Table 1.
Demographic, hematologic, and clinical characteristics of microfilaremic subjects (Fil+) and uninfected controls (Fil-).
Fig 1.
Antigen-driven lymphocyte proliferation is not suppressed in Mansonella ozzardi infection.
PBMC from microfilaremic (Fil+) and uninfected (Fil-) subjects were labeled with CellTrace Violet and incubated with filarial (B. malayi adult worm extract [BmA]) and unrelated antigens (Staphylococcus aureus enterotoxin B [SEB], Toxoplasma gondii taquizoite extract [TgT] and red blood cells infected with Plasmodium vivax schizonts [PvS]), mitogen (phytohemagglutinin [PHA-P]), or medium alone (“unstimulated”). The net % of divided cells (stimulated minus unstimulated) was estimated by flow cytometry and shown in all panels, except for the lower right panel, which shows % of unstimulated divided cells. Data are presented as medians and interquartile ranges for 20 Fil+ and 10 Fil- subjects (TgT, PvS, and PHA-P) or 36 Fil+ subjects and 23 Fil- subjects (BmA, SEB, and medium alone) and were compared using the Mann-Whitney U test. The only significant P value after controlling for a false discovery rate (q) set at 0.10 is shown (number of comparisons m = 6).
Table 2.
Frequency of CD4+ T cells expressing regulation and activation markers in microfilaremic subjects (Fil+) and uninfected controls (Fil-).
Table 3.
Frequency of CD4+CD25hiCD127-FoxP3+ Treg cells expressing regulation and activation markers in microfilaremic subjects (Fil+) and uninfected controls (Fil-).
Fig 2.
The expansion of CD4+CD39+ T-cell populations in microfilaremics is directly proportional to their microfilarial load.
The frequencies of CD4+CD39+ T cells (% of all CD4+ T cells that express CD39; upper panel) and CD39+ Treg cells (% of all CD4+CD25hiCD127-FoxP3+ T cells that express CD39; lower panel) were plotted against the number of Mansonella ozzardi-specific ITS-2 amplicon copies obtained by quantitative real-time PCR, a proxy of microfilarial load. Data analyzed for 48 subjects using the Spearman rank correlation test.
Fig 3.
CD39 blocking markedly inhibits antigen-driven lymphocyte proliferation.
PBMC from microfilaremic (Fil+) and uninfected (Fil-) subjects were labeled with CellTrace Violet and incubated with Staphylococcus aureus enterotoxin B (SEB). The net % of divided cells was compared in the presence or absence of anti-CD39 antibody (“anti-CD39” and “control”, respectively; panel A), in the presence or absence of anti-IL-10 antibody (“anti-IL-10” and “control”, respectively; panel B), in the presence of anti-CD39 antibody with or without 2 mM adenosine added to the culture medium (“adenosine” and “control”, respectively; panel C), and in the presence of anti-CD39 antibody with or without 2 mM ATP added to the culture medium (“ATP” and “control”, respectively; panel D). Individual data are presented for 36 Fil+ and 21 Fil- subjects (panel A), 10 Fil+ and 4 Fil- subjects (panel B) or 13 Fil+ and 5 Fil- subjects (panels C and D); they were compared using the Wilcoxon signed rank test. Significant P values after controlling for a false discovery rate (q) set at 0.10 are underlined (number of comparisons m = 4 for each group, Fil+ and Fil-).
Fig 4.
CD39 blocking alters antigen-driven cytokine production by CD4+ T cells.
PBMC from microfilaremic (Fil+) and uninfected (Fil-) subjects were stimulated with Staphylococcus aureus enterotoxin B (SEB) in the presence or absence of anti-CD39 antibody and stained for intracellular cytokines. The % of CD4+ T cells producing each cytokine was estimated by flow cytometry. Individual data are presented for 40 Fil+ and 28 Fil- subjects and were compared using the Wilcoxon signed rank test. Significant P values after controlling for a false discovery rate (q) set at 0.10 are underlined (number of comparisons m = 5 for each group, Fil+ and Fil-).
Fig 5.
CD39 blocking alters antigen-driven cytokine secretion in PBMC culture supernatants.
PBMC from microfilaremic (Fil+) and uninfected (Fil-) subjects were stimulated with Staphylococcus aureus enterotoxin B (SEB) in the presence or absence of anti-CD39 antibody. After 72 h of culture, supernatants were harvested and assessed for cytokines. Data are presented as individual cytokine concentrations (pg/mL) for 27 Fil+ and 15 Fil- subjects and were compared using the Wilcoxon signed rank test. Significant P values after controlling for a false discovery rate (q) set at 0.10 are underlined (number of comparisons m = 5 for each group, Fil+ and Fil-).
Fig 6.
Reduced Ki67 expression in CD4+CD39+ T cells following CD39 blocking.
PBMC from microfilaremic (Fil+) and uninfected (Fil-) subjects were stained for intracellular Ki67, an indicator of T-cell proliferation. We first compared the proportion of Ki67+ cells among CD4+CD39+ and CD4+CD39- T cells (panel A) and CD39+ and CD39- Treg (CD4+CD25hiCD127-FoxP3+) cells (panel B). Next, we compared the proportion of Ki67+ cells among CD4+ T cells (panel C) and Treg cells (panel D) in the presence or absence of anti-CD39 antibody (“anti-CD39” and “control”, respectively). Individual data are shown for 11 Fil+ and 5 Fil- subjects (panels A and B) or 9 Fil+ and 5 Fil- subjects (panels C and D) and were compared using the Wilcoxon signed rank test. Significant P values after controlling for a false discovery rate (q) set at 0.10 are underlined (number of comparisons m = 5 for each group, Fil+ and Fil-).