Negative Checkpoint Regulatory Molecule 2B4 (CD244) Upregulation Is Associated with Invariant Natural Killer T Cell Alterations and Human Immunodeficiency Virus Disease Progression

Abstract

The CD1d-restricted invariant natural killer T (iNKT) cells are implicated in innate immune responses against human immunodeficiency virus (HIV). However, the determinants of cellular dysfunction across the iNKT cells subsets are seldom defined in HIV disease. Herein, we provide evidence for the involvement of the negative checkpoint regulator (NCR) 2B4 in iNKT cell alteration in a well-defined cohort of HIV-seropositive anti-retroviral therapy (ART) naïve, ART-treated, and elite controllers (ECs). We report on exaggerated 2B4 expression on iNKT cells of HIV-infected treatment-naïve individuals. In sharp contrast to CD4^-iNKT cells, 2B4 expression was significantly higher on CD4⁺ iNKT cell subset. Notably, an increased level of 2B4 on iNKT cells was strongly correlated with parameters associated with HIV disease progression. Further, iNKT cells from ART-naïve individuals were defective in their ability to produce intracellular IFN-γ. Together, our results suggest that the levels of 2B4 expression and the downstream co-inhibitory signaling events may contribute to impaired iNKT cell responses.

Keywords: 2B4; CD4; IFN-γ; human immunodeficiency virus; inhibitory; invariant natural killer T cells.

Figure 1

Frequency of CD1d-restricted invariant natural killer T (iNKT) cells and their subsets. Frozen peripheral blood mononuclear cells (PBMCs) from healthy controls (HCs, n = 15), human immunodeficiency virus-seropositive (ART-naïve, n = 23), ART-treated (ART, n = 19), and elite controllers (ECs, n = 6) were stained with PBS-57 loaded/CD1d tetramer, anti-CD3, and anti-CD4 antibodies before acquisition by flow cytometer. A detailed gating strategy is described in Figure S1 in Supplementary Material. (A) Representative FACS plot from an individual of each cohort, numbers indicate percent CD1d Tet⁺ iNKT cells. (B) Representative plot showing percent of CD4⁺ and CD4⁻ sub-population within the CD1d tet⁺ iNKT cell-gated population. (C) Scatter plot showing a summary of CD1d tet⁺ iNKT cells frequency across the various study cohorts. (D) Frequency of CD4⁺ iNKT cells and CD4⁻iNKT cells (E) in various cohorts. *P < 0.05, **P < 0.01, ***P < 0.001, and n.s. stands for non-significant.

Figure 2

Expression pattern of 2B4 molecule on CD3, CD4, and CD8 T cells. (A) Representative FACS plots showing percent 2B4 expression on total CD3 (upper panel), CD4 (middle panel), and CD8 (lower panel) from different cohorts. Fluorescence-minus-one (FMO) staining was used to determine the threshold values for the expression of specific markers. (B) Scatter plot showing the mean percentage 2B4 expression on CD3⁺ T cells of various cohorts, HCs (n = 9), ART naïve (n = 15), ART (n = 11), and ECs (n = 6); whereas (C,D) represent the mean percentage of 2B4 expression levels on CD4⁺ and CD8⁺ T cells, respectively. *P < 0.05, ***P < 0.001, and n.s. stands for non-significant value.

Figure 3

Expression levels of 2B4 on invariant natural killer T (iNKT) cells and their subsets. (A) Representative zebra plot showing percent 2B4 expression on iNKT cells of representative individuals from various cohorts. iNKT cells (PBS-57 loaded/CD1d-tetramer⁺ CD3⁺) gated population was looked for surface 2B4 levels (upper panel). While 2B4 expression on CD4⁺ (middle panel) and CD4⁻iNKT cell subset (lower panel) were first gated on CD4⁺ and CD4⁻ population then presented with FSC-A (Y-axis). (B) Scatter plot showing the mean percentage of 2B4 levels on bulk iNKT cells of HCs (n = 15), ART naïve (n = 23), ART (n = 19), and ECs (n = 6). Mean percentage of 2B4 levels on CD4⁺ (C) and CD4⁻iNKT cell subsets (D). Fluorescence-minus-one (FMO) was taken to exclude false-positive values for 2B4. *P < 0.05, ***P < 0.001, and n.s. stands for non-significant value.

Figure 4

Invariant natural killer T (iNKT) cell production of IFN-γ and their association with 2B4 levels. (A) Representative plot from each cohort showing percentage of intracellular IFN-γ expression. α-GalCer-stimulated peripheral blood mononuclear cells (PBMCs) were surface-labeled with PBS-57 loaded/CD1d tetramer and CD3 followed by intracellular staining with anti-IFN-γ antibody as described in the section “Materials and Methods.” iNKT cell-gated population were looked for IFN-γ production. (B) Scatter plots show mean percentages of IFN-γ production by each cohort HCs (n = 8), ART naïve (n = 14), ART (n = 10), and ECs (n = 6). **P < 0.01 and n.s. stands for non-significant. (C) Plot showing the correlation between 2B4⁺ iNKT cells and IFN-γ production by ART-naive cohort (n = 14).

Figure 5

Correlation between frequency of 2B4⁺ iNKT cells and human immunodeficiency virus (HIV) disease progression. (A) Correlation plot between frequency of 2B4⁺ iNKT cells versus HIV viral load (n = 23) (B) CD4 counts (n = 23) and (C) CD4/CD8 ratio (n = 23).