Impaired in vitro Interferon-γ production in patients with visceral leishmaniasis is improved by inhibition of PD1/PDL-1 ligation

Abstract

Visceral leishmaniasis (VL) is a neglected tropical disease that causes substantial morbidity and mortality and is a growing health problem in Ethiopia, where this study took place. Most individuals infected with Leishmania donovani parasites will stay asymptomatic, but some develop VL that, if left untreated, is almost always fatal. This stage of the disease is associated with a profound immunosuppression, characterised by impaired production of Interferonγ (IFNγ), a cytokine that plays a key role in the control of Leishmania parasites, and high expression levels of an inhibitory receptor, programmed cell death 1 (PD1) on CD4+ T cells. Here, we tested the contribution of the interaction between the immune checkpoint PD1 and its ligand PDL-1 on the impaired production of IFNγ in VL patients. Our results show that in the blood of VL patients, not only CD4+, but also CD8+ T cells express high levels of PD1 at the time of VL diagnosis. Next, we identified PDL-1 expression on different monocyte subsets and neutrophils and show that PDL-1 levels were significantly increased in VL patients. PD1/PDL-1 inhibition resulted in significantly increased production of IFNγ, suggesting that therapy using immune checkpoint inhibitors might improve disease control in these patients.

Fig 1. Expression of PD1 on T cells.

The Median Fluorescence Intensity (MFI) of PD1 was measured ex vivo by flow cytometry on CD4⁺ T cells (A) and CD8⁺ T cells (B) in the PBMCs from VL patients (n = 16) and controls (n = 10). PBMCs were isolated from whole blood as described in Material and Methods. The gating strategy is detailed in S1 Fig. Statistical differences were determined by a Mann-Whitney test. Each symbol represents the value for one individual, the straight lines represent the median.

Fig 2. Monocytes and neutrophils express PDL-1.

Ex vivo PDL-1 MFI was measured by flow cytometry on monocytes from PBMCs of VL patients n = 16) and controls (n = 10). PBMCs were isolated from whole blood as described in Material and Methods. The gating strategy is detailed in S2 Fig. A. PDL-1 expression on classical monocytes (CD14⁺/CD16^low). B. PDL-1 expression on intermediate monocytes (CD14⁺/CD16⁺). C. PDL-1 expression on non classical monocytes (CD14^low/CD16⁺). Statistical differences were determined by a Mann-Whitney test. D. Ex vivo PDL-1 MFI was measured by flow cytometry on neutrophils in the PBMCs from VL patients (n = 16) and controls (n = 8). Neutrophils were purified from whole blood as described in Materials and Methods. The gating strategy is detailed in S3 Fig. Statistical differences were determined by a Mann-Whitney test. Each symbol represents the value for one individual, the straight lines represent the median.

Fig 3. Interfering with the PD1/PDL-1 pathway results in increased production of IFNγ.

Whole blood cells from VL patients at ToD (n = 14) were stimulated with A. SLA in the presence (1μg) or absence of anti-PD-1 mAb; or B. SLA in the presence (1μg) or absence of an isotype control. IFNγ was measured by ELISA in the supernatant after 24hrs. Statistical differences were determined by a Wilcoxon test. Each symbol represents the value for one individual.