An exploratory trial of cyclooxygenase type 2 inhibitor in HIV-1 infection: downregulated immune activation and improved T cell-dependent vaccine responses

Abstract

Chronic HIV infection is characterized by chronic immune activation and dysfunctional T cells with elevated intracellular cyclic AMP (cAMP), which inhibits the T cell activation capability. cAMP may be induced by prostaglandin E(2) following lipopolysaccharide (LPS)-induced upregulation of cyclooxygenase type 2 (COX-2) in monocytes due to the elevated LPS levels in patients with chronic HIV infection. This hypothesis was tested using celecoxib, a COX-2 inhibitor, for 12 weeks in HIV-infected patients without antiretroviral treatment in a prospective, open, randomized exploratory trial. Thirty-one patients were randomized in the trial; 27 completed the study, including 13 patients on celecoxib. Celecoxib reduced chronic immune activation in terms of CD38 density on CD8(+) T cells (-24%; P = 0.04), IgA levels (P = 0.04), and a combined score for inflammatory markers (P < 0.05). Celecoxib further reduced the inhibitory surface receptor programmed death 1 (PD-1) on CD8(+) T cells (P = 0.01), including PD-1 on the HIV Gag-specific subset (P = 0.02), enhanced the number of CD3(+) CD4(+) CD25(+) CD127(lo/-) Treg or activated cells (P = 0.02), and improved humoral memory recall responses to a T cell-dependent vaccine (P = 0.04). HIV RNA (P = 0.06) and D dimers (P = 0.07) tended to increase in the controls, whereas interleukin-6 (IL-6) possibly decreased in the treatment arm (P = 0.10). In conclusion, celecoxib downmodulated the immune activation related to clinical progression of chronic HIV infection and improved T cell-dependent functions in vivo.

Fig. 1.

Gating strategies for CD38 density and the CD4⁺ CD25⁺ CD127^lo/− T cell subset. (A) Scatter plots on CD38 and PD-1 after primary gating on the lymphocyte live gate in forward scatter (FSC)-side scatter (SSC) plots and secondary gating on CD3⁺ CD8⁺ T cells in CD3-CD8 scatter plots for two different patients with relatively high and low CD38 densities. FMO cutoffs are indicated by horizontal and vertical lines. CD38 densities within the CD3⁺ CD8⁺ PD-1⁺ CD38⁺ T cells are indicated in quadrant 2 (2,095 and 10,054 molecules/cell). PBMC were stained with Quantibrite PE–CD38 MAb having one PE molecule per MAb. A PE standard curve was obtained with a mixture of Quantibrite PE bead batches, each with a defined number of PE molecules per bead. The CD38 density in a cell population was determined from extrapolation of the median fluorescence intensity of the anti-CD38–Quantibrite PE signal to the standard curve according to the manufacturer's instructions. (B) One- and two-parameter histograms after primary gating on the lymphocyte live gate in FSC-SSC plots, demonstrating the identification of CD3⁺ CD4⁺ CD25⁺ CD127^lo/− T cells.

Fig. 2.

Reduced expression of CD38 following celecoxib treatment. Changes in CD38 density (molecules/cell) in celecoxib (COX-2i)-treated and control groups from weeks 0 to 12 are shown for different T cell subsets, including total CD8⁺ and CD8⁺ PD-1⁺ T cells (top panels) and the PD-1-positive subsets of CCR7⁺ CD27⁺ and CCR7⁻ CD27⁻ memory CD8⁺ T cells (bottom panels). Median values and interquartile ranges (25th to 75th percentiles) are indicated. P values of <0.10 are shown in the relevant panels (Wilcoxon matched-pair test). Groupwise differences between the COX-2i-treated and control groups at week 12 were assessed by MWU and are indicated by asterisks. n.s., not significant.

Fig. 3.

Celecoxib causes downregulation of surface-expressed PD-1 on T cells. (A) MFI of PD-1 on CD4⁺ and CD8⁺ T cells (top row), activated CD8⁺ T cells (HLA-DR^+/− CD38⁺; second row), and CCR7⁻ CD27⁻ and CCR7⁺ CD27⁺ CD8⁺ memory T cell subsets (third row). (B) Percentages of PD-1-positive cells among Gag- and Env-specific CD8⁺ T cells at 0 and 12 weeks in both study arms, as indicated. Median values and interquartile ranges (25th to 75th percentiles) are indicated.

Fig. 4.

Increases in absolute numbers and frequencies of regulatory T cells upon celecoxib treatment. The relative proportions (percentages of T cells) (left) and absolute numbers (×10⁶ cells/liter) (right) of Tregs increased in the celecoxib treatment study arm. Median values and interquartile ranges (25th to 75th percentiles) are indicated. P values of <0.10 are shown in the relevant panels (Wilcoxon matched-pair test). Groupwise differences (MWU) between the COX-2i-treated and control groups at week 12 are indicated by asterisks.

Fig. 5.

Celecoxib treatment potentiates the effects of indomethacin on T cell responsiveness in vitro. We measured the effects of indomethacin on the ability of CD4⁺ and CD8⁺ T cells to produce the proinflammatory cytokines IFN-γ and TNF-α in response to stimulation with CD2/3/28-coated microbeads (compared to beads alone). PBMC were pretreated with indomethacin overnight prior to stimulation with beads. Celecoxib (COX-2i) treatment for 12 weeks potentiated the effects of indomethacin with respect to production of both IFN-γ and TNF-α in CD4⁺ as well as CD8⁺ T cells. Individual effects of indomethacin pretreatment are indicated for baseline and the study end at week 12.

Fig. 6.

Results of vaccine substudy. The two left panels show changes in antibody levels from weeks 6 to 12 after vaccination with TT and PP vaccines. For TT responses, only recall responses are shown, i.e., responses for those who had measurable anti-TT at baseline and for whom a single vaccine would be expected to be sufficient to induce a recall response. The right panel shows the groupwise variations in TT/PP response ratios, with a relative dominance of TT responses in the celecoxib group. Medians and interquartile ranges are indicated.