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

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.

Introduction

Visceral leishmaniasis (VL) is one of the most neglected tropical diseases [1]. An estimated 550 million individuals are at risk of VL in high-burden countries: 17,082 new cases of VL were reported in 2018, with Brazil, Ethiopia, India, South Sudan and Sudan–which each reported more than 1000 VL cases–represented 83% of all cases globally in that year [2]. The remote location of VL endemic areas and the lack of surveillance make its likely that this is a significant underestimate of the real burden of VL in endemic areas. VL imposes a huge pressure on low and middle income countries and delays economic growth, with an approximate annual loss of 2.3 million disability-adjusted life years [3]. In Ethiopia, VL is caused by Leishmania (L.) donovani and is one of the most significant vector-borne diseases, with over 3.2 million people at risk of infection [4]. VL is a growing health problem, with spreading endemic areas and a steady increase in incidence since 2009 [5].

The majority of infected individuals control the parasite replication and do not progress to disease, they remain asymptomatic. In contrast, some individuals will progress and develop visceral leishmaniasis that is characterised by hepatosplenomegaly, fever, pancytopenia and wasting; this stage of the disease is generally fatal if left untreated [6,7]. One of the main immunological characteristic of VL patients is their profound immunosuppression [8]: these patients do not respond to the leishmanin skin test, their peripheral blood mononuclear cells (PBMCs) have an impaired capacity to produce IFNγ and proliferate in response to Leishmania antigen; this dysfunctional response to antigen challenge is restored following successful chemotherapy [9-11]. These findings show that T cell responses are impaired in VL patients, but the mechanisms leading to this impairment remain to be fully understood.

Using a whole blood assay (WBA), we have previously shown that whole blood cells from VL patients from Northern Ethiopia displayed an impaired capacity to produce IFNγ in response to stimulation with soluble Leishmania antigens (SLA) at time of diagnosis; but that these cells gradually regained their capacity to produce IFNγ over time after successful treatment [12,13].

We have recently shown that high levels of PD1 on CD4+ T cells–an inhibitory receptor that can be expressed on exhausted T cells–was a hallmark of VL patients at time of diagnosis and that this was associated with low production of IFNγ [13]. The interaction of PD1 with its ligand PDL-1 contribute to T cell dysfunction [14]. Therefore, in this study we aimed to identify which cells express PDL-1; and determine whether the impaired production of IFNγ can be improved by interfering with the PD1/PDL1 pathway.

Methods

Ethics statement

This study was approved by the Institutional Review Board of the University of Gondar (IRB, reference O/V/P/RCS/05/1572/2017), the National Research Ethics Review Committee (NRERC, reference 310/130/2018) and Imperial College Research Ethics Committee (ICREC 17SM480). Informed written consent was obtained from each patient and control.

Subjects and sample collection

For this cross-sectional study, 10 healthy male non-endemic controls were recruited among the staff of the University of Gondar, Ethiopia, these individuals had not travelled to endemic areas and all tested negative by rk39; as well as 16 male patients with visceral leishmaniasis (VL patients) were recruited from the Leishmaniasis Treatment and Research Center, University of Gondar. The exclusion criterion was age <18 years. The diagnosis of VL was based on positive serology (rK39) and the presence of Leishmania amastigotes in spleen aspirates [15]. All patients were treated with a combination of sodium stibogluconate (20mg/kg body weight/day), and paromomycin (15mg/kg body weight/day) injections for 17 days according to the Guideline for Diagnosis and Prevention of Leishmaniasis in Ethiopia [16].

Sample collection and processing

8ml of blood was collected in heparinised tubes and was used as follows: 3ml for the whole blood assay (WBA) and 5ml to purify PBMC and neutrophils as described in [17] for flow cytometry.

