HIV-Specific Antibody-Dependent Cellular Cytotoxicity (ADCC) -Mediating Antibodies Decline while NK Cell Function Increases during Antiretroviral Therapy (ART)

Abstract

Understanding alterations in HIV-specific immune responses during antiretroviral therapy (ART), such as antibody-dependent cellular cytotoxicity (ADCC), is important in the development of novel strategies to control HIV-1 infection. This study included 53 HIV-1 positive individuals. We evaluated the ability of effector cells and antibodies to mediate ADCC separately and in combination using the ADCC-PanToxiLux assay. The ability of the peripheral blood mononuclear cells (PBMCs) to mediate ADCC was significantly higher in individuals who had been treated with ART before seroconversion, compared to the individuals initiating ART at a low CD4+ T cell count (<350 cells/μl blood) and the ART-naïve individuals. The frequency of CD16 expressing natural killer (NK) cells correlated with both the duration of ART and Granzyme B (GzB) activity. In contrast, the plasma titer of antibodies mediating ADCC declined during ART. These findings suggest improved cytotoxic function of the NK cells if initiating ART early during infection, while the levels of ADCC mediating antibodies declined during ART.

Fig 1. Increased ADCC-mediated activity in the PBM effector cells.

The individuals in the study started on ART either before seroconversion (triangles), at CD4⁺ T cell counts above 350 cells/μl blood (gray quadrants), at CD4⁺ T cell counts below 350 cells/μl blood (filled circles), or were either ART-naïve (open quadrants) or HIV-negative controls (open circles). The ADCC mediated by the PBM effector cells was defined as the highest percentage of GzB activity after background subtraction at a given dilution. A) PBM effector cells are mediating significantly higher ADCC in individuals treated before seroconversion compared to individuals initiating ART at CD4⁺ T cell counts below 350 cells/μl blood, ART-naïve individuals and to the HIV-negative control group. B) Seven out of the 8 individuals treated before seroconversion have been in treatment for 5 years or more. C) The duration of ART in years in correlation to the relative percentage of GzB activity for the PBM effector cells. D) The ADCC activity mediated by plasma from individuals treated before seroconversion could be significantly less diluted compared to ART-naïve individuals. This was also the case for individuals who had initiated ART at CD4⁺ T cell counts above 350 cells/μl blood. E) The ADCC activity mediated by plasma from individuals who were treated could be significantly less diluted compared to the ART-naïve group. F) A significant inverse correlation was observed between the dilution factor and the duration of ART in years. G) Combining individuals’ PBM effector cells and plasma showed no significant difference between the HIV-positive individuals independent of when ART was initiated. H-I) The ADCC activity of PBM effector cells and plasma was independent of the duration of ART in years. In A-C and G-I GzB activity was normalized to the frequency of NK cells in each individual, in order to evaluate the activity per NK cell. The statistical significance is indicated in each Figure. Linear regression curves with 95-confidence intervals are indicated in C and I and non-linear regression curve in F. Not significant (NS) mean a p≥0.05; * means 0.01<p<0.05; ** means 0.001<p<0.01; *** means 0.0001<p<0.001 and; **** means p<0.0001.

Fig 2. The frequency of CD16^pos NK cells increases with the duration of ART.

A-B) The frequency of CD16^pos NK cells correlates with the duration of ART in years and with relative GzB activity. The total NK cell percentages and NK cell subsets are expressed as a percentage of the total lymphocytes. Linear regression curves with 95-confidence intervals are indicated. C) The individuals who had been treated for 5 years or more had significantly increased frequencies of CD16^pos NK cells compared to the individuals who had been treated for less than 5 years.

Fig 3. The frequency of CD16^pos NK cells expressing CCR7 correlates with the relative percentage of GzB activity.

Correlation between the relative GzB activity mediated by PBM effector cells to the five phenotypic NK markers CCR7, NKp46, CD57, CD70 and CD27. A) The frequency of CD16^pos NK cells expressing CCR7 correlated inversely with relative GzB activity. B-E) No correlation was found between the frequency of CD16^pos NK cells expressing NKp46, CD57, CD70 and the relative GzB activity. The total NK cell percentages and NK cell subsets are expressed as a percentage of the total lymphocytes. Linear regression curves with 95-confidence intervals are indicated in each Figure.