We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.
The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of 'unconventional' T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women's Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.
We developed methodology of using endocervical brush samples to evaluate intraepithelial GD T cells. The endocervical canal is lined by a single layer of columnar epithelium. Intraepithelial lymphocytes represent frontline lymphocytes residing within the epithelial layer that can rapidly initiate immune responses upon encountering pathogenic microbes7,8. Understanding the immunological events of endocervical intraepithelial compartment is important for the design of effective strategies to prevent infections, including HIV.
Our method can be applied for freshly collected human endocervical samples as well as frozen endocervical cells to further explore the role of intraepithelial GD T cells in women with HIV infection or at risk for HIV infection. The main goal of this protocol is to discover novel immunological events in endocervical intraepithelial compartment and to evaluate GD T cell responses as a marker of mucosal vulnerability in women with HIV infection.
The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. Furthermore, the complex heterogeneity of mucosal immune cells, and their interconnectedness with each other, are major challenges to identifying clinically relevant measurements that reflect the state and capability of the immune system. We developed a method to obtain highly purified intraepithelial GD T cells that can be further analyzed using highly multiplexed, single-cell technologies that may be critical for identifying the underlying mechanisms of FRT immunity.
Study activities took place at University of Miami HIV Research Unit in collaboration with the Miami Women's HIV Interagency Study (WIHS) and the Miami Center for AIDS Research (CFAR).Institutional Review Board (University of Miami Miller School of Medicine) approval was obtained prior to recruitment and any assessment or study related procedures.
1. Endocervical Brush Sample Collection
NOTE: Participants underwent a vaginal examination performed by trained MD or gynecologist and collection of intraepithelial lymphocytes were performed under speculum examination by inserting cytobrush.
2. Endocervical Cytobrush Sample Processing
3. Flow Cytometry
4. Endocervical Gamma delta T Cell Isolation
Recently, we analyzed endocervical intraepithelial GD T cells and we were the first one to report about the loss of endocervical gamma delta T cells in HIV infected women9,11. Here, we describe the protocol to isolate and analyze the subset of endocervical gamma delta T cells. As shown in Figure 1, GD T cells can be readily detected in the human endocervical cell samples.
A unique population of endocervical gamma delta V1 (GD1) T cells was described using multiparametar flow cytometry-based immunophenotyping (Figure 1). A representative gating strategy for HIV uninfected endocervical sample is shown in Figure 1. Analysis was performed on the total endocervical cells by setting the electronic gate on Forward/Side scatter (Figure 1A) followed by the exclusion of cell doublets (Figure 1B). Viable cells were defined by exclusion of dead cells using LIVE/DEAD Fixable Yellow Amine Dead Cell Stain (Figure 1C). Analysis of CD45 expression was performed on live gated cells (Figure 1D), followed by the analysis of CD3 expression (Figure 1E). Gamma delta 1 (GD1) or gamma delta 2 (GD2) positive cells were defined as a subpopulation of CD3+ cells that express GD TCR (TCR gamma delta V1 or TCR gamma delta V2). Furthermore, it was confirmed that only CD3+ T cells express TCR gamma delta since gated CD3– T cells do not express TCR gamma delta V1 or delta V2. Phenotypic analysis of gated CD3+GD1+ cells revealed (Figure 1G), that majority of GD1 cells (~74%) are CD4–CD8–.
In the present study, feasibility of utilizing endocervical brush samples to purified intraepithelial GD T cells by positive magnetic selection was demonstrated (Figure 2).
Figure 1: Gamma delta (GD) T cell subpopulation in endocervical mucosa analyzed by flow cytometry. Following gating strategy was used to identified endocervical T cell populations: (A) endocervical cytobrush cells were gated according the size and granularity (FSC, forward side scatter vs SSC, side scatter). (B) Cell doublets were excluded by setting the electronic gates in the FSC-A (area) vs SSC-W (width). (C) Live cells are gated on the viability dye-negative population. (D) Electronic gate was set on live, CD45 positive cells. (E) Electronic gate for CD45 positive cells was set on CD3 positive cells (F) TCRVD1 (GD1) and TCRVD2 (GD2) expression was analyzed on CD3+ cells. (G) CD4 and CD8 expression was analyzed on GD1+ cells. (H) TCRVD1 and TCRVD2 expression was analyzed on gated CD3 negative cells. Please click here to view a larger version of this figure.
Figure 2: Isolation of endocervical gamma delta T cells (GD) by positive magnetic separation. Frequency of endocervical GD T cells was confirmed before and after positive magnetic separation. Endocervical cells were first labeled with anti-TCR gamma delta antibody and then with fluorescently stained with Anti-Hapten microbeads-FITC. Percent of TCR GD+ T cells was reported before and after magnetic separation. Please click here to view a larger version of this figure.
Assessment of female lower genital tract mucosal vulnerability is an essential component of clinical studies addressing risk of HIV acquisition and transmission. Typically FGT vulnerability to HIV infection is evaluated by measuring soluble immune modulators in genital secretions (cytokines, chemokines and antimicrobial peptides)12,13. However, these biomarkers are only indicative of vaginal inflammation, are affected by physiological and pathological events, and do not always represent direct mucosal damage. In order to improve our understanding of how mucosal factors increase the risk of acquiring and transmitting HIV, cellular markers of mucosal damage need to be developed. GD intraepithelial T cells are present in the FGT, intercalate between epithelial cells, and are thought to contribute to homeostasis as well as to a first line of defense against environmental challenges. GD T cells have the potential to constitute an additional biomarker to assess mucosal vulnerability in the FGT.
In this protocol, it is shown how to prepare intraepithelial lymphocytes from human endocervical cytobrush sample and is easily mastered. The most critical point of this process is to maintain cell viability by fast processing of the sample as well as by keeping the cells on ice during the entire procedure. The cell yield will depend on cytobrush collection technique, handling skill as well as the infectious status (HIV infection or other viral infections, bacterial or fungal infections). Our group and others have published various studies describing different lymphocyte populations in human genital tract9,14,15. In our hands, one endocervical cytobrush sample can provide 0.5-5 x 106 cells with 80-90% viability. In comparison with the commonly used isolation method14,15, by our method the viability and cell yield is increased.
This method provides a powerful and an efficient tool to study endocervical intraepithelial gamma delta T lymphocytes and could provide insight into other immune cells that migrate to this compartment during infection and/or inflammation. The in vivo cellular composition of mucosal tissues is often difficult to investigate in a comprehensive and quantitative way. Techniques such as immunohistochemistry and flow cytometry are limited by the availability of antigen specific monoclonal antibodies and by the small number of parallel measurements that can be performed on each individual cell. Traditional high-throughput assays, such as gene-expression arrays, when performed on whole tissues, provide information on average gene expression level, and can be only indirectly correlated to quantitative modifications in cellular subpopulations. These limitations become particularