Pilot study protocol for determining differences in quantitative and functional aspects of dendritic cell subsets in early onset pre-eclampsia patients- a feasibility study for identication of biomarker(s) and immunotherapeutic target(s)

Background: Pre-eclampsia (PE) is a pregnancy associated pro-inammatory disorder. The only known treatment is to deliver the placenta and fetus. PE is clinically identied by hypertension and proteinuria post-20th week of gestation. Early onset pre-eclampsia (EOPE), a severe form of PE is dened as when the clinical symptoms are observed before 34 weeks of gestation. There are no denite biomarkers available for early diagnosis of EOPE. Human dendritic cell (DC) subsets (CD1c + , CD141 + myeloid DCs and plasmacytoid DCs) are intricately involved with the process of inammation and are signicantly altered (quantitatively and functionally) in several proinammatory disorders. These changes offer value for monitoring DC subsets as potential biomarker(s) and as targets for immunotherapeutic treatment. DC subsets play a critical role in normal pregnancy by mediating ecient migration and invasion of trophoblasts and maintaining anti-inammatory environment of immunotolerance. In contrast, the status of DC subsets in the proinammatory microenvironment of EOPE pregnancy requires thorough evaluation. In this direction, the designed study protocol aims to understand how DC subsets are altered (quantitatively and functionally) in EOPE patients, compared to normal pregnant women. Methods: Study protocol is designed to determine changes in the prole of DC subsets in the blood and decidua of EOPE patients by multiparametric ow cytometry approach. Normal pregnant women are included as controls. Discussion: Human DC subsets are altered both quantitatively and functionally in the pro-inammatory microenvironment. EOPE is a pro-inammatory disorder and changes in the composition and function of DC subsets in these patients, compared to normal pregnant women is unclear. In this study, the blood sample-based analysis will determine the feasibility for identication of DC subsets associated novel immune biomarkers for early diagnosis of EOPE. In addition, changes in the prole of DC subsets in the decidua of EOPE patients will determine the feasibility for developing novel immunotherapeutic and decidual leukocytes are subjected to stimulation with TLR-9 ligand, CpG. Intracellular cytokine staining procedure (permeabilization and xation) is performed. Nxt ow (Thermo Fisher Scientic) being used to uorochrome HLA-DR, CD11c, CD123) used DC

available for early diagnosis of EOPE. Human dendritic cell (DC) subsets (CD1c + , CD141 + myeloid DCs and plasmacytoid DCs) are intricately involved with the process of in ammation and are signi cantly altered (quantitatively and functionally) in several proin ammatory disorders. These changes offer value for monitoring DC subsets as potential biomarker(s) and as targets for immunotherapeutic treatment. DC subsets play a critical role in normal pregnancy by mediating e cient migration and invasion of trophoblasts and maintaining anti-in ammatory environment of immunotolerance. In contrast, the status of DC subsets in the proin ammatory microenvironment of EOPE pregnancy requires thorough evaluation. In this direction, the designed study protocol aims to understand how DC subsets are altered (quantitatively and functionally) in EOPE patients, compared to normal pregnant women.
Methods: Study protocol is designed to determine changes in the pro le of DC subsets in the blood and decidua of EOPE patients by multiparametric ow cytometry approach. Normal pregnant women are included as controls.
Discussion: Human DC subsets are altered both quantitatively and functionally in the pro-in ammatory microenvironment. EOPE is a pro-in ammatory disorder and changes in the composition and function of DC subsets in these patients, compared to normal pregnant women is unclear. In this study, the blood sample-based analysis will determine the feasibility for identi cation of DC subsets associated novel immune biomarkers for early diagnosis of EOPE. In addition, changes in the pro le of DC subsets in the decidua of EOPE patients will determine the feasibility for developing novel immunotherapeutic strategies targeting distinct DC subsets or their products for the treatment of EOPE. Overall, the current study protocol and ndings will help develop future large scale, prospective design clinical trials focussing on formulating strategies for early diagnosis and treatment of EOPE among pregnant women.

