Inversed Ratio of CD39/CD73 Expression on γδ T Cells in HIV Versus Healthy Controls Correlates With Immune Activation and Disease Progression

Abstract

Background: γδ T cells are unconventional T cells that have been demonstrated to be crucial for the pathogenesis and potentially for the cure of HIV-1 infection. The ectonucleotidase CD39 is part of the purinergic pathway that regulates immune responses by degradation of pro-inflammatory ATP in concert with CD73. Few studies on the expression of the ectoenzymes CD73 and CD39 on human γδ T cells in HIV have been performed to date.

Methods: PBMC of n=86 HIV-1-infected patients were compared to PBMC of n=26 healthy individuals using 16-color flow cytometry determining the surface expression of CD39 and CD73 on Vδ1 and Vδ2 T cells in association with differentiation (CD45RA, CD28, CD27), activation and exhaustion (TIGIT, PD-1, CD38, and HLA-DR), and assessing the intracellular production of pro- and anti-inflammatory cytokines (IL-2, TGF-ß, TNF-α, Granzyme B, IL-10, IFN-γ) after in vitro stimulation with PMA/ionomycin.

Results: CD39 and CD73 expression on γδ T cells were inversed in HIV infection which correlated with HIV disease progression and immune activation. CD39, but not CD73 expression on γδ T cells of ART-treated patients returned to levels comparable with those of healthy individuals. Only a small subset (<1%) of γδ T cells co-expressed CD39 and CD73 in healthy or HIV-infected individuals. There were significantly more exhausted and terminally differentiated CD39+ Vδ1 T cells regardless of the disease status. Functionally, IL-10 was only detectable in CD39+ γδ T cells after in vitro stimulation in all groups studied. Viremic HIV-infected patients showed the highest levels of IL-10 production. The highest percentage of IL-10+ cells was found in the small CD39/CD73 co-expressing γδ T-cell population, both in healthy and HIV-infected individuals. Also, CD39+ Vδ2 T cells produced IL-10 more frequently than their CD39+ Vδ1 counterparts in all individuals regardless of the HIV status.

Conclusions: Our results point towards a potential immunomodulatory role of CD39+ and CD73+ γδ T cells in the pathogenesis of chronic HIV infection that needs further investigation.

Keywords: CD39; CD73; HIV-1; IL-10; T cell; Vδ2; elite controllers; γδ T cells.

Figure 1

The relative frequency of CD39+ γδ T cells is increased in viremic and HIV patients on ART compared to healthy controls (A) while the frequency of CD73+ γδ T cells is decreased in HIV infection regardless of the disease status (B). (C) Frequency of CD39+CD73- γδ T cells in PBMC. (D) Frequency of CD39-CD73+ γδ T cells in PBMC. (E) Frequency of double-positive CD39+CD73+ γδ T cells in PBMC. Data from 26 healthy individuals, 32 viremic, 36 HIV patients on ART, 8 EC, and 10 LTNP. ns, non-significant p≥0,05; *p<0,05; **p≤0,01; ****p≤0,0001.

Figure 2

Correlation of CD39+ and CD73+ γδ T cells with disease progression markers. The frequency of CD39+ γδ T cells in HIV correlates negatively with CD4+ T-cell count (A) and positively with HIV viral load (B). The frequency of CD73+ γδ T cells in HIV correlates positively with CD4+ T-cell count (C) and negatively with HIV viral load (D). Graphs contain pooled data from HIV-infected patients including viremic and individuals on ART, EC, and LTNP.

Figure 3

Frequency of CD39+ Vδ1 and Vδ2 cells among total and activated γδ T cells. (A) Frequency of CD39+ Vδ1 versus Vδ2 γδ T cells. (B) Frequency of activated (HLA-DR+CD38+) CD39+ Vδ1 versus Vδ2 γδ T cells. ns, non-significant p≥0,05; *p<0,05; **p≤0,01; ***p≤0,001; ****p≤0,0001.

Figure 4

Frequency of exhausted and terminally differentiated cells among CD39+ Vδ1 versus CD39+ Vδ2. (A) Frequency of exhausted (TIGIT+PD-1+) CD39+ Vδ1 versus Vδ2 γδ T cells (B) Frequency of CD39+ Vδ1 versus Vδ2 γδ T cells at a late stage of differentiation (CD27-CD28-). *p<0,05; **p≤0,01; ****p≤0,0001.

Figure 5

CD39+ γδ T cells produce more IL-10 upon in vitro stimulation than CD39- γδ T cells. (A) Frequency of IL-10 producing total γδ T cells (B) Frequency of IL-10 producing CD39+ versus CD39- γδ T cells. (C) Frequency of IL-10 producing CD73+ versus CD73- γδ T cells. (D) Frequency of IL-10 producing CD39+CD73+ versus CD39-CD73- γδ T cells. ns, non-significant p≥0,05; *p<0,05; **p≤0,01; ***p≤0,001.

Figure 6

Multidimensional comparison of the cytokine profiles of CD39+/- Vδ2 γδ T cells via SPICE analysis. Samples from (A) healthy controls (B) HIV-infected viremic patients (C) HIV-infected patients on ART (D) elite controllers. Overlapping arcs denote cell populations that co-express the respective cytokines. See also Supplementary Figure 10 for a detailed pie figure legend.