Journal: Cell Reports Medicine

Article Title: Dominant CD4 + T cell receptors remain stable throughout antiretroviral therapy-mediated immune restoration in people with HIV

doi: 10.1016/j.xcrm.2023.101268

Figure Lengend Snippet: Experimental workflow (A) A5248 participants were sampled 0–504 days following antiretroviral therapy (ART) initiation. In the long-term (LT)-ART group, participants were sampled 3 times over 17–26 months. (B) Participant peripheral blood mononuclear cells (PBMCs) from A5248 and LT-ART (n = 10/group) were phenotyped by mass cytometry and analyzed using unsupervised and manual gating methods, and changes were quantified. TCRβ clonotype sequencing of isolated CD4 + T cells was performed over time in A5248 (n = 4, 5 time points) and LT-ART (n = 3, 3 time points). Proliferative capacity of CD4 + and CD8 + T cells was also assessed in both cohorts using flow cytometry (n = 8/cohort). (C and D) HIV VL, absolute CD4 + and CD8 + T cell count, and CD4:CD8 ratio of A5248 participants (C) and LT-ART participants (D).

Article Snippet: CD4 RPA-T4 145ND , Standard BioTools , Cat# 3145001B; RRID:AB_2661789.

Techniques: Mass Cytometry, Sequencing, Isolation, Flow Cytometry, Cell Counting

Journal: Cell Reports Medicine

Article Title: Dominant CD4 + T cell receptors remain stable throughout antiretroviral therapy-mediated immune restoration in people with HIV

doi: 10.1016/j.xcrm.2023.101268

Figure Lengend Snippet: ART initiates immune restoration across multiple immune lineages (A) EN regression analysis showing the descriptive mass cytometry immune features from unsupervised metaclusters. Shown are immune features that have a non-zero coefficient (median with whiskers reaching the minimum and maximum values across cross-validation folds) and are predicted to be associated with time post ART initiation. A positive coefficient indicates an increase and a negative coefficient indicates a decrease over time. Δ = difference between time point > 0 from time point = 0. (B) Manual gating analysis of total NK, γδ T, CD4 + T, CD8 + T, and CD19 + B cells, showing average %frequency of the indicated markers from day 0 to day 504 post ART initiation; n = 10 mean ± SD. Note: NK cell data are combined CD16 + CD56 − , CD16 + CD56lo, CD16 − CD56 bright populations (see Table 1 ).

Article Snippet: CD4 RPA-T4 145ND , Standard BioTools , Cat# 3145001B; RRID:AB_2661789.

Techniques: Mass Cytometry, Biomarker Discovery

Journal: Cell Reports Medicine

Article Title: Dominant CD4 + T cell receptors remain stable throughout antiretroviral therapy-mediated immune restoration in people with HIV

doi: 10.1016/j.xcrm.2023.101268

Figure Lengend Snippet: Quantification of ART-induced changes in leukocyte populations over time in PWH

Article Snippet: CD4 RPA-T4 145ND , Standard BioTools , Cat# 3145001B; RRID:AB_2661789.

Techniques:

Journal: Cell Reports Medicine

Article Title: Dominant CD4 + T cell receptors remain stable throughout antiretroviral therapy-mediated immune restoration in people with HIV

doi: 10.1016/j.xcrm.2023.101268

Figure Lengend Snippet: Quantification of ART-induced changes in CD4 + and CD8 + T cell populations in PWH

Article Snippet: CD4 RPA-T4 145ND , Standard BioTools , Cat# 3145001B; RRID:AB_2661789.

Techniques:

Journal: Cell Reports Medicine

Article Title: Dominant CD4 + T cell receptors remain stable throughout antiretroviral therapy-mediated immune restoration in people with HIV

doi: 10.1016/j.xcrm.2023.101268

Figure Lengend Snippet: CD4 + T cell immune reconstitution in PWH following ART (A) Mean %frequency (±SD) of selected CD4 + T cell subpopulations in the first 90 days post ART in A5248 (n = 10). %Frequencies at day 0 versus day 84 were compared using Wilcoxon signed-rank test. (B) Mean log2 fold change in mean signal intensity (MSI) of markers on CD4 + T cells over time in A5248 relative to day 504 post ART (day 504 = 0/gray). Average %frequency is denoted by circle size. On the right, significance is indicated for two comparisons: MSI on day 0 versus day 84 (Wilcoxon signed-ranked test) and %change in MSI between day 0 to day 504 (GFE regression; see Table S5 ). (C) Mean %frequency (±SD) of CD127 + cells in CD4 + T cell subsets post ART in A5248 (black) compared with LT-ART (n = 10, gray) over time points T1–T3 spanning 17–26 months. (D) %Frequency of CD4 + T cell subpopulations in A5248 participants on day 504 post ART (light blue) compared with the LT-ART cohort (dark blue, average of 3 longitudinal time points, T1–T3, collected over 17–26 months). (E) Correlation between years virally suppressed and %CD127 + on CD4 + T cell subsets in the LT-ART cohort. All data were gated manually. In (C) and (D), %frequencies on day 504 post ART and LT-ART (average T1–T3) were compared using a Mann-Whitney U test. In (E), associations were tested using Spearman’s rank correlation. For all analyses: (A)–(D), ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001. For (B), -- is p >0.05, elsewhere p > 0.5 left blank.

Article Snippet: CD4 RPA-T4 145ND , Standard BioTools , Cat# 3145001B; RRID:AB_2661789.