Background
Pre-eclampsia (PE) is a pro-in ammatory, pregnancy-associated disorder and is one of the major causes of maternal and fetal associated morbidity and mortality in India and worldwide [1]. In addition, there is an increased risk for the mother and her child to develop serious complications later in life including cardiovascular diseases and diabetes mellitus [2]. PE pathogenesis suggests a bias towards proin ammatory immune response triggering impaired trophoblast invasion and incomplete spiral arteries remodeling in the uterus, ultimately resulting in placental ischemia. Therefore, placental vascularity and blood supply to the fetus is reduced. The placental oxidative stress leads to the release of placental factors that trigger more in ammatory cytokine release and endothelial dysfunction causing downstream clinical symptoms such as hypertension [3].
Currently, the only known de nitive treatment for PE is to deliver the placenta and the fetus [4]. Diagnosis of PE is made on the basis of clinical symptoms that appear after 20 gestational weeks; characterized by a) hypertension with a systolic blood pressure of 140 mm Hg and a diastolic blood pressure of 90 mm Hg, measured twice over four hours apart and b) proteinuria (> 0.3 g per 24 hours) [3]. Based on the onset of clinical symptoms, PE is divided into early onset (EOPE), if symptoms develop before 34 weeks of gestation, and late onset preeclampsia if symptoms develop at or after 34 weeks of gestation. Although EOPE is more severe and high risk for mother and fetus [5], currently, there are no de nite biomarkers for early diagnosis.
Pro-in ammatory disorders including lupus, Crohn's disease, systemic sclerosis, atopic dermatitis, rheumatoid arthritis, in ammatory bowel disease and multiple sclerosis are associated with drastic changes in circulating DC subsets that suggest evaluation of their value as biomarkers [7,8]. In addition to diagnostic potential, monitoring circulating DC subsets can offer insight into the progression of in ammatory disease and response to treatment [9,10]. Along these lines, several tolerogenic DCs based clinical trials for treating pro-in ammatory diseases are ongoing [11]. Other treatment strategies include targeting the pathogenic cytokines produced by DC subsets [12].
DC subsets have been well characterized in normal pregnancy [13,14]. CD141 + mDCs predominate in the decidua of rst trimester of pregnancy, with reduction in CD1c + mDCs and no changes in pDCs [13]. CD141 + mDCs are known to mediate differentiation of anti-in ammatory T-helper 2 (Th2) type of cells [15] and are tolerogenic in nature-expressing immunoglobulin like transcript-3 (ILT-3) [13]. Such functions of DCs are critical for a) e cient invasion and remodeling of spiral arteries by trophoblasts, subsequent successful establishment of feto-placental unit and b) maintenance of immune tolerance towards fetus [16].
In contrast, it is unclear how the pro-in ammatory microenvironment in EOPE patients alters the pro le (quantitative and functional) of DC subsets in circulation and decidua. Identi cation of changes in the pro le of DC subsets in EOPE could offer novel strategies for identi cation of biomarkers and immunotherapeutic treatment. In support of this thought process, studies indicate both quantitative and functional changes in DCs among PE patients. For example, in preeclamptic decidua, the total DCs are increased and the chemokines involved in DC migration to the decidua are also increased [17]. In addition, GM-CSF expression (mediator of DC differentiation and activation) has been shown to increase in preeclamptic decidua [18] causing enhanced local differentiation and activation of DCs. Density of DCs drastically affects antigen presentation in uencing the outcome of T cell responses. The CD4 T cell pro le in PE patients is altered as the number of Th2 cells is decreased in the pre-eclamptic decidua, compared to normal pregnancy associated decidua [19]. Along these lines, the ability of DCs to induce regulatory T cells is impaired in PE patients [20], disrupting the tolerogenic environment characteristic of a normal pregnancy. There is a bias towards the generation of pro-in ammatory Th1 and Th17 cells, and pro-in ammatory cytokine production in DCs isolated from PE patients suggesting functional alterations in DC subsets [21][22][23] (Fig. 2).
Overall, the main objective of the study is to delineate how the three DC subsets are altered quantitatively and functionally in blood and decidua of EOPE patients, compared to normal pregnant women. This study will provide signi cant insight into the role of DC subsets in EOPE pathogenesis. Importantly, the ndings of this explorative pilot study will determine feasibility for designing future prospective clinical trials focussing on a) identifying blood-based DC subsets associated biomarkers for early diagnosis of EOPE and b) developing immunotherapeutic interventions targeting distinct DC subsets or their products in decidua (placenta) for the treatment of EOPE (Fig. 3).