Four millilitres of blood (median white blood cells: 2.05 ± 0.18 x 106 cells/ml) from 14 VL patients at time of diagnosis were collected in heparinised tubes and 4 x 1ml aliquots were distributed in 4 tubes and stimulated with SLA (10μg/ml) alone, SLA + anti-PD1 (Nivolumab, Pb3, BioVision, 1μg/ml), SLA + isotype control (QA16A15, Biolegend, 1 μg/ml) and with PBS as a negative control. The tubes were incubated for 24 hours at 37°C, and supernatants were collected and stored at -20°C until further analysis.

Soluble Leishmania antigen (SLA) was prepared using Leishmania donovani clinical isolates [18] collected from splenic aspirates of VL patients. To grow Leishmania parasites from spleen aspirations, the following culture media was used: 500ml of M199 medium (Sigma, USA) which was enriched with 25mM hepes, 0.2μM folic acid, 5ml vitamin mix, 1mM hemin, 1mM adenine, 800μM Biopterin, 5ml of Penicillin streptomycin and 50ml fetal bovine serum (Sigma, USA). Stationary-phase promastigotes were harvested and centrifuged at 4500 rpm for 20 minutes, the pellet was washed three times with cold PBS (Sigma, USA). The pellet was adjusted to 2 x 109/ml and resuspended in the following reagent: 50mM of EDTA, 50mM of HCL, 100mM of Phenylmethanesulfonyl fluoride and 5mg/ml of Leupeptin (Sigma, USA). The SLA suspension was sonicated 4–5 times for 15 seconds at 10 Hz and centrifuged at 27,000xg for 30 minutes at 4°C. The lipid layer was removed from the surface of the supernatant. The remaining supernatant was ultra-centrifuged at 100,000xg for 4hrs at 4°C. The supernatant was collected and was stored at -20°C.

IFNγ levels were measured in the supernatant of the WBA using IFN gamma ELISA Kit (Invitrogen) according to the manufacturer’s instructions.

Flow cytometry: the following antibodies were used directly ex vivo: CD4FITC (OKT-4), CD8PE CY7 (RPA-T8), PD1PE (J105), PDL-1PE(MIH1), CD15APC (MMA) (eBioscience), CD14APC (M5E2) and CD16FITC (B73.1) (Biolegend) as described in [19].

Acquisition was performed using a BD Accuri C6 flow cytometer, at least 30,000 lymphocytes, 10,000 neutrophils and 5,000 monocytes were acquired, and data were analysed using BD Accuri C6 analysis software.

Statistical analysis

Data were evaluated for statistical differences as specified in the legend of each figure. The following tests were used: Mann-Whitney or Wilcoxon tests. Differences were considered statistically significant at p<0.05. Unless otherwise specified, results are expressed as median±SEM. * = p<0.05, ** = p<0.01, *** = p<0.001 and **** = p<0.0001.

Results

Clinical and haematological parameters

The cohort of 16 VL patients and 10 healthy non-endemic controls that were recruited for this study were all male and aged-matched (Table 1). Leishmania amastigotes were present in all splenic aspirates from VL patients (parasite grade (+): 2.5±0.5). All VL patients presented with low BMI, fever, splenomegaly, and pancytopenia (Table 1).

Table 1

Clinical and haematological parameters.

	ToD	Controls	p values
Clinical parameters
Age (years)	25±2.1	26±2.2	0.5057
Parasite grade (+)	2.5±0.5	nd	na
BMI (kg/m 2 )	16.2±0.4	21.0±1.0	<0.0001
Fever ( o C)	37.4±0.2	36.0±0.1	<0.0001
Spleen size (cm)	8.5±1.0	0.0±0.0	<0.0001
Haematological parameters
WBC (μl of blood x 103)	1.8±0.1	5.8±0.7	<0.0001
RBC (μl of blood x 106)	3.0±0.2	5.4±0.2	<0.0001
Platelets (μl of blood x 104)	5.7±1.2	24.8±2.0	<0.0001

A cohort of VL patients (n = 16) were recruited at time of diagnosis (ToD). Clinical and haematological parameters were assessed as described in Materials and Methods and compared to those of a cohort of age- and sex-matched non-endemic healthy controls (n = 10). Spleen sizes were measured below the left costal margin. nd = not done, na = not applicable.

