HIV-specific CD8+ T-cell proliferative response 24 weeks after early antiretroviral therapy initiation is associated with the subsequent reduction in the viral reservoir
- Department of Experimental Immunology, Amsterdam UMC, Netherlands
- Amsterdam Institute for Infection and Immunity, Amsterdam UMC, Netherlands
- Department of Medical Microbiology and Infection Prevention, Laboratory of Experimental Virology, Amsterdam UMC, Netherlands
- Department of Medical Microbiology, Translational Virology, University Medical Center Utrecht, Netherlands
- Department of Biochemistry and Department of Pathology, Erasmus Medical Center, Netherlands
- Department of Internal Medicine and Radboud Center for Infectious Diseases, Radboud University Medical Center, Netherlands
- Department of Internal Medicine and Department of Medical Microbiology and InfectiousDiseases, Erasmus University Medical Center, Netherlands
- Department of Internal Medicine, section Infectious Diseases, Amsterdam UMC, location AMC, Netherlands
- Apheresis Unit,Dianet, location Amsterdam UMC, Netherlands
- Department of Internal Medicine, section Nephrology, Amsterdam UMC, location AMC, Netherlands
Figures
Figure 1
Plasma viral load, CD4+ T-cell count, and CD4/CD8 ratio at baseline, 24 and 156 weeks post-antiretroviral therapy (ART) initiation.
Participant X (green dot) had detectable loads of 200 and 100 copies/mL at 24 and 156 weeks. Participant Y (pink dot) had detectable loads of 74 and 98 copies/mL at 24 and 156 weeks. Lower limit of quantification of the assays used ranged between 40 copies (earlier) and 20 copies (later time points) (see Methods). Numerical data provided in Figure 1—source data 1.
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Figure 1—source data 1
Numerical data corresponding to Figure 1.
- https://cdn.elifesciences.org/articles/106402/elife-106402-fig1-data1-v1.xlsx
Figure 2 with 1 supplement
Correlation matrix of viral reservoir measures at 24 and 156 weeks after start of antiretroviral therapy (ART).
Correlation coefficients (rho) determined by Spearman correlation analyses are shown. Strength of positive and negative correlations is indicated by the color shade displayed in the legend. Significant correlations are indicated by *** if p<0.001 or by ** if p<0.01. All significant correlations remained significant after corrections for multiple comparisons. Numerical data provided in Figure 2—source data 1.
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Figure 2—source data 1
Numerical data corresponding to Figure 2.
- https://cdn.elifesciences.org/articles/106402/elife-106402-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Comparison of intact HIV DNA levels at 24 weeks antiretroviral therapy (ART) between participants with positive (n=6) and negative (n=14) quantitative viral outgrowth assay (QVOA).
Mann-Whitney test was used to determine statistical significance. *, 0.01<p<0.05. Numerical data provided in Figure 2—figure supplement 1—source data 1.
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Figure 2—figure supplement 1—source data 1
Numerical data corresponding to Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/106402/elife-106402-fig2-figsupp1-data1-v1.xlsx
Figure 3
Paired comparisons of viral reservoir measures at 24 and 156 weeks of antiretroviral therapy (ART).
Open symbols represent values below the detection limits of the assays. The four participants with a positive quantitative viral outgrowth assay (QVOA) at 24 weeks are marked with colors (purple, blue, green, or red). A paired Wilcoxon test was performed to test the significance of the differences between the time points (* if p<0.05 or ** if p<0.01). Pairs where both values were undetectable, or where one was undetectable and its detection limit was higher than the value of the detectable partner, were excluded from the analysis. Exact p-values are indicated below the graphs. Numerical data provided in Figure 3—source data 1.
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Figure 3—source data 1
Numerical data corresponding to Figure 3.
- https://cdn.elifesciences.org/articles/106402/elife-106402-fig3-data1-v1.xlsx
Figure 4
Longitudinal analysis of immunological parameters.
(A) Longitudinal analysis of frequencies of activated and naive, memory and effector subsets within the CD4+ and CD8+ T-cell populations. Wilcoxon signed-rank test (p<0.05) was used to determine statistical significance of the differences between the time points. Numerical data provided in Figure 4—source data 1. (B) Left panel: the frequency of dextramer+CD8+ T-cells at 24 and 156 weeks after antiretroviral therapy (ART) initiation. Right panel: the expression of exhaustion markers (upregulation of PD-1, CTLA-4, and CD160 expression; loss of CD28 expression) on HIV-dextramer-specific CD8+ T-cells at 24 and 156 weeks after ART initiation. For participants that were HLA-A*2 and HLA-B*7 positive, HLA-A*2 and HLA-B*7 dextramer staining was included separately. HLA-A*2 dextramer is marked as a triangle and HLA-B*7 dextramer as a square in both panels. Numerical data provided in Figure 4—source data 2. (C) HIV-specific T-cell responses upon HIV-peptide stimulation (Env, Gag, Nef, Pol) at 24 and 156 weeks after ART: interferon gamma release assay (IGRA)/IFN-γ release, activation-induced marker (AIM) reactive CD8+ T-cells, precursor frequency, and the proportion of proliferating CD8+ T-cells. Frequencies of proliferating cells in response to Env, Gag, Nef, and Pol peptides were combined. Numerical data provided in Figure 4—source data 3.
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Figure 4—source data 1
Numerical data corresponding to Figure 4A.
- https://cdn.elifesciences.org/articles/106402/elife-106402-fig4-data1-v1.xlsx
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Figure 4—source data 2
Numerical data corresponding to Figure 4B.
- https://cdn.elifesciences.org/articles/106402/elife-106402-fig4-data2-v1.xlsx
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Figure 4—source data 3
Numerical data corresponding to Figure 4C.
- https://cdn.elifesciences.org/articles/106402/elife-106402-fig4-data3-v1.xlsx
Figure 5 with 1 supplement
Correlations of reservoir measures and HIV-specific CD8+ T-cell responses as determined by IFN-γ release assay, activation-induced marker (AIM), and proliferation assay (proliferating cells and precursor cells).
The immune responses are defined as the sum of the responses to Env, Gag, Nef, and Pol combined at 24 and 156 weeks. Correlation coefficients (rho) determined by Spearman correlation analyses are shown. Strength of positive and negative correlations is indicated by the color shade displayed in the legend. Significant correlations are indicated by *** if p<0.001 or by ** if p<0.01. All significant correlations remained significant after corrections for multiple comparisons. Numerical data provided in Figure 5—source data 1.
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Figure 5—source data 1
Numerical data corresponding to Figure 5.
- https://cdn.elifesciences.org/articles/106402/elife-106402-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Correlation matrix of reservoir measurements and the frequency of dextramer+CD8+ T-cell responses at 24 and 156 weeks after antiretroviral therapy (ART) initiation.
The correlation coefficient (rho) determined by Spearman correlation is shown. Positive and negative correlations (rho) are indicated by the color shade displayed in the legend. Significant correlations are indicated by * if p<0.05 or ** if p<0.01. Numerical data provided in Figure 5—figure supplement 1—source data 1.
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Figure 5—figure supplement 1—source data 1
Numerical data corresponding to Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/106402/elife-106402-fig5-figsupp1-data1-v1.xlsx
Tables
Table 1
Characteristics of the study participants*.
| Total (n=22) | Paired analysis (n=12)† | ||
|---|---|---|---|
| Sex at birth, male | 100% | ||
| Age at inclusion, years‡ | 38 (28–48) | 40.5 (25.5–49) | |
| Country of birth | The Netherlands | 18 | 12 |
| Other | 4 | 0 | |
| Fiebig stage at diagnosis§ | II | 1 | 0 |
| III – IV | 12 | 7 | |
| V | 5 | 4 | |
| VI | 4 | 1 | |
| Plasma viral load at baseline, 106 copies/mL | 0.4 (0.04–10) | 0.61 (0.28–10) | |
| CD4+ T-cell count at baseline, cells/mm3 | 445 (308–593) | 435 (295–555) | |
| HIV subtype | B | 15 | 10 |
| CRF02_AG | 1 | 0 | |
| AE | 3 | 1 | |
| F | 2 | 1 | |
| Unknown | 1 | 0 | |
| Time between diagnosis and start of ART | Same day | 15 | 8 |
| 1 day | 3 | 1 | |
| 2 days | 2 | 1 | |
| Unknown | 2 | 2 |
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*
Numerical data provided in Table 1—source data 1.
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†
Participants with available samples from both 24-week and 156-week time points.
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‡
Medians and interquartile ranges are shown for continuous variables.
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§
Fiebig, E. W. et al. Dynamics of HIV viremia and antibody seroconversion in plasma donors: implications for diagnosis and staging of primary HIV infection. AIDS 17, 1871–1879 (2003).
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Table 1—source data 1
Numerical data corresponding to Table 1.
- https://cdn.elifesciences.org/articles/106402/elife-106402-table1-data1-v1.xlsx
Table 2
Variables measured at 24 weeks’ antiretroviral therapy (ART) associated with the changes in HIV reservoir markers between 24 and 156 weeks of ART*.
| Variable | Total HIV DNA† | Intact HIV DNA | Defective HIV DNA | |||
|---|---|---|---|---|---|---|
| Effect size (95% CI) | p‡ | Effect size (95% CI) | p | Effect size (95% CI) | p | |
| Age, per year | –2.99 (−28.5 to 22.5) | 0.60 | –0.49 (−5.00 to 4.02) | 0.86 | 1.19 (−14.3 to 16.7) | 0.66 |
| NRTI backbone, ABC+3 TC vs. FTC +TDF/TAF§ | –1.10 (−4.80 to 2.60) | 0.56 | –0.67 (−4.56 to 3.22) | 0.74 | 2.67 (−0.94 to 6.27) | 0.15 |
| IFN-γ release, per pg/mL | 2.40 (−13.2 to 18.0) | 0.79 | –1.32 (−4.04 to 1.40) | 0.59 | –5.29 (−14.4 to 3.81) | 0.14 |
| CD8+ T-cell reactivity, per % | –537 (−1677 to 603) | 0.27 | –85.6 (−298 to 127) | 0.25 | –361 (−1073 to 351) | 0.15 |
| CD8+ T-cell proliferation, per % | 62.7 (28.7–96.7) | 0.014 | 2.84 (−4.62 to 10.3) | 0.56 | 37.8 (18.5–57.1) | 0.0017 |
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*
Numerical data provided in Table 2—source data 1.
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†
Total, intact, and defective HIV DNA are in copies/106 PBMC.
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‡
Calculated by fitting generalized linear models (GLM), type III tests were used.
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§
Six participants were treated with ABC+3TC and six participants – with FTC+TDF or FTC+TAF as an NRTI backbone.
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Table 2—source data 1
Numerical data corresponding to Table 2.
- https://cdn.elifesciences.org/articles/106402/elife-106402-table2-data1-v1.xlsx
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Anti-CD4-FITC (Mouse monoclonal) | BD Biosciences | Cat# 345768; RRID:AB_2868797 | 1:100 |
| Antibody | Anti-OX40-PE (Mouse monoclonal) | BioLegend | Cat# 350004; RRID:AB_10645478 | 1:20 |
| Antibody | Anti-CD69-PE-Cy7 (Mouse monoclonal) | BioLegend | Cat# 310912; RRID:AB_314847 | 1:20 |
| Antibody | Anti-CD137-APC Fire 750 (Mouse monoclonal) | BioLegend | Cat# 309834; RRID:AB_2734280 | 1:20 |
| Antibody | Anti-CD8-PB (Mouse monoclonal) | BioLegend | Cat# 301023; RRID:AB_493110 | 1:50 |
| Antibody | Anti-CD3-BV510 (Mouse monoclonal) | BioLegend | Cat# 300447; RRID:AB_2563467 | 1:200 |
| Antibody | Anti-CD4-PerCP-Cy5.5 (Mouse monoclonal) | BD Biosciences | Cat# 332772; RRID:AB_2868621 | 1:25 |
| Antibody | Anti-CD8-APC (Mouse monoclonal) | BioLegend | Cat# 344722; RRID:AB_2075388 | 1:50 |
| Antibody | Anti-CD3-FITC (Mouse monoclonal) | E-bioscience | Cat# 11-0036-42 | 1:25 |
| Antibody | Anti-CD3-V500 (Mouse monoclonal) | BD Biosciences | Cat# 561416; RRID:AB_10612021 | 1:50 |
| Antibody | Anti-CD4-APC-H7 (Mouse monoclonal) | BD Biosciences | Cat# 641407; RRID:AB_1645733 | 1:50 |
| Antibody | Anti-CCR7-BV786 (Mouse monoclonal) | BD Biosciences | Cat# 564355; RRID:AB_2738765 | 1:30 |
| Antibody | Anti-CD45RA-BV605 (Mouse monoclonal) | BioLegend | Cat# 304133; RRID:AB_11126164 | 1:50 |
| Antibody | Anti-CTLA4-BV711 (Mouse monoclonal) | BioLegend | Cat# 369631; RRID:AB_2892450 | 1:50 |
| Antibody | Anti-CD160-PE-Cy7 (Mouse monoclonal) | BioLegend | Cat# 143009; RRID:AB_2562677 | 1:50 |
| Antibody | Anti-CD28-AF700 (Mouse monoclonal) | BioLegend | Cat# 302919; RRID:AB_528785 | 1:50 |
| Antibody | Anti-PD1-PE (Mouse monoclonal) | eBioscience | Cat# 12-9969-42 | 1:20 |
| Antibody | Anti-HLA-DR-FITC (Mouse monoclonal) | BD Biosciences | Cat# 347400; RRID:AB_2868846 | 1:60 |
| Antibody | Anti-CD38-PE (Mouse monoclonal) | BD Biosciences | Cat# 345806; RRID:AB_2868828 | 1:100 |
| Antibody | Anti-CD27-APCeFluor 780 (Mouse monoclonal) | eBioscience | Cat# 47-0271-82 | 1:20 |
| Antibody | Anti-CD28-PerCP Cy5.5 (Mouse monoclonal) | BD Biosciences | Cat# 337181; RRID:AB_513211 | 1:20 |
| Antibody | Anti-CD4-PE-Cy7 (Mouse monoclonal) | BD Biosciences | Cat# 348809; RRID:AB_2783789 | 1:250 |
| Antibody | Anti-CD57-APC (Mouse monoclonal) | BD Biosciences | Cat# 560845; RRID:AB_10563760 | 1:500 |
| Commercial assay or kit | DNA-free DNA Removal Kit | Thermo Fisher Scientific | Cat# AM1906 | |
| Chemical compound, drug | Platinum Quantitative PCR SuperMix-UDG | Thermo Fisher Scientific | Cat# 11730–025 | |
| Commercial assay or kit | TaqMan β-Actin Detection Reagents | Thermo Fisher Scientific | Cat# 401846 | |
| Commercial assay or kit | TaqMan Ribosomal RNA Control Reagents | Thermo Fisher Scientific | Cat# 4308329 | |
| Chemical compound, drug | SuperScript III reverse transcriptase | Thermo Fisher Scientific | Cat# 18080-085 | |
| Chemical compound, drug | Random primers | Thermo Fisher Scientific | Cat# 48190-011 | |
| Chemical compound, drug | RNaseOUT Recombinant Ribonuclease Inhibitor | Thermo Fisher Scientific | Cat# 10777-019 | |
| Chemical compound, drug | ddPCR Supermix for Probes (No dUTP) | Bio-Rad | Cat# 1863024 | |
| Commercial assay or kit | Puregene Cell Kit | QIAGEN | Cat# 158043 | |
| Commercial assay or kit | Human IFN-γ DuoSet ELISA | R&D Systems | DY285B | |
| Commercial assay or kit | CellTrace Violet Cell Proliferation Kit | Thermo Fisher Scientific | C34557 | |
| Commercial assay or kit | CD4+ T-Cell Isolation Kit, human | Miltenyi Biotec | 130-096-533 | Isolation of untouched CD4 T-cells |
| Other | HLA-A*0201 SLYNTVATLY APC-labeled MHC class I dextramers | Immudex | WB03338 | 50 tests |
| Other | HLA-B*0702 GPGHKARVL APC-labeled MHC class I dextramers | Immudex | WH03590 | 50 tests |
| Peptide, recombinant protein | HIV subtype B consensus Env peptide pool | NIH AIDS Reagent Program | HRP-12540 | 2 µg/mL |
| Peptide, recombinant protein | HIV subtype B consensus Gag peptide pool | NIH AIDS Reagent Program | HRP-12425 | 2 µg/mL |
| Peptide, recombinant protein | HIV subtype B consensus Nef peptide pool | NIH AIDS Reagent Program | HRP-12545 | 2 µg/mL |
| Peptide, recombinant protein | HIV subtype B consensus Pol peptide pool | NIH AIDS Reagent Program | ARP-6208 | 2 µg/mL |
| Software, algorithm | Prism 10.6.0 | GraphPad Software | https://www.graphpad.com/ RRID:SCR_002798 | Statistics |
| Software, algorithm | IBM SPSS Statistics 28.0.1.0 | IBM | https://www.ibm.com/products/spss-statistics RRID:SCR_016479 | Statistics |
| Software, algorithm | QuantaSoft 1.7.4 | Bio-Rad | RRID:SCR_025696 | ddPCR data analysis |
| Software, algorithm | Rotor-Gene 2.3.5 | QIAGEN | RRID:SCR_015740 | qPCR data analysis |
| Software, algorithm | FlowJo 10.8.1 | Becton Dickinson | https://www.flowjo.com/ RRID:SCR_008520 | Flow cytometry data analysis |
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HIV-specific CD8+ T-cell proliferative response 24 weeks after early antiretroviral therapy initiation is associated with the subsequent reduction in the viral reservoir
eLife 14:RP106402.
https://doi.org/10.7554/eLife.106402.3