Methods
Study design: This is an exploratory, pilot and feasibility study to determine quantitative and functional differences among DC subsets of early onset pre-eclampsia patients based on well established experimental protocols. Section A criteria: Patients answering 'NO' for the following conditions will be ELIGIBLE for the study.
1. Multiple pregnancy (pregnancy with more than one fetus).
2. Women pregnant for the second or more time.
4. Chronic hypertension (≥140/90mm Hg) diagnosed before pregnancy or in the rst half of pregnancy (<20 weeks) and continued for >12 weeks after delivery.
5. Atypical pre-eclampsia (pre-eclampsia symptoms <20 weeks of gestation or > 48hrs after delivery. 6. Positive for SARS-CoV-2 infection (currently or in the past). 9. Any "other" obstetric complications. "Other" term for pre-eclampsia patient group includes obstetric complications except for the early-onset pre-eclampsia condition. Examples include hemorrhage, obstructed labor, amniotic uid embolism.

Pregnant via assisted reproductive technology (ART).
Factors such as multiple pregnancy, chronic hypertension diagnosed before pregnancy, atypical preeclampsia, stated medical complications and infections (including COVID-19), smoking, higher maternal age, increased BMI and other obstetric complications can introduce biological mechanisms unrelated to the true representation of pre-eclampsia pathogenesis. Therefore, subjects with these scenarios are excluded from the study. Additionally, women who got pregnant via ART procedure are excluded from this study as these women demonstrated increased risk of PE, compared to women with spontaneous pregnancy [24]. It is not clear if the technique of assisted reproductive technology itself in uences the placental biology [25]. Among spontaneous pregnancy, the risk of pre-eclampsia is much lower in women who are getting pregnant for the second or more time [26]. Therefore, these women are excluded from the study. As the study focus is on early-onset pre-eclampsia patients, pregnant women with late-onset preeclampsia are excluded from the study. Obtaining informed consent: Based on the eligibility criteria, 2 groups of pregnant women are created: a) Early-onset pre-eclampsia (EOPE) patients and b) Normal pregnant women. These pregnant women are given the patient information sheet and sample collection details are clearly explained to them by the obstetrician. After explanation of the study, informed consent from the subjects are obtained.
Sample collection: Blood Collection: Around 2 ml of blood is collected from each pregnant woman at the time of parturition. Experienced hospital staff/phlebotomy team are performing the blood collection by venipuncture. The sample is collected in sterile blood collection tubes coated with an anticoagulant such as EDTA, stored at room temperature and brought to research lab at IIPHG for further processing.
Placenta collection: Placenta is collected during parturition, placed in a sterile tissue collection bag enclosed in an ice box and brought to the research lab at IIPHG for further processing. Proper and approved biosafety practices for handling and disposal of biological materials is followed.
Case report form (CRF): Clinical data is collected from the pregnant women recruited into the study.
These data are entered into the CRF during sample collection (blood and placenta) and post-partum. Brie y, general details of the subject are entered into CRF, including name, date of birth and BMI. In addition, participant and their family history are collected. Clinical parameters are recorded in the CRF; including gestation age, diastolic & systolic blood pressure, mean arterial pressure, proteinuria, any ongoing medical treatments, presence of HELLP/IUGR/atypical pre-eclampsia, recently taken hemoglobin levels, platelet levels and complete blood counts, doppler examination of uterine arteries and any other obstetric complication. Other parameters such as delivery date and gestation, placenta weight, type of delivery, systolic and diastolic blood pressure, any medical treatments done, and any other obstetric complications are also recorded.
The collected clinical parameters are compared between pre-eclampsia patients and normal pregnant women. Data is presented as mean +/-standard deviation (SD) and range. Differences are considered signi cant when the p value will be equal to or less than 0.05. All statistical analyses assume a 2-sided signi cance level. Mann-Whitney U non-parametric test is used for comparisons between groups.
Experimental work plan: An overview of the complete experimental plan with blood and placenta samples is shown in gure 5.
A. Blood sample: A portion of blood sample is processed for direct immuno uorescence surface staining procedure and another portion for DC subset speci c TLR stimulation.
Direct immuno uorescence staining of whole blood: This is a well-established methodology for directly detecting dendritic cell subsets in blood samples from normal pregnant women and healthy non-pregnant individuals [27][28][29][30][31][32]. This method is more e cient compared to other methods as it is shown to improve assay reproducibility and is less likely to show loss of lymphocyte subsets [33][34][35][36]. All monoclonal antibodies are titrated for determining optimum antibody concentration for usage.