PD1 expression on T cells

Efficient effector functions of specific T cells are of crucial importance for the clinical outcome of visceral leishmaniasis. One of the main immunological features of VL patients in Ethiopia is their impaired ability at time of diagnosis (ToD) to produce antigen-specific IFNγ in a whole blood assay (WBA); after successful treatment the impaired IFNγ production is restored over time [12,19]. Our recent work [19] showed that the gradual increase in antigen-specific IFNγ production during follow-up is accompanied by a gradual decrease of PD1 expression on CD4+ T cells. Results presented in Fig 1A and 1B show that directly ex vivo, not only CD4+ T cells, but also CD8+ T cells expressed PD1. In both T cell subsets, the expression levels of PD1 on CD4+ T cells and CD8+ T cells were significantly higher as compared to controls (p<0.0001 and 0.0004, respectively).

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.

Monocytes and neutrophils express PDL-1

We have hypothesised that the high levels of PD1, via its interaction with PDL-1, is a major contributor of T cell hyporesponsiveness in VL patients [19]. However, the phenotypes of PDL-1 expressing cells have not yet been identified in VL patients.

Depending on the expression levels of CD14 and CD16, monocyte can be subdivided in three distinct subsets: classical (CD14+/CD16low), intermediate (CD14+/CD16+) and non-classical (CD14low/CD16+). Each subset can display different functions: broadly, classical monocytes exhibit strong phagocytosis abilities; intermediate monocytes are characterised by their abilities to induce T cell stimulation and high ROS production, as well as pro-angiogenic abilities; and non-classical monocytes are characterised by their patrolling behaviour of the vascular endothelium [20]. Importantly, the role of these different subsets in VL patients is poorly characterised [21].

Here we tested whether the different monocyte subsets isolated from VL patients express PDL-1, and if so, if they express it differentially. Results presented in Fig 2 and S1 Table show that directly ex vivo, monocytes in the PBMCs of VL patients express PDL1. All three monocyte subsets: classical (CD14+/CD16low, Fig 2A), intermediate (CD14+/CD16+, Fig 2B) and non-classical (CD14low/CD16+, Fig 2C) expressed significantly more PDL-1 at ToD compared to controls (p>0.0001, p>0.0001 and p = 0.0061, respectively).

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+/CD16low). B. PDL-1 expression on intermediate monocytes (CD14+/CD16+). C. PDL-1 expression on non classical monocytes (CD14low/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.

Next, we assessed if neutrophils also expressed PDL-1 at ToD. Results presented in Fig 2D show that directly ex vivo, neutrophils expressed significantly higher levels of PDL-1 at ToD than controls (p = 0.0022).

We also tested for correlations between the parasite grades and the PD1 and PDL-1 MFI, but none of these correlations were significant (p>0.05).

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

Based on the increased expression levels of PD1 on T cells and PDL-1 on monocytes and neutrophils and the low levels of IFNγ produced in the WBA [19], we tested if blockade of the PD1/PDL-1 interaction improves IFNγ production. Results presented in Fig 3A show that interfering with the PD1/PDL-1 ligation resulted in significantly higher levels of IFNγ (p = 0.0006). As expected, there was no significant difference between the levels of IFNγ produced in response to SLA alone and those produced in response to SLA in the presence of an isotype control (Fig 3B).

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.

These results suggest that the impaired ability of whole blood cells to produce IFNγ efficiently can be improved by PD1/PDL-1 blockade.

Discussion

Severe immune suppression has been previously documented in VL patients [9], however, it is still poorly understood. Here we show that at the time of diagnosis of VL both CD4+ and CD8+ T cells express high levels of PD1. The interaction of inhibitory receptors such as PD1 with their ligand play a crucial role in controlling autoreactivity and immunopathology. These receptors are also upregulated during T cell activation, but this is transient. In contrast, chronic stimulation of T cells results in the maintenance of high levels of PD1 expression on T cells; the duration and degree of this chronic stimulation are key to T cell exhaustion and dysfunction [22]. VL patients present at the Leishmaniasis Treatment and Research Center with severe disease and on average have had VL symptoms for around 2 months [19]. It is therefore likely that the chronic stimulation by Leishmania antigen plays a major role in the maintenance of high expression levels of PD1 on T cells in these patients. Several inflammatory mediators have been shown to result in the upregulation of PDL-1 on different cell types such as neutrophils and monocytes [23]. It is well established that high levels of inflammation are common in VL patients [9] and indeed, we found high levels of TNFα, IL-6, IL-8, IFNγ [19], as well as IL-17 [24] in the plasma of VL patients at the time of diagnosis. IL-10 has also been shown to upregulate PDL-1 on monocytes [25] and indeed, high levels of IL-10 in plasma are a hallmark of VL patients [9,19]. Furthermore, IFNγ is also a central driver of PDL-1 expression [26]; and indeed, our results have shown that IFNγ is high at ToD in VL patients. Therefore, we propose that in VL patients, the chronic inflammation and antigenic stimulation results in the upregulation of both PD1 and PDL-1, that results in T cell exhaustion; that is manifested in the whole blood assay by impaired production of IFNγ. Exhausted T cells become hypofunctional, they maintain some of their effector functions such proliferation [14,22,27]. Exhausted T cells have been shown to contribute to the control of chronic infections and limit immunopathology [14,22,27]. It is therefore possible that during the acute state of infection, exhausted T cells might still contribute to the control of parasite replication, as well as limit tissue damage.

In addition to inflammatory mediators, parasites can upregulate PDL-1 on immune cells. A study showed that Leishmania amazonensis, a parasite causing cutaneous leishmaniasis, can upregulate PDL-1 on both mouse and human neutrophils, these neutrophils have the ability to suppress the production of IFNγ by CD8+ T cells [28].

Our data show that the levels of PD1 expression of CD4+ and CD8+ T cells and PDL-1 expression on monocytes and neutrophils are significantly increased at ToD as compared to control. The controls in our study were all individuals living in Gondar, an area of Ethiopia that is not endemic for VL, they had not travelled to an endemic area and they all tested negative for rk39. However, we cannot exclude that these individuals might have been infected with L. donovani and were asymptomatic.

Whereas PDL-2 is another co-signalling molecule that can bind to PD1 and contribute to T cell suppression, our knowledge of its functional role is still poor [29]. Since we did not test for the expression levels of PDL-2 on monocytes and neutrophils, we cannot exclude that it contributes to further T cell suppression in VL patients; and that additional blockade of PDL-2/PD1 pathway might have further improved the production of IFNγ in the whole blood assay. Indeed, a study in a hamster model showed that interfering with the PDL-2 pathway resulted in decrease in parasite burden [30].

PD-L1/PD1 interaction is a powerful antagonist of TCR signal transduction, as well as CD28 and ICOS costimulatory signalling. This interaction results in impaired cytokine production and cell cycle arrest; as well as in the reduction of the transcription of Bcl-XL, a mitochondria transmembrane molecule with anti-apoptotic properties. The ligation of PDL-1 and PD1 abrogates the phosphorylation of phosphorylation of various signaling molecules, such as ERK, Vav, PLCγ, and PI3K [31,32]. A recent study showed that T cell suppression via PD-1 ligation is a result of the inactivation CD28 signaling by PD1 ligation [33]. However, the mechanism of PD-1-mediated T cell inhibition are still poorly understood.