Techniques: MANN-WHITNEY

Journal: Cell Reports Medicine

Article Title: Dominant CD4 + T cell receptors remain stable throughout antiretroviral therapy-mediated immune restoration in people with HIV

doi: 10.1016/j.xcrm.2023.101268

Figure Lengend Snippet: Memory and functional phenotypes of CD8 + T cells restore earlier than CD4 + T and NK cells in PWH following ART (A) Mean (±SD) %frequency of CD8 + T cell subpopulations in the first 90 days post ART initiation in A5248 (n = 10). %Frequency on day 0 versus day84 was compared using Wilcoxon signed-rank test. (B) Mean log2 fold change in MSI (yellow to blue) of markers on CD8 + T cells over time in A5248 (n = 10) relative to day 504 post ART. Average %frequency is denoted by circle size. On the right, significance is indicated for two comparisons: MSI on day 0 versus day 84 using Wilcoxon signed-ranked test and %change in MSI between day 0 to day 504, examined using gamma fixed-effects regression with log link (full details in Table S5 ). (C) Comparison of the %frequency of CD8 + T cell subpopulations in A5248 participants on day 504 post ART (light blue, n = 10 participants) compared with durably suppressed (average 6.7 years) participants in the LT-ART cohort (dark blue, n = 10 average of 3 longitudinal time points, T1–T3, collected over 17–26 months). (D) Mean frequency (±SD) of CD127 + cells in naive and memory CD8 + T cell subsets post ART in A5248 compared with LT-ART (n = 10, gray) over time points T1–T3 spanning 17–26 months. (E) Average frequency (±SD) of NK cell subsets post ART in A5248 compared with LT-ART (n = 10, gray) over visits T1–T3. %Frequencies on day 504 post ART and LT-ART (average T1–T3) in (C)–(E) were compared using a Mann-Whitney U test. For all analyses (A–E), ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001. For (B), -- is p > 0.05, elsewhere p > 0.5 left blank.

Article Snippet: CD4 RPA-T4 145ND , Standard BioTools , Cat# 3145001B; RRID:AB_2661789.

Techniques: Functional Assay, Comparison, MANN-WHITNEY

Journal: Cell Reports Medicine

Article Title: Dominant CD4 + T cell receptors remain stable throughout antiretroviral therapy-mediated immune restoration in people with HIV

doi: 10.1016/j.xcrm.2023.101268

Figure Lengend Snippet: CD4 + TCR clonotypes and immunodominance hierarchies are maintained over time on ART (A–D) Personal Identifier (PID) 611183 (A), 363043 (B), 363044 (C), and 291374 (D). Left: correlation between frequencies of TCRβ clonotypes on days 0 and 504 post ART in A5248 participants. Data were transformed prior to graphing. Percentages identify clonotypes detected on day 0 or 504 only (fall on dotted orange lines); clonotypes were detected at frequencies of less than 10 −1 or 10 −1 or greater. Spearman rank (r) correlation provided for clonotypes of less than 10 −1 or 10 −1 or greater. ns, non-significant. Center: frequency of dominant TCRβ clonotypes (≥0.1% at first and last visit) over time in A5248 participants. Right: matrix displaying correlations between frequencies of dominant TCRβ clonotypes on days 0, 7/10, 21, 84, and 504 in A5248. The absolute CD4 count (cells/mm 3 ) post ART at each time point is displayed below. (E–G) PIDs 728 (E), 674 (F), and 861 (G). Left: correlation between frequencies of TCRβ clonotypes at T1 and T3 in the LT-ART group. Center: dominant TCRβ clonotypes (≥0.1% at the first and last time point) over time in LT-ART participants. Right: matrix displaying correlations between frequencies of dominant TCRβ clonotypes at T1–T3 in LT-ART. The absolute CD4 count (cells/mm 3 ) at each time point is displayed below. (H and I) CD4 + and CD8 + T cell proliferation following stimulation with PHA, HCMV, pp65/IE1 peptides, or HIV Gag/Nef peptides in A5248 (H) and LT-ART (I). (J) Day 420 T cell proliferation in A5248 and average proliferation in LT-ART for each participant. Proliferation data (% CellTrace Violet [CTV] lo ) are shown as fold change between PHA/peptide-stimulated and mock-stimulated cultures (n = 8/group), showing mean ± SD. Spearman’s rank correlations were computed between frequencies of TCRβ clonotypes on different days (A–G), and log2 fold changes from A5248 and LT-ART cohorts in (J) were compared using a Mann-Whitney U test, where ∗p ≤ 0.05, ∗∗p ≤ 0.01, and ∗∗∗p ≤ 0.001.

Article Snippet: CD4 RPA-T4 145ND , Standard BioTools , Cat# 3145001B; RRID:AB_2661789.

Techniques: Transformation Assay, MANN-WHITNEY

Journal: Cell Reports Medicine

Article Title: Dominant CD4 + T cell receptors remain stable throughout antiretroviral therapy-mediated immune restoration in people with HIV

doi: 10.1016/j.xcrm.2023.101268

Figure Lengend Snippet:

Article Snippet: CD4 RPA-T4 145ND , Standard BioTools , Cat# 3145001B; RRID:AB_2661789.

Techniques: Recombinant, Antibody Labeling, Staining, Cell Isolation, Control, Mass Cytometry, Sequencing, Software