cells (eTreg cells in red and rTreg cells in black). Numbers indicate the percentage of proliferating Treg cells (including eTreg and rTreg). (B) The percentages of proliferating Treg cells (including eTreg and rTreg) from different HIV-infected study groups are given as indicated. Each dot represents the result from one HIV-infected individual. The mean ± SEM (standard error of the mean) is shown. Significant differences were determined by a Mann-Whitney U test, corrected for multiple comparisons using Bonferroni method, and indicated by asterisks (**p <0.01; ***p <0.001). Panels C to E show correlations between the percentage of proliferating Treg cells and CD4 T cell counts (C), HIV viral load (D), and PD-1 expression on Treg cells before stimulation (E), respectively. Spearman’s rank correlation coefficients (r) and p values (P) are indicated.</p

Virus reactivation upon PD-L1 blockade is related to increased percentage of Treg cells.

<p>(A) PBMCs were stimulated with Gag peptides in the presence of a PD-L1 blocking antibody or an isotype control antibody. After 4 days in culture, supernatants were harvest to quantify the p24 HIV core antigen by ELISA. The dashed line indicates the ELISA cut-off. Significant differences were determined by a Wilcoxon matched pairs test (*p <0.05; **p <0.01). Ild: inferior to the limit of detection. Panels B to E show correlations between fold change (FC) in p24 and fold change in percentage of proliferating CD4 T cells (B), percentage of Treg cells (including eTreg and rTreg) (C), fold change in the CD4 T cell to Treg cell (CD4/Treg) percentage ratio (D), and fold change in the CD8 T cell to Treg cell (CD8/Treg) percentage ratio (E); respectively. Spearman’s rank correlation coefficients (r) and p values (P) are indicated.</p

PD-L1 blockade differentially increases Treg and CD8 T cell proliferative capacity depending on host viremia.

<p>PBMC from HIV-infected individuals were stimulated with Gag peptides for 6 days in the presence of a PD-L1 blocking antibody or an isotype control antibody. Proliferation was determined by CFSE dilution (A) and alternatively by Ki67 staining (B and C). FC (fold change) in proliferation is calculated as the ratio between PD-L1 blockade conditions and isotype control conditions. Each symbol represents the result for one individual. In panels A and B, the dashed line (FC = 1) indicates no change due to PD-L1 blockade. The means of fold changes in proliferation are shown. (A) Given are the fold changes in the proliferation of Treg (including eTreg and rTreg) (black circles), CD4- (empty squares) and CD8- (empty triangles) T cell populations of different HIV-infected study groups as indicated. Significant differences in fold change of proliferation of Treg, CD4- and CD8- T cells among the 4 HIV study groups were determined using the Kruskal-Wallis test. Significant differences were found in the fold change of proliferation of Treg cells across the 4 HIV- study groups (*p <0.05) but not of CD4- and CD8- T cells. (B) Fold changes in proliferation of Treg, CD4- and CD8- T cell populations upon PD-L1 blockade were measured longitudinally in 7 individuals before and after >2 years of antiretroviral treatment (pre-cART and on-cART, respectively). Significant differences in fold change of proliferation between Treg, CD4- and CD8- T cells were determined by a Wilcoxon matched pairs test (*p <0.05; ns: non significant). (C) Ratio of the FC in proliferation of CD8 T cells and the FC in proliferation of Treg cells upon PD-L1 blockade. The dashed line (FC = 1) indicates the same FC in proliferation for CD8 T and Treg cells upon PD-L1 blockade. Significant differences between the ratios before and after antiretroviral treatment were determined by Wilcoxon matched pairs test (*p <0.05).</p