Adenosine deaminase enhances the immunogenicity of human dendritic cells from healthy and HIV-infected individuals

Abstract

ADA is an enzyme implicated in purine metabolism, and is critical to ensure normal immune function. Its congenital deficit leads to severe combined immunodeficiency (SCID). ADA binding to adenosine receptors on dendritic cell surface enables T-cell costimulation through CD26 crosslinking, which enhances T-cell activation and proliferation. Despite a large body of work on the actions of the ecto-enzyme ADA on T-cell activation, questions arise on whether ADA can also modulate dendritic cell maturation. To this end we investigated the effects of ADA on human monocyte derived dendritic cell biology. Our results show that both the enzymatic and non-enzymatic activities of ADA are implicated in the enhancement of CD80, CD83, CD86, CD40 and CCR7 expression on immature dendritic cells from healthy and HIV-infected individuals. These ADA-mediated increases in CD83 and costimulatory molecule expression is concomitant to an enhanced IL-12, IL-6, TNF-α, CXCL8(IL-8), CCL3(MIP1-α), CCL4(MIP-1β) and CCL5(RANTES) cytokine/chemokine secretion both in healthy and HIV-infected individuals and to an altered apoptotic death in cells from HIV-infected individuals. Consistently, ADA-mediated actions on iDCs are able to enhance allogeneic CD4 and CD8-T-cell proliferation, globally yielding increased iDC immunogenicity. Taken together, these findings suggest that ADA would promote enhanced and correctly polarized T-cell responses in strategies targeting asymptomatic HIV-infected individuals.

Figure 1. ADA enhances CD83, CD80 and CD86 expression on iDCs.

iDCs, obtained as indicated in the Materials and Methods, were cultured for 48 h in medium in the absence (−ADA) or in the presence (+ADA) of 2 µM ADA or in the presence of maturating cocktail (mDCs). Expression of CD83 (A and B), CD80 (C and D) or CD86 (E and F) in the DCs gate was assessed by flow cytometry. In A, C and E, histogram overlays and the percentage of positive cells (A and B) or geometric mean (C) for a representative healthy donor and HIV-infected subject are shown. In B, D and F, values obtained from 16 to 19 healthy donors (circles) or 12 to 16 HIV-infected individuals (triangles) in the absence (open symbols) or in the presence (filled symbols) of 2 µM ADA are plotted. Each pair of linked symbols represents results from a particular individual. *P<0.05; ***P<0.001.

Figure 2. ADA effect on CD40 and HLA expression on iDCs.

iDCs, obtained as indicated in the Materials and Methods, were cultured for 48 h in medium in the absence (−ADA) or in the presence (+ADA) of 2 µM ADA or in the presence of maturating cocktail (mDCs). Expression of HLA-DR (A and B), HLA-ABC (C and D) or CD40 (E and F) in the DCs gate was measured by flow cytometry. In A, C and E, histogram overlays and the geometric mean from a representative healthy donor and HIV-infected patient are shown. In B, D and F, values obtained from 6 to 10 healthy donors (circles) or 9 to 10 HIV-infected subjects (triangles) in the absence (open symbols) or in the presence (filled symbols) of 2 µM ADA. Values obtained from 6 to 10 healthy donors (circles) or 9 to 10 HIV-infected subjects (triangles). * P<0.05.

Figure 3. ADA increases cytokines and chemokines secretion.

iDCs, obtained as indicated in the Materials and Methods, from 9 healthy (circles) and 8 HIV-infected (triangles) donors were cultured during 48 h in medium in the absence (iDCs) or in the presence of 2 µM ADA and the indicated cytokines and chemokines were determined in the supernatant as described in the Materials and Methods. Values are expressed as the ratio (in-fold) of cytokine or chemokine levels obtained in the presence of ADA versus levels obtained in the absence of ADA (iDCs, the reference value of 1 is represented by a dotted line). For each group, the median is indicated by a thick line. *P<0.05; **P<0.01 with respect to iDCs.

Figure 4. Enzymatic and non-enzymatic activities are implicated on ADA-mediated effects.

iDCs, obtained as indicated in the Materials and Methods, from 4 to 8 healthy donors were cultured for 48 h in medium in the absence (iDC) or in the presence of 2 µM ADA (+ADA) or 2 µM HgCl₂ inactivated ADA (ADA-Hg) or iDCs were pre-incubated with the mAb anti-CD26 TA5.9 and incubated with 2 µM ADA (ADA+TA5.9). In (A) and (B), the expression of CD83 and CD80 was assessed in the DCs gate by flow cytometry. In C) the indicated cytokines and chemokines were determined in the supernatants after 48 h of cell culture. Each pair of linked symbols represents results from a particular individual. Results are expressed as the ratio (in-fold) of the values obtained in the presence of ADA, ADA-Hg or ADA+TA5.9 versus untreated cells (iDCs, the reference value of 1 is represented by a dotted line). *P<0.05; **P<0.01 with respect to iDCs.

Figure 5. ADA enhances DCs immunogenicity in iDC-T-cell Allogeneic cocultures.

In A and B, iDCs, obtained as described in the Materials and Methods, from a representative healthy donor were cultured during 48 h in medium in the absence of ADA (iDCs), in the presence of 2 µM ADA (iDCs+ADA) or in the presence of maturating cocktail (mDCs). DCs were washed and cocultured with allogeneic (upper contour plots) or autologous (lower contour plots) T-cells (1∶20 DCs:T-cells ratio). After 7 days, the percentage of CD4⁺ (A) and CD8⁺ (B) T-cell proliferation was assessed by flow cytometry using the CFSE method. In (C) percentages of CD4⁺ (circles) and CD8⁺ (squares) T-cell proliferation in the absence (open symbols) or the presence (filled symbols) of 2 µM ADA in allogeneic cocultures from 6 to 7 healthy donors are shown. Each pair of linked symbols represents results from a particular healthy donor. *P<0.05. In (D) bars indicate IFN-γ, IL-4 and IL-17 levels in ADA-treated iDC co-culture supernatants. Results are the mean ± SD (pg/ml) of 3 independent experiments.

Figure 6. Effect of ADA on CCR7 expression and iDC migration.

iDCs, obtained as described in the Materials and Methods, from healthy donors were cultured during 48 h in medium in the absence (−ADA) or in the presence (+ADA) of 2 µM ADA or in the presence of maturating cocktail (mDCs). In (A) and (B), CCR7 expression in the CD40⁺ DCs gate was measured by flow cytometry. Histogram overlays and the percentage of CCR7 expression for a representative donor are shown in (A). Values obtained from 7 healthy donors in the absence (open symbols) or in the presence (filled symbols) of 2 µM ADA are shown in (B). In (C) and (D), CCL19/CCL21 chemokine-induced migration assays were performed as described in the Materials and Methods. The percentage of cell migration to CCL19 (C) or CCL21 (D) in the absence (−ADA) or in the presence (+ADA) of 2 µM ADA or in the presence of maturating cocktail (mDCs), in relation to the initial cell input is shown. *P<0.01, ***P<0.001.

Figure 7. Effect of ADA on the iDCs viability.

iDCs, obtained as described in the Materials and Methods, from healthy or HIV-infected donors were cultured during 48 h in the absence (−ADA) or in the presence (+ADA) of 2 µM ADA. Cell viability was assessed through DIOC₆ and propidium iodide (PI) staining and measured by flow cytometry. In (A), contour plots showing the percentage of viable (bright DIOC₆ and negative propidium iodide staining), apoptotic (low DIOC₆ and negative propidium iodide staining) and necrotic (low DIOC₆ and positive propidium iodide staining) populations from a representative healthy or HIV-infected donor are shown. Percentage of viable (B), apoptotic (C) and necrotic (D) DCs from 7 different healthy and 6 HIV-infected donors are shown.*P<0.05.