Around 0.2ml of blood sample is stained with monoclonal antibodies against surface markers (Table 1), followed by RBC lysis. These samples are run on the ow cytometer (Thermo Fisher Attune Nxt) to characterize DC subsets. The 9-color multiparametric ow panel has been designed using FluoroFinder2.0 software (Table 1), with careful consideration given for minimal spectral spill-over values between uorochromes so that automatic compensation can be easily performed using FlowJo software.
Abc TM Anti-Mouse Bead Kit (Thermo Fisher Scienti c) is used to set up ow cytometry compensation. The 9-color multiparametric ow panel is designed to identify the 3 DC subsets, simultaneously determining other phenotypic changes, such as activation (CD80), maturation (CD83) and tolerogenic properties (ILT-3). ILT3 (immunoglobulin-like transcript 3), also known as CD85K is highly expressed on myeloid DCs in the decidua of normal pregnant women [13]. ILT3 is involved in the induction of immune tolerance in DCs via interaction with HLA-G on extra villous trophoblasts (EVTs) [37]. Therefore, ILT-3, along with activation and maturation markers are included in the panel to monitor their expression changes in the pro-in ammatory environment of EOPE.
TLR stimulation of whole blood: This procedure has been well established for directly analyzing DC subsets functional responses in whole blood post stimulation/activation with TLR ligands [32]. Brie y, 0.5ml of blood sample is subjected to TLR stimulation, by using LPS 100ng/ml (for TLR-4 stimulation on CD1c+ mDCs) and CpG 2216 30ug/ml (for TLR-9 stimulation on plasmacytoid DCs) and poly I:C 30ug/ml (for TLR-3 stimulation on CD141+ mDCs) along with brefeldin A 10 ug/ml (protein transport inhibitor) for 5hrs at 37c, 5% CO 2 . Selection of TLRs for each DC subset is based on differential expression of TLRs on these cells [38]. Thereafter, post surface staining, intracellular cytokine staining procedure (permeabilization and xation) is performed-speci cally for IL-12, TNF-A (for myeloid DCs) and IFN-A, TNF-A (for plasmacytoid DCs) (Tables 2-4). These samples are run on the ow cytometer (Thermo sher Attune Nxt). Production of these cytokines by DC subsets is drastically altered in several proin ammatory disorders [7,8]. Therefore, these cytokines are being included in the ow-panel to assess their production in EOPE patients and to determine feasibility of including them as biomarkers for diagnosis and/or immunotherapeutic intervention.
B. Placenta (decidua) samples: Procedures for the isolation of decidua, decidual cells and leukocytes are being adapted from well-established studies [39,40]. Decidua basalis (part of decidua in contact with placenta) and decidua parietalis (rest of the decidua on the maternal myometrium end) are isolated from the placenta. Collected decidua are subjected to mechanical processing to obtain decidual cells. Decidual leukocytes are isolated by Ficoll-Paque density gradient centrifugation method [41]. The leukocytes settled at the interface are carefully collected and washed for immunophenotyping DC subsets. The total yield of leukocytes from this protocol is up to 30x10 6 cells per decidual tissue per study participant.
Immunophenotyping and functional analysis of decidual DC subsets: A portion of freshly isolated decidual leukocytes is used for cell-surface antigen staining using monoclonal antibodies and another portion of cells will be subjected to speci c TLR stimulation. Appropriate mouse anti-human isotype controls are included. In addition, appropriate uorescence minus one (FMO) controls are used to eliminate any spill-over-induced background.
Around 2 x10 6 live cells are used per sample (dead cells are excluded by trypan blue counting with a hemocytometer). All monoclonal antibodies are titrated for determining optimum antibody concentration for usage. For surface staining, the cells are stained with appropriate monoclonal antibodies forming the 9-color panel ( Table 2). TLR stimulation of decidual cells are performed similar to blood samples and optimal concentration of TLR ligands to stimulate decidual DC subsets are being optimized. Similar to blood samples, surface staining (including activation markers) and intracellular cytokine staining (permeabilization & xation) are performed for detecting IL-12, TNF-A (myeloid DCs) and IFN-A, TNF-A (plasmacytoid DCs) (Tables 2-4).
Data analyses: At least 200 000 events within the combined lymphocyte-monocyte gate, based on the FSC and SSC parameters per sample is collected in the ow cytometer and data is analyzed using FlowJo software. Appropriate mouse anti-human isotype controls are included to rule out any nonspeci c background signal caused by primary antibodies. In addition, appropriate uorescence minus one (FMO) controls is used to eliminate any spill-over-induced background.
Data is analyzed using FlowJo software as follows (Fig-6). This is followed by determining frequencies and absolute numbers of a) Plasmacytoid DCs: CD11c -CD123 + , b) CD1c + Myeloid DCs: CD11c + CD1c + and c) CD141 + Myeloid DCs: CD11c + CD141 + . In addition, the frequencies and mean uorescence intensity (MFI) of the cytokines and activation, tolerogenic markers expressed by each of the 3 DC subsets is calculated. All the data are compared between normal pregnant women(n=30) and pre-eclampsia patients (n=30).
Statistical analyses: A standard non-parametric test (Mann-Whitney U-test) is used to determine statistical differences of blood and decidua derived DC subsets between the two groups of pregnant women. Differences at P<0.05 is considered statistically signi cant. IBM SPSS 20 software is used to perform statistical analyses.

Discussion
EOPE is a severe form of pre-eclampsia and currently, no de nite biomarkers or treatment strategies are available. Human DC subsets are one of the key players in modulating the pro-in ammatory changes in the microenvironment. Towards this direction, the study is aimed at identifying changes in the pro le (quantitative and functional) of DC subsets in EOPE patients, compared to normal pregnant women. This pilot study will determine the feasibility of identifying potential DC subsets associated biomarker(s) and immunotherapeutic target(s).
The experimental techniques chosen to characterize DC subsets in the study protocol are well established, reproducible and are being easily adapted. This study protocol allows the collection, processing, sample acquisition on ow cytometer and subsequent data analysis to be completed in the same day. An e cient collaboration of study sites has been established and recruitment of subjects has been initiated from January 2020. However, due to the recent emergence of COVID-19 pandemic, there have been unanticipated delays in the process of recruitment and sample collection. These issues are being confronted, and it has been made mandatory among recruited pregnant women to be tested for SARS-CoV-2 before obtaining informed consent and sample collection. Appropriate changes have also been re ected in the eligibility criteria.
Human DC subsets present as ideal and novel cellular markers that could be monitored for pathogenic changes in early pregnancy as they are one of the key players involved in the decidualization and angiogenesis process leading to implantation and placentation. They play a critical role during pregnancy by modulating remodeling of decidual tissue and producing chemokines for the migration and invasion of trophoblast cells into the endometrium during placentation [16,43]. These features are signi cantly altered leading to the pathogenesis associated with EOPE patients. These immunological cellular changes could be combined with other risk factors to develop a comprehensive panel of biomarkers for early diagnosis of EOPE.
Multiparametric ow cytometry approach for diagnosis of pregnancy complications offers signi cant advantages. Multilevel changes in immune cells can be identi ed simultaneously in a small volume of biological sample within a short period of time. For example, one study identi ed signi cant quantitative and functional changes in DC subsets among pregnant women with intrauterine growth restriction (IUGR), without PE symptoms [44]. Therefore, timely intervention and care could be provided to women with pregnancy complications. However, the cost per test through ow cytometer could be higher, compared to other testing approaches. It is likely that the speci c changes observed in DC subsets and their products through ow cytometry approach in EOPE patients in the study could be modi ed in future studies by detecting the same biomolecules through parallel lower cost methods such as ELISA assays.
A prospective study design would be most effective to identify appropriate biomarker(s) for early diagnosis and immunotherapeutic target(s) for early treatment. However, the current study explores a novel direction and a pilot study is essential to determine DC subset pro le changes in established cases of EOPE, and large-scale prospective design studies involving early pregnancy can be followed up.
Overall, we believe that the blood and decidua samples based analysis in the study will identify DCsubsets pro le changes in EOPE patients that could be translated into practical and tangible biomarker(s) and immunotherapeutic target(s) for early diagnosis and treatment or intervention strategies. Informed consent is being obtained from study participants.