In cutaneous leishmaniasis, it was also shown that blocking PD-1 resulted in increased production of IFNγ by circulating T cells [34]. The authors speculate that PD-1 blockade may result in improved T cell effector functions and thereby reduce the pathology. Furthermore, in a mouse model of infection with Leishmania amazonensis, mice treated with anti-PD1 and anti-PDL-1 displayed larger lesions, that contained significantly lower parasite [35].

Our results that show that interfering with the PD1/PDL-1 pathway results in increased production of IFNγ disagree with the results presented by Gautam et al. [36]. This study was performed in India, where VL patients present at a significantly earlier stage of the disease, with less severe symptoms; and indeed, the results of their study show that the levels of IFNγ measured in the whole blood assay were not impaired at time of diagnosis as compared to those detected after successful treatment [37]. As previously discussed, [12,19], we propose that the impaired production of IFNγ we measured in the WBA from the Ethiopian patients is closely related to the fact that they present late, often in a critical state. Of note, we cannot exclude that the parameters we measured in the blood in the present study might be different in other organs affected by VL, such as spleen, bone marrow or lymph nodes.

In Ethiopia, the first line of treatment against visceral leishmaniasis is a combination therapy of Sodium Stibogluconate and Paromomycin [16]. While this treatment shows a good safety profile and a good efficacy, there are still severe side effects, such as cardiotoxicity and nephrotoxicity [38]. In our latest study, four individuals from our cohort of 50 VL patients died during treatment [19]. PD1/PDL-1 blockade has emerged as a front-line treatment for several types of cancer [39]. However, little is known about its potential use in chronic infectious diseases. In a non-human primate model of simian immunodeficiency virus blockade of the PD1 pathway improved T cell effector functions and resulted in more efficient viral control [40]. Paradoxically, in a 3D cell culture model of tuberculosis despite the increased levels of both PD1 and PDL-1, blockade of PD1 promoted the replication of M. tuberculosis [41]. Several studies in experimental models have suggested that immune checkpoint blockade maybe relevant to treat several infectious diseases [42]. Even though immune checkpoint blockade can cause organ-specific immune-related adverse events, such as hepatitis and colitis, as well as systemic inflammation [43]; further studies are needed to determine whether blockade of the PD1/PDL1 pathway can be used to improve therapies against infectious diseases. In the case of visceral leishmaniasis, it might be particularly useful in tackling the severe form of the disease, to prevent the treatments’ adverse side effects, by allowing the use of shorter courses or reduced doses of current anti-leishmanial treatments.

PDL-1 MFI was measured ex vivo 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. Statistical differences were determined by a Mann-Whitney test.

(DOCX)

Click here for additional data file.

PBMCs were isolated from whole blood as described in Material and Methods. The gating strategy is detailed in S2 Fig. Statistical differences between the 3 different subsets were determined by a Kruskal-Wallis test.

(DOCX)

Click here for additional data file.

Flow cytometry analysis of PD1 expression on CD4+ and CD8+ T cells.

PBMCs were purified as described in Materials and Methods and the expression levels (Median Fluorescence Intensity [MFI]) of PD1 on CD4+ and CD8+ T cells were measured by flow cytometry. A. FSC and SSC of the lymphocyte gate (P1). B. CD4+ T cells in the lymphocyte gate (P1). C. CD8+ T cells in the lymphocyte gate (P1). D. PD1 (M2) on CD4+ T cells gate (M7). E. PD1 (M3) on CD8+ T cells gate (M1).

(TIFF)

Click here for additional data file.

Flow cytometry analysis of PDL-1 expression on Classical CD14++CD16-, Intermediate CD14++CD16+ and Non-classical CD14+CD16++ Monocytes.

PBMCs were purified as described in Materials and Methods. PBMCs were stained with anti-human CD14APC, CD16FITC and PDL-1PE and the expression levels (Median Fluorescence Intensity [MFI]) of PDL-1 on the three subsets of monocytes were measured by flow cytometry. A. FSC and SSC of the monocyte gate (P4). B. Different monocyte subsets based on the expression levels of CD14 and CD16: classical (R18), Intermediate (R19) and Non-classical (R20) monocytes. C, D and E. PDL-1 MFI on the Classical (M31), Intermediate (M32) and Non-classical monocytes (M33).

(TIFF)

Click here for additional data file.

Flow cytometry analysis of PDL-1 expression on neutrophils.

Neutrophils were purified as described in Materials and Methods and the expression of PDL-1 on neutrophils was measured by flow cytometry. A.FSC and SSC of the neutrophil gate (P3). B. CD15+ neutrophils in P3. C. PDL-1 MFI (M10) on neutrophils in M1.

(TIFF)

Click here for additional data file.

18 Jan 2022

Dear Dr. Kropf,

Thank you very much for submitting your manuscript "Impaired in vitro Interferon-γ production in patients with visceral leishmaniasis is improved by inhibition of PD1/PDL-1 ligation" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

The reviewers raise a number of methodological issues that must be addressed. The gating strategies for flow cytometry must be provided. The role of neutrophils in PD-1 mediated IFNg suppression should be addressed. Thee effect of PD1 blockade on individual cell populations (intracellular cytokines) should be addressed.

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***********************

The reviewers raise a number of methodological issues that must be addressed. The gating strategies for flow cytometry must be provided. The role of neutrophils in PD-1 mediated IFNg suppression should be addressed. Thee effect of PD1 blockade on individual cell populations (intracellular cytokines) should be addressed.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: The objectives of the study were clearly described and articulate with the hypothesis of the study. The work was designed in a way to address the objectives and the population was clearly described and is appropriate to the aim of the investigation. The sample size is sufficient to test the hypothesis of the work. The statistical analysis was appropriate. The study protocol was approved by research ethics committees.

Reviewer #2: The authors had previously shown that in VL patients, CD4+ T cells from whole blood stimulated with SLA express high levels of PD-1, and that this is associated with a reduced secretion of IFN-gamma.

In the current study, the authors aim to determine which cells produce PD-L1, the ligand of PD-1, and if the reduction in IFN-gamma secretion can be abolished by cellular treatment with the PD-1 blocker Nivolumab.

Overall, the study provides conclusive results on the stated objectives, and we are aware that the number of experiments that can be conducted with precious patient’s material (8ml of whole blood) is limited. However, the amount of data shown to prove the link between the single observations is a little sparse.

With 16 VL patients and 10 control individuals, the sample size seems reasonable to reveal data for answering the main questions. The statistical analysis of the data is appropriate.

Reviewer #3: About Figure 1: Are T cells stimulated? Is the difference of PD-1 expression in the presence or absence of SLA?

About Figures 2 and 3: are they stimulated with SLA? Are there diferrences?

About figure 4: It is missing isotype control.

It is missing the production of SLA

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The analysis presented by the author match with the analysis plan, and the results are clearly presented. The figures are of sufficient quality.

Reviewer #2: The experiments in this study are well chosen to provide information on the objectives.

Table 1 summarizes the clinical and haematological parameters of the VL patients and the non-infected control individuals.

Could the authors distinguish between healthy individuals (not infected until then) and individuals that might have had an asymptomatic Leishmania infection before?

It might be beneficial to graphically depict the parasite grade of the different infected individuals for better insight into the variance in parasite load, which might correlate with the extent of the later analyzed PD-1 and PD-L1 expression as well as the IFN-y secretion.

Fig. 1: Besides the already previously published PD-1 expression on CD4+ T cells (A), the authors now additionally determine PD-1 expression on CD8+ T cells (B), which is significantly increased in comparison to the healthy controls.

Data in Panel 1a was already published previously, so why show it again, new donors? Please specify.

Please show the gating strategy that was applied to measure the PD-1 MFI of the T cell subpopulations. Did the authors set a gate on cell size (FSC/SSC), single cells, did they check for cell viability? Information should be provided in the material and methods section or graphically in the results section.

