Journal: Cell Reports Medicine

Article Title: Distinct viral reservoirs and immune signatures in individuals on long-term antiretroviral therapy with perinatally acquired HIV-1

doi: 10.1016/j.xcrm.2025.102150

Figure Lengend Snippet: HIV-specific immune responses of the LeukoHIV cohort (A) IFN-ɣ/IL-2 FluoroSpot T assay on a representative LeukoHIV subject. (B) Scatterplots with bar (mean ± SEM) depict IL-2 (Cy3/red) or IFN-ɣ (FITC, green) spot-forming cells (SFCs) per 10 6 peripheral blood mononuclear cells (PBMCs) after 24-h in vitro stimulation of thawed PBMCs, isolated from the LeukoHIV cohort, with HIV Gag or Env peptide pools, keyhole limpet hemocyanin (KLH, negative control), or staphylococcal enterotoxin B (SEB, positive control) on anti-IFN-ɣ and IL-2-coated plate. (C) HIV-specific CD8 + T cell responses in participants of focus. ELISPOT plates and the subsequent spot counts reflect the IFN-ɣ secretion in response to HLA-optimal HIV peptides or control treatments. CFSE dilution was used to measure CD8 + T cell proliferation (%CFSE-low), highlighting observed responses among 53 total peptides screened. LeukoHIV09 responses were recorded in response to HLA-B∗14-restricted Gag epitope DA9 (DRFYKTLRA), HLA-B∗14-restricted Env epitope (ERYLKDQQL), and HLA-B∗51-restricted Pol epitope (LPPVVAKEI); these are outlined in red, the proliferative response annotated. (D) Western blot (WB) score tabulated findings for the LeukoHIV cohort. Plasma samples collected in 2017 (left) and 2022 (right) were tested for antibodies against 9 different HIV-1 viral proteins (gp160, gp120, p66, p55/p51, gp41, p39, p31, p24, and p17). The band intensity for each viral antigen was calculated using the ImageJ software. A WB score was assigned to each participant by adding up the number of positive (1, black) and weak (0.5, gray) responses (from 0 to 9). (E) Scatterplots for WB score (2022) against Env-specific peripheral T follicular helper (pTfh) cell frequency (%) (left), Gag-specific pTfh cell frequency (%) (middle), as well as of Env-specific CD4 + T cell frequency (%) against the time needed to reach viral suppression in months (right), with linear regression and correlation analyses performed. Linear regression R 2 and p values presented. Spearman’s correlation rho and p values presented. Gray area represents the confidence interval.

Article Snippet: T cells secreting single IFN-ɣ (FITC/green spots) single IL-2 (Cy3/red spots) and dual IFN-ɣ/IL-2 (merged/yellow spots) were counted using AID EliSpot v7 software (Autoimmun Diagnostika GmbH, Straβberg, Germany) normalized for the number of plated PBMCs and expressed as spot-forming cells/million of PBMCs.

Techniques: In Vitro, Isolation, Negative Control, Positive Control, Enzyme-linked Immunospot, Control, Western Blot, Clinical Proteomics, Software