Lymphocyte activation gene-3 expression defines a discrete subset of HIV-specific CD8+ T cells that is associated with lower viral load

Abstract

Mechanisms leading to the observed immune dysregulation in chronic HIV infection are not well understood. The MHC-II class ligand, lymphocyte activation gene-3 (LAG-3, CD223), has been implicated in the complex regulation mechanism of immune functions. In this study, we describe a new population of HIV-specific CD8(+) T cells expressing LAG-3. These LAG-3(+)CD8(+) T cells do not display immunophenotypic patterns traditionally attributed to regulatory T cells. The LAG3(+)CD8(+) T cells are CCR7(+),CD127(-), and display heterogeneous surface expressions of CD45RA and CD25. Interestingly, HIV-specific LAG-3(+)CD8(+) T cells do not substantially express CTLA-4 and LAG-3 expression does not correlate with interleukin (IL)-10 or tumor growth factor (TGF)-β production. In addition, HIV-specific LAG3(+)CD8(+) T cells do not produce interferon (IFN-γ) or express CD107a. The frequency of HIV-specific LAG3(+)CD8(+) T cells negative correlated with plasma viral load. Our study introduces a new population of HIV-specific CD8(+) T cells and proposes additional mechanisms of immune regulation in chronic HIV infection.

FIG. 1.

Lymphocyte activation gene-3 (LAG-3⁺) expression on HIV-specific CD8⁺ T cells does not express CD107a or produce interferon (IFN)-γ. (A) Peripheral blood mononuclear cells (PBMCs) were stimulated for 6 h with HIV antigens Gag B, Nef B, or CMV pp65 in the presence of anti-CD107a PE and Golgi-Stop or (B) restimulated following an initial 24-h stimulation, as described in Materials and Methods, then stained with Vivid viability dye for dead/live cell exclusion, anti-CD3 AmCyan, anti-CD4 PerCP Cy5.5, anti-CD8 APC-Cy7, anti-LAG-3 FITC, and IFN-γ APC, and analyzed by flow cytometry. Samples were gated on the CD3⁺/CD8⁺ lymphocyte population and then the percent of CD107a and IFN-γ-positive CD8⁺ T cells was determined. (C) Representative plots of the frequency LAG-3 on Gag- or Nef-specific CD8⁺ T cells and CMV pp65-specific CD8⁺ T cells expressing CD107a and IFN-γ are shown. Staphylococcus enterotoxin B (SEB) is used as a positive control. No significant overlap between Lag-3 expression and CD107a or IFN-γ was observed. (D) LAG-3⁺CD8⁺ T cell expression after antigen peptide stimulation compared to no antigen-stimulated control. Gag B-specific LAG-3⁺CD8⁺ T cells were statistically significant compared to Nef B or CMV pp65 (p<0.0001 and p=0.0002, respectively). Results were expressed as the percentage of CD107a or IFN-γ-positive CD8⁺ T cells (percent positive=percent Ag-specific – percent negative control). Responses ≥0.1% and two times the background were considered positive. p values <0.05 were considered significant (n=35).

FIG. 2.

HIV-specific LAG-3⁺CD8⁺ T cells express a variable surface immunophenotype. PBMCs were stimulated with HIV peptides then stained with Vivid for live/dead cell exclusion, LAG-3 FITC, CTLA-4 PE-Cy5, CD3 AmCyan, CD8 APC H7, CD25 PE, CD127 Alexa Fluor 647, and CD45RA PE-Cy7 and analyzed by flow cytometry. Samples were first gated on SSC/FSC, followed by SSC/CD3⁺, and then on the CD3⁺/CD8⁺ lymphocyte population; the percent of LAG-3⁺CD8⁺ T cells was then determined. (A) Two representative plots of HIV-specific LAG-3⁺CD8⁺ T cells after Gag B peptide stimulations that express CD25⁺ and are CD127 low. (B) HIV-specific LAG-3⁺CD8⁺ T cells with variable expression of CD45RA and CCR7. Box inserts within each quadrant represent the indicated cell subpopulations. Dotted arrows indicate further subgating of indicated cell subpopulations.

FIG. 3.

LAG-3⁺CD8⁺ T cell populations are distinct from PD1⁺, CTLA4⁺, or 2B4⁺ CD8⁺ T cells. PBMCs were stimulated with HIV-specific peptide Gag B peptides then stained with Vivid for live/dead cell exclusion, LAG-3 FITC, 2B4 PE, CD3 AmCyan, PD-1 APC, CTLA-4 PE-Cy5, CD4 PE-Cy7, and CD8 APC-H7. Samples were first gated on the CD3⁺/CD8⁺ lymphocyte population; the percent of LAG3⁺, PD-1⁺ CTLA4⁺, or 2B4⁺ cells was then determined. Representative plots of the frequencies of the LAG-3⁺ Gag-specific CD8⁺ T cells and expression of PD-1⁺ CTLA4⁺ or 2B4⁺ are shown. Responses ≥0.1% and two times the background were considered positive.

FIG. 4.

Negative correlation between the frequency of HIV-specific LAG-3⁺CD8⁺ T cells and HIV plasma RNA. The percentage of Gag-specific LAG-3 is plotted against the HIV plasma RNA. A significant negative correlation between HIV plasma RNA and Gag-specific LAG-3⁺CD8⁺ T cells in (A) was observed (r_s=−0.51, p=0.0008). (B) Increased LAG-3 expression is also observed in antiretroviral treatment (ART)-mediated viral suppression compared to samples from individuals with CD4 <200 plasma viral load (pVL) >100 and CD4 >200 pVL >100 (p=0.02 and p=0.008, respectively); CD4 >200 (n=19), CD4 <200 (n=9), ART (n=5). Spearman rank correlations with p values<0.05 were considered significant.