Fig. 2: As the binding of PD-L1 to PD-1 can elicit a T cell exhaustion phenotype which might explain a pronounced disease development. Thus, the authors measured the relative amount of PD-L1 that is expressed on blood monocytes.

For all three examined monocyte subtypes, there is a significant difference between PD-L1 expression in healthy controls and VL patients.

However, it’s not clearly stated why exactly those three monocyte subtypes were chosen for the analysis. What is their individual role in the recognition of Leishmania parasites or in activation of the adaptive immune system?

Can the authors confirm that these blood monocytes were (partially) infected with Leishmania? If not, what would be the trigger to make them express PD-L1. How would they get in contact with the parasites?

Would it be an option to additionally investigate PD-L1 expression on macrophages from splenic aspirates, or even more specific from infected macrophages?

Figure 2D and 2E are redundant to Fig 2A-C as this is just another combination of the graphs. This is not correct, please change accordingly and put all 6 datasets into one chart in order to show all relevant comparisons and significances.

Fig. 3: Here the authors show the PD-L1 expression by neutrophils. As in Fig 2, it is not clear if these neutrophils are (partially) infected with Leishmania in the blood, which would explain their activation. Once recruited to the affected organs (spleen, liver) neutrophils might fulfill their function as local drivers of an immune response but they might not get back into the blood stream.

The authors should also state in the figure legend or results text which surface markers were used for identifying the cells as neutrophils (CD15, CD16)

Fig.4: Treatment of whole blood cells from VL patients with SLA in the presence of the anti-PD-1 mAB Nivolumab increases IFN-gamma secretion as shown by ELISA.

How much SLA was used (concentration) and from which strain was it produced, please specify.

According to the methods section, cells were treated with 1µg Nivolumab. What was the total volume of the assay and/or the (approx.) number of cells treated?

The authors should also provide data on whole blood cells derived from healthy patients in this assay.

Reviewer #3: Figure 1: Please include the representative dot plot

Figure 2: Please include the representative dot plot

What is the difference in figure 2D? The median is very similar.

Figure 3: Please include the representative dot plot

Figure 4:

Is there a possibility to do a correlation of expression of PD1 versus IFNg production?

What is the level of PD-1 before and after the treatment with anti-PD1.

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: The conclusion are supported by the data provided. Yet, study limitations are not so well described. The subject is of highly public health relevance and the authors have clearly emphasized the importance of the current study.

Reviewer #2: no

no

yes

yes

Reviewer #3: It is missing intracellular citokines IFNg on CD4 and CD8 T cells to see the impact of anti-PD1 in each population.

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: Minor revision.

Reviewer #2: -

Reviewer #3: Figure 2 and 3 could be combined.

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The authors in this study investigated the expression levels of PD-1 on CD4 and CD8 T cells of visceral leishmaniasis patients, and of its ligand PDL-1 on monocytes and neutrophils. The authors have found that anti-PD-1 treatment of whole blood cells of VL patients increases the production of INFg.

General comments:

The article requires minor modification that can be addressed through revision. The study addressed the interruption of PD-1/PDL-1 pathway, but do not mention the other ligand PDL-2 which has been shown to contribute as well to the success of anti-PD-1 treatment. It would be interesting to introduce PDL-2 as another ligand of PD-1 and discuss whether PDL-2 has been addressed in VL or not and the consequences.

Methods:

- How SLA is produced? Could the authors describe on that as well?

- What are the clones of the monoclonal antibodies used for FACS? This information should be in the paper.

- What is the clone of the commercial anti-PD-1 used for blocking assays? Is this information possible to add in the paper?

- The authors inform that acquisition was performed on a BD Accuri B6, how many events were acquired per sample? How was the gating strategy used for the analyses?

- Why the authors did not use CD3 or CD45 in the panels to better define the cell types investigated?

- The authors should explain why they did not use the same antibody isotype of PD-1 as control for the antibody blocking assay. This is a critical part of the methods used in the paper.

Figures:

Table 1. should be reformatting.

Fig 1. It would be interesting to provide all the gating strategy used to define CD4 and CD8 T cells, and the other cell types. This information could be as supplementary material to the article. Did the authors observed a frequency difference as well? Do the authors imagine that these T cells are exhausted? It would be interesting to discuss this as well.

The information on the markers to define each cell type evaluated should be in figure legends as well.

Fig 2. Do the authors have any data on the levels of PDL-1 of in situ macrophages from biopsies? It would be interesting for the discussion.

Fig 4. Do the authors have an idea if blocking affects the production of other cytokines that could be relevant for VL as well? I understand that the main hypothesis was drafted on the impairment of IFNg production ToD, but it would be scientifically relevant to show the data on other cytokines as well, such as IL-10, IL-4, IL-2 and TNF.

Discussion:

The authors could at least discuss how the interaction of PD-1/PDL-1 mechanistically contribute to IFNg impairment during VL.

Reviewer #2: -

Reviewer #3: It is missing important references

- PD-1 Blockade Modulates Functional Activities of Exhausted-Like T Cell in Patients With Cutaneous Leishmaniasis

DOI: 10.3389/fimmu.2021.632667

- Leishmania Parasites Drive PD-L1 Expression in Mice and Human Neutrophils With Suppressor Capacity

DOI: 10.3389/fimmu.2021.598943

- Immunotherapy using anti-PD-1 and anti-PD-L1 in Leishmania amazonensis-infected BALB/c mice reduce parasite load

DOI: 10.1038/s41598-019-56336-8

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Reviewer #1: Yes: Rafael de Freitas e Silva

Reviewer #2: No

Reviewer #3: No

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22 Apr 2022

Submitted filename: Response to reviewers Takeleetal210422.docx

Click here for additional data file.

30 May 2022

Dear Dr. Kropf,

We are pleased to inform you that your manuscript 'Impaired in vitro Interferon-γ production in patients with visceral leishmaniasis is improved by inhibition of PD1/PDL-1 ligation' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Peter C. Melby, M.D.

Associate Editor

PLOS Neglected Tropical Diseases

Charles Jaffe

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

The authors have sufficiently addressed the concerns of the reviewers

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: The objectives of the current work articulate with the hypothesis and the authors have chosen the correct tools to address it. In this new version the population studied was appropriately described and the number of samples is enough to draw the conclusions. It seems that proper statistics has been employed and there are no ethical concerns in this study.

**********

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: In this work the analysis presented matched with the analysis planned. The results were clearly and completely presented and the figures of sufficient quality.

**********

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: The conclusions are supported by the data presented and the limitations or questions have been clarified in this new version and in the rebuttal letter. The authors discussed how their data can advance the understanding of the role of PD1/PDL1 pathway during visceral leishmaniasis. The topic of this study is of utmost importance in worldwide public health.

**********

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: Accept.

**********

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The data presented in this work is really important to understand the role of PD1/PDL1 during T cell disfunction/exhaustion in visceral leishmaniasis. So far, VL is a public health issue in many countries and the current drugs available are limited and the ones available are extremely toxic. I believe the study addresses a new perspective in the field. Yet, it lacks a more comprehensive panel of effector molecules involved in T cell disfunction and how interruption of PD1/PDL1 also impact on the production of other molecules and genes. Even tough the study is extremely significant for the field of parasite immunology and also helps paving the way for new treatments against VL.

**********

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Reviewer #1: Yes: Rafael de Freitas e Silva

20 Jun 2022

Dear Dr. Kropf,

We are delighted to inform you that your manuscript, "Impaired in vitro Interferon-γ production in patients with visceral leishmaniasis is improved by inhibition of PD1/PDL-1 ligation," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

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PLOS Neglected Tropical Diseases

Paul Brindley

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PLOS Neglected Tropical Diseases