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. 2022 Aug 4:14:365-379.
doi: 10.2147/HIV.S374369. eCollection 2022.

Flow-Cytometry Intracellular Detection and Quantification of HIV1 p24 Antigen and Immunocheckpoint Molecules in T Cells among HIV/AIDS Patients

Belay Tessema  1   2   3 , Andreas Boldt  2 , Brigitte König  3 , Melanie Maier  4 , Ulrich Sack  2
Affiliations

Flow-Cytometry Intracellular Detection and Quantification of HIV1 p24 Antigen and Immunocheckpoint Molecules in T Cells among HIV/AIDS Patients

Belay Tessema et al. HIV AIDS (Auckl). .

Abstract

Introduction: HIV p24 antigen-positive T cells measured by flow cytometry (FCM) correlate directly with HIV viral load, inversely with CD4 + T cells, and decrease with antiretroviral therapy (ART). However, the sensitivity of FCM assays depends on the protocol of intracellular staining. Therefore, this study aimed to evaluate the diagnostic performance of our FCM protocol for detection of HIV p24-positive T cells and measure the level of immunocheckpoint molecules (PD1 and TIM3) in T cells.

Methods: The study was conducted at the University of Leipzig hospital between January 2020 and November 2020. Viremic and ART-suppressed HIV-positive patients and negative controls were included in this study. HIV1 p24 KC57-, p24 28B7-, PD1-, and TIM3-positive CD4 and CD3 T cells were analyzed from whole blood using a BD FACS Canto II flow cytometer equipped with FACSDiva software. HIV1 p24 antigen FCM results were compared with HIV1 RNA viral load results measured by Alinity M assays on the fully automated random-access platform. We analyzed the data using SPSS 20.

Results: The absolute CD4 + and CD4 +:CD8 + T-cells ratio showed a significant inverse correlation with HIV1 viral load. Moreover, the absolute CD4+ T-cells count showed a significant inverse correlation with p24 KC57-positive CD4 T cells. The percentage of p24 KC57, p24 28B7, and double-positive CD4 T cells showed significant correlation with HIV1 viral load. PD1 expressing CD4 T cells were higher in ART-viremic cases than controls, while TIM3-expressing CD4 T cells were lower in ART-viremic cases than controls. Sensitivity, specificity, PPV, and NPV of p24 KC57-positive CD4 T cells were 64%, 82%, 78%, and 69%, respectively, for the diagnosis of HIV infection and 55%, 73%, 40%, and 83%, respectively, for treatment monitoring.

Conclusion: Our protocol showed moderate performance for the diagnosis of HIV infection and treatment monitoring. Therefore, the p24 KC57 but not the p24 28B7 clone could be considered as a simple alternative method for rapid diagnosis of HIV infections and treatment monitoring, particularly in low- and middle-income countries.

Keywords: HIV p24 28B7; HIV p24 KC57; PD1; TIM3; diagnosis; flow cytometry; human immunodeficiency virus; treatment monitoring.

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Conflict of interest statement

The authors declare no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Demonstration of the stepwise gating strategy for sorting and identification of HIV p24-positive CD4+ T cells using an HIV-positive (HIV viremic) patient’s blood (A–E) and HIV-negative control blood sample (F–J). (A and F) (P1) = total lymphocytes; (B and G) (CD3+)= CD3+ T cells; C and H (Q1) = CD3+ CD4- T cells; C and H (CD4+) = CD4+ T cells; D (Q1-2) and I (Q1-3) = gates for HIV p24 (clone 28B7) positive CD4+ T cells; D (Q4-2) and I (Q4-3) = gates for HIV p24 (clone KC57) positive CD4+ T cells; D (Q2-2) and I (Q2-3) = gates for both HIV p24 28B7 and HIV p24 KC57 positive (double-stained) CD4+ T cells; F = analysis output of HIV-positive patient’s blood that shows the percentage of HIV p24-positive CD3+CD4– T cells (Q1-1 = p24 28B7, Q4-1 = p24 KC57, Q 2–1 = double-stained cells) and CD4+ T cells (Q1-2 = p24 28B7, Q4-2 = p24 KC57, Q 2–2 = double-stained cells); J = analysis output of HIV-negative person’s blood that shows the percentage of HIV p24-positive CD3+CD4– T cells (Q1-1 = p24 28B7, Q4-1 = p24 KC57, Q2-1 = double-stained cells) and CD4+ T cells (Q1-3 = p24 28B7, Q4-3 = p24 KC57, Q2-3 = double-stained cells).
Figure 2
Figure 2
Demonstration of the objective-based gating strategy for sorting and quantification of immunocheckpoint molecules (ICMs) (PD1 and TIM3)-positive CD3+ and CD4+ T cells from an HIV-positive ART-suppressed patient’s whole blood. The objective gating and quantification of ICM-positive cells were done based on the fluorescence intensity of PD1 or TIM3-positive CD3+ and CD4+ T cells in a stepwise approach. P1 = total lymphocytes; CD3+ = CD3+ T cells; Q1 = CD3+ CD4– T cells; Q2 = CD4+ T cells; P3 = PD1+ CD3+ T cells; P7= the top 99.8% of PD1+CD3+ T cells after outliers removed (trimmed cells); P8 = the top 90% of the fluorescence range of PD1+CD3+ T cells of the trimmed cells. P4 = TIM3+ CD3+ T cells; P9 = the top 99.8% of TIM3+CD3+ T cells after outliers removed; P10 = the top 90% of the fluorescence range of PD1+CD3+ T cells of the trimmed cells. P5 = PD1+ CD4+ T cells; P11 = the top 99.8% of PD1+CD4+ T cells after outliers removed; P12 = the top 90% of the fluorescence range of PD1+CD4+ T cells of the trimmed cells. P6 = TIM3+ CD3+ T cells; P13 = the top 99.8% of TIM3+CD3+ T cells after outliers removed; P14 = the top 90% of the fluorescence range of TIM3+CD3+ T cells of the trimmed cells.
Figure 3
Figure 3
Receiver-operating characteristic (ROC) curves for HIV1 p24 KC57- and p24 28B7-positive CD4+ T cells drawn from sensitivity versus 1 – specificity using HIV-positive cases and controls showing the performance of a test at all classification thresholds. AUC = area under the ROC curve. The AUC provides an aggregate measure of performance of a test across all possible classification thresholds, and here indicates the accuracy of HIV1 p24 KC57 and p24 28B7 antigens to correctly classify ART-viremic and ART-suppressed HIV-positive cases from controls as well as ART-viremic from ART-suppressed HIV-positive cases. An AUC of 1 represents a perfect test; 0.9–1 = excellent; 0.8–0.9 = good; 0.7–0.8 = fair; 0.6–0.7 = poor; 0.5–0.6= fail, and 0.5 represents a worthless test.
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Grants and funding

Financial support has been provided by the Institute of Clinical Immunology and Alexander von Humboldt Foundation to purchase the reagents, chemicals, antibodies, and supplies used for this study.