Consent for publication
Not applicable

Availability of data and materials
Data is available from corresponding author upon reasonable request.

Competing interests
The authors declare that they have no competing interests. Tables Table-1: Surface markers for the generation of 9-color ow panel to determine quantitative and phenotypic differences among DC subsets in blood and decidua of EOPE and normal pregnant women.
Attune Nxt ow cytometer (Thermo Fisher Scienti c) is being used to acquire uorochrome labeled samples. The surface markers (Lineage, HLA-DR, CD11c, CD1c, CD141, CD123) are used to identify the 3 DC subsets. CD80, CD83, ILT-3 markers are used as representatives of activation, maturation and tolerogenic marker respectively. All antibodies are purchased from BD Biosciences, except for CD1c, respective isotype control (Miltenyi Biotec) and ILT-3, respective isotype control (BioLegend). The dashes indicate unused channels.      Implications of ongoing pilot study. The ongoing pilot study protocol objective is to de ne dendritic cell (DC) subsets associated quantitative and functional differences in blood and decidua of early onset preeclampsia (EOPE) patients, compared to normal pregnant women. The study ndings will determine the feasibility of identi cation of DC subsets-based a) biomarkers for early diagnosis of EOPE through blood samples and b) immunotherapeutic targets for treatment of EOPE through decidual samples. Ongoing pilot study ndings will be tested in a large scale, prospective design setting.

Figure 4
An overview of processes involved in each study site. The recruitment of study subjects based on eligibility criteria, obtaining informed consent, collection of blood and placenta samples and completion of case report form for each participant is being done at the Civil hospital, Gandhinagar. The blood and placenta samples are processed at the Indian Institute of Public Health Gandhinagar (IIPHG), followed by acquisition of samples on ow cytometer (Attune Nxt, ThermoFisher Scienti c) and data analysis at the Institute of Science, Nirma University, Ahmedabad.

Figure 5
An overview of research laboratory associated study protocol. At the time of parturition, blood and placenta are collected from EOPE patients (n=30) and normal pregnant women (n = 30). A portion of blood samples are subjected to direct immuno uorescence staining involving surface markers to identify, quantitate DC subsets and to assess activation, maturation and tolerogenic marker differences between EOPE and normal pregnant women. Another portion of blood sample is subjected to DC subsetassociated Toll-like Receptor (TLR) stimulation to assess speci c cytokine production differences between the two groups of women. Decidua is isolated from placenta and through mechanical, nonenzymatic approach, decidual leukocytes are isolated. Similar to blood samples, decidual leukocytes are subjected to surface markers immunostaining for identi cation, quantitation of DC subsets and assessment of activation, maturation and tolerogenic marker differences between EOPE and normal pregnant women. The decidual leukocytes are also subjected to DC subset associated TLR stimulation, similar to blood samples to assess speci c cytokine production differences between the two groups of women. (maturation marker), ILT-3 (Tolerogenic marker) are determined. The surface staining of DC subsets encompasses a 9-color ow panel being compared between EOPE and normal pregnant women. In order to assess functional differences in DC subsets between EOPE and normal pregnant women, the blood and decidual cells are subjected to indicated DC subset associated TLR stimulation. Intracellular staining is done to determine changes in the indicated DC-subset associated cytokine expression. Expression changes are determined through mean uorescence intensity (MFI) calculation and through frequencyabsolute numbers of DC subsets expressing the markers.