Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Summary

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

Abstract

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

Introduction

The majority of new HIV infections worldwide arise through heterosexual transmission, with women representing 47% of new infections in 2011 (UNAIDS¹). Understanding the female genital tract (FGT), one of the main entry portals for HIV and other sexually transmitted pathogens, is of high importance on the path to finding efficient strategies to prevent infection. Immune responses at the genital mucosa are clearly unique and differ from those measured in peripheral blood². However, current knowledge of the immune dynamics at the FGT is limited at best. To date, studies of the mucosal immune environment have largely focused on the gut-associated lymphoid tissues (GALT), where it has become clear that the early events in mucosal tissues following infection have a strong impact on subsequent disease progression^3,4. Collecting samples from the genital mucosa represents a great challenge and is at least partially responsible for the lack of understanding of the immunology of the FGT. Solving the puzzle of the immune dynamic between host and pathogen in the context of the distinct environment that is the FGT necessitates efficient methods for collecting and analyzing samples from this locale.

The FGT is divided into two sections: the upper reproductive tract that includes the fallopian tubes, endometrium and endocervix, and the lower tract which contains the ectocervix and the vagina (reviewed by Kaushic et al⁵). It is still unclear what the relative contribution of these different sites is to HIV infection, but it is believed that both sites could contribute to HIV entry⁶. T cells represent 40-50% of the leucocytes in the upper and lower reproductive tracts, while macrophages comprise approximately 10% (reviewed in Rodriguez-Garcia et al²). T cells can be detected in the vagina, cervix, and endometrium. Macrophages are more strongly localized in the endometrium and myometrial connective tissue than the cervix, although they can be detected in both tissues. Finally, plasmacytoid dendritic cells (pDCs) and Langerhans cells can also be detected in FGT tissues. The phenotype and proportions of immune populations and their susceptibility to HIV infection may vary importantly according to hormonal cycles, the use of hormonal contraceptives, bacterial vaginosis or sexual activities^5,7-9.

Diverse methods have been developed to study the immune populations and environment of the FGT. Cervical biopsy, cervical cytobrushes and cervicovaginal lavages (CVL)^10-12 are the most commonly used across the literature. CVL collection by PBS lavage is the simplest method and allows the study of immune modulatory proteins but results in extremely low cell yield, and is therefore not suitable for studying the immune cell populations of the FGT¹³. CVL samples are, on the other hand, very useful for evaluating the immune environment of the FGT by measuring the expression of various cytokines, chemokines or antimicrobial factors using methods such as ELISA, cytokine bead array¹⁴ or mass spectrometry^15,16. Characterization of immune cell frequencies, phenotypes and functions can be achieved by collecting cervical mononuclear cells (CMC) by cervical cytobrush or by cervical biopsy sampling.

Cervical biopsy sampling is an invasive method that increases the discomfort and risk of bleeding and takes 2 to 11 days to heal following the procedure depending on the immune status of the woman¹². On the other hand, cervical cytobrushes, despite the lower yield of cells collected, is a less invasive and more convenient method to collect immune cells from the FGT. Both methods can reach the same yield of CD45+ leucocytes, but two sequential cervical cytobrushes are necessary to obtain the same amount of cells contained in one biopsy¹³. Nonetheless, cytobrush sampling still provides an acceptable number of cells (about 5,000 CD45+ cells/cytobrush) for further ex vivo phenotyping by flow cytometry¹⁴. Also, functional characterization can be carried out on these samples, as stimulation and intracellular flow cytometry or qPCR have been performed using cytobrush-derived CMCs to identify HIV-specific immune responses¹⁷ or Th cell polarization¹⁸. Expansion of the T cells population may also facilitate functional studies with CMCs¹⁹.

It is important to note that biopsies and cytobrushes sample distinct portions of the FGT. Biopsies are derived from the superior portion of the epithelium and stroma of the ectocervix^12,13, while cervical cytobrushes sample the cervical os, collecting cells derived from the epithelium of endocervix and presumably the transformation zone. Cytobrush samples therefore sample a region composed of a single layer of columnar epithelium, while biopsies, include a region lined by a squamous stratified epithelium⁵. As a result, the nature of the leucocyte populations collected by cervical biopsy and cytobrush differs. Biopsies collect a higher proportion of CD3+ T cells, whereas cytobrushes result in collection of a higher proportion of CD14+ monocytes/macrophages¹³.

Studying the immunology of the FGT has been an interest for many years^20-22 and we have accumulated a great deal of expertise with the study of cytobrush-derived CMCs. Our studies focus mostly on the study of HIV-infected, uninfected and HIV-exposed seronegative (HESN) female sex workers from Nairobi, Kenya. HIV preferentially replicates in activated T cells²³ and lower numbers of activated cells that can be targeted by HIV in the FGT could contribute to protection against HIV acquisition. In line with this hypothesis, several studies have described lower immune activation among HESN sex workers who are highly exposed to HIV yet remain uninfected^24,25, and this quiescent phenotype is also observed in the FGT¹⁴. Here, we describe methodology for processing and assessing T cells activation in CMC samples derived from cervical cytobrushes by ex vivo flow cytometry.

Protocol

Ethics statement: The research ethics boards of both the University of Manitoba and Kenyatta National Hospital/University of Nairobi approved this study and written informed consent was obtained from all study participants.

1. Preparation of Media and CMC Collection Tubes

1. Prepare phosphate buffered saline (PBS) solution (137.93 mM NaCl, 2.67 mM KCl, 8.1 mM Na₂HPO₄, 1.47 mM KH₂PO₄). Autoclave for sterility. This can be stored at 4 ºC for several months.
2. Pre-aliquot 5 ml PBS into 50 ml falcon tube (one per sample to be collected) and store at 4 ºC until sample collection time.
3. Label 2-4 cryovials or 1.5 ml microcentrifuge tubes per sample to collect lavage for storage.
4. Prepare cell culture media: RPMI 1640 + 1% penicillin/streptomycin/amphotericin B (final concentration 100 units/ml, 100 μg/ml and 250 ng/ml, respectively, with no FCS/FBS added.

2. Collection of Cytobrush Samples

Collection of cytobrush samples is a non-invasive procedure that must be performed by a trained MD or gynecologist.

1. Collect cervical mononuclear cells (CMC) using both a cytobrush and a wooden or plastic scraper. Collect CMC from the participant under speculum examination by inserting the scraper and rotating around the cervical os (Figure 1). Insert the cytobrush into the endocervical os, rotate 360°, and immediately place both the cytobrush and scraper into the 50 ml falcon tube containing 5 ml of PBS.
2. Keep samples on ice/at 4 ºC until processing. To maintain cell viability, process samples within 2 hr of collection.
3. If possible, collect matched blood samples in green top heparin vacutainers for peripheral blood mononuclear cell (PBMC) isolation to serve as a comparator/control for CMC analysis.

3. Isolation of CMCs

Perform sample processing in a biosafety level 2 laboratory, in a sterile biosafety cabinet with double gloves.

1. During collection, CMC samples can become contaminated with blood. Exclude samples with visible blood contamination from the study in order to prevent confounding inclusion of PBMC in the final sample. Due to the low number of lymphocytes isolated from CMC samples, minor blood contamination can easily overwhelm the CMC lymphocyte component.
2. Vortex falcon tubes containing both the cytobrush and scraper for 45 sec to dissociate cells from the brushes. Samples may become foamy during this process; this is normal.
3. Use the cytobrush to scrape any remaining material off the scraper, and discard the scraper into a bleach solution. To collect any cells remaining attached to the brush/scraper, use a fresh glove to squeeze the scraper between the thumb and forefinger. Slide down the brush, allowing the cells and PBS to be collected in the same 50 ml tube. Discard the cytobrush into a bleach solution, and discard the glove.
   IMPORTANT: Use a new glove for each sample.
4. Fit a 100 μm nylon cell strainer to a fresh 50 ml falcon tube. Using a transfer pipette, collect the PBS-cell suspension from the sample tube and filter through the strainer into the fresh tube.
5. Add 5 ml of RPMI 1640 to the original sample tube. Using the transfer pipette, wash the sides of the sample tube with the RPMI, and transfer through the filter to the new collection tube. Wash the bottom of the nylon filter with the RPMI as well.
6. Centrifuge the samples for 10 min at 514 x g. It is crucial to leave the centrifuge brake off during this step, in order to prevent dislodging of the CMC pellet.
7. Gently remove the supernatant from the tube, taking care to not disturb the pellet. Pellet size will be highly variable; in some samples, the pellet is quite large due to the presence of large numbers of epithelial cells, while in other cases, the pellet is barely visible and highly transparent.
8. Resuspend the pellet by gentle agitation of the falcon tube. Add 5 ml of PBS to wash the sample.
9. Centrifuge again for 10 min at 514 x g with no centrifuge brake.
10. Carefully discard the supernatant and re-suspend the pellet as above. The cells are ready for either cryopreservation (using a PBMC cryopreservation protocol), stimulation or flow cytometry phenotyping.

4. CMC Surface Staining and Flow Cytometry

1. Resuspend the pellet from step 3.10 in 100 μl of blocking solution and transfer either into a 96-well plate or FACS tube. The blocking solution (to block Fcγ receptors) recipe is as follows: 1.8 μl mouse IgG (final concentration 0.2 μg/μl), 5 μl FBS and 93.2 μl FACS wash (PBS+2%FBS). Staining can be performed in a 96 well plate if pellet sizes are small enough, but may need to be performed in FACS tubes if pellets are routinely large. It is important to titrate antibodies accordingly.
2. Block the samples for 10 min on ice/at 4 °C.
3. Wash the cells with 100 μl of FACS Wash (PBS+2% FBS) in 96-well plates, or 500 μl in FACS tubes. Centrifuge at 600 x g for 10 min at 4 °C with the brake on low (or off, if a low brake setting is unavailable).
4. Remove the supernatant and re-suspend the cell pellet by agitation. Add viability Stain (Live Dead fixable viability dye). Prepare viability dye according to manufacturer’s instructions. It may need to be titrated for use in each lab/cytometer setup. Incubate for 30 min in the dark at 4 °C.
5. Wash the cells 100 μl/500 μl of PBS (for 96-well plate/FACS tubes). Centrifuge at 600 x g for 10 min with low/no brake. Remove supernatant and resuspend cells.
6. Incubate the cells with the cocktail of surface marker antibodies, in a total volume of 100 μl. IMPORTANT: Optimize and titrate each flow cytometry panel prior to sample staining. Incubate cells for 30 min in the dark at 4 °C.
7. Repeat step 4.5. if staining in a 96-well plate, transfer cells to a FACS tube and dilute to a final volume of 750 µl of FACS Wash containing 1% paraformaldehyde (PFA) (or CytoFix, diluted 1 in 4). Proceed to data acquisition on a flow cytometer.

5. Collection and Preparation of Cervical Vaginal Lavage (CVL)

1. Prior to cytobrush sampling, use a syringe to wash the endocervix with 2 ml of sterile PBS and aspirate the lavage from the posterior fornix.
2. Collect the aspirated lavage sample into a 15 ml falcon tube and keep on ice/at 4 ºC until processing.
3. Centrifuge sample at 400 x g for 7 min to remove cellular debris.
4. Collect the supernatant, aliquot the sample into 1 ml or 500 μl aliquots, and store at -70 °C. Aliquots can be thawed for ELISA or bead array analysis of CVL proteins, but avoid multiple freeze/thaw cycles.
5. If desired, resuspend the pellet of cellular debris in RNA Later to measure RNA expression by following the manufacturer’s protocol.

6. Data Acquisition

1. Prepare a set of compensation tubes that matches the antibody panel. Run the compensation tubes to adjust spectral overlap. Run the samples on any multicolor flow cytometer that is configured for the fluorochrome conjugated antibodies used in the panel.
2. Acquire a PBMC sample first to adjust Forward Scatter (FSC) and Side Scatter (SSC) voltage in order to detect the lymphocyte population. Try to keep them at 100 on each axis on a linear scale. Running a PBMC control will make it significantly easier to find the CMC lymphocyte population.
3. The FSC threshold for CMC samples may need to be increased relative to PBMC samples due to smear up the SSC axis at low FSC values.
4. Vortex each tube before acquiring. Acquire the entire tube to maximize the number of lymphocyte gate events collected. Monitor the machine carefully to avoid clogging.
5. Export the data into a flow cytometric analysis program such as FlowJo.

7. Gating Strategy

1. Select Forward side scatter high (FSC-H)/Forward side scatter area (FSC-A) to determine the singlet population.
2. Select Time versus Fluorescent marker (as example FITC) to control for the quality of the acquisition. Change in flow rate may introduce artifacts in the data. Exclude any area that shows discrepancy in flow rate.
3. Identify the lymphocyte population on SSC-A/FSC-A plot. Use a PBMC control to facilitate the identification. Note that the CMC lymphocyte population might not be as clear as the PBMC control.
4. Gate on SSC-A/viability dye to exclude dead cells from live cells. Dead cells can non-specifically incorporate the antibodies, which will introduce artifacts.
5. Gate on the SSC-A/CD3 to identify the T cell population. From this gate, identify the CD4+ and CD8+ populations.

Results

Multiparameter flow cytometry is a powerful tool to dissect the phenotypes and functions of cell subsets in previously uncharacterized tissues. Analysis of CMC samples can yield information on both lymphocyte and monocyte populations with appropriate gating strategies.

A representative CMC gating strategy, compared to a matched PBMC profile, is shown in Figure 2. The FSC-A versus FSC-H plot allows for the exclusion of cell doublets, which are highly prevalent in CMC samples co...

Discussion

Given the large gaps in knowledge with respect to immunity at the female genital tract (FGT), phenotypic analysis of CMCs can provide a wide array of insights into multiple lymphocyte populations at the cervix. Coupled with proteomic analysis and viral load measurements in cervical lavage, immunity to sexually transmitted infections (STI)s and other pathogens can be dissected in various populations.

Technical considerations - CMCs: The isolation and successful staining of CMC ...

Materials

Name	Company	Catalog Number	Comments
Name of Material/ Equipment	Company	Catalog Number	Comments/Description
100uM Cell Strainer for 50 ml Falcon tube	BD	352360	CMC processing
RPMI 1640	Hyclone	SH30027.01	CMC processing
Fetal Bovine serum	Life technology	16000044	CMC processing
Fungizone	Life technology	15290-018	CMC processing
Penicillin/streptomycin	Sigma	P4333-20ml	CMC processing
50ml Falcon tube	Fisher	14-959-49A	CMC processing
Blood Bank disposable transfer pipette	Fisher	13-711-6M	CMC processing
Cytobrush plus	Cooper surgical	C0121	CMC sampling
Disposable cervical scraper	Quick medical	2183	CMC sampling
15 ml Falcon tube	Fisher	14-959-70c	CVL processsing
1.5ml tube ependroff	Fisher	05-402-18	CVL storage
LIVE/DEAD Fixable Cell Stain Kit	Invitrogen	Various	Flow cytometry reagent
Fixation Buffer (4% PFA)	BD	554655	Flow cytometry regeant
IgG mouse	Sigma	I8765	Flow cytometry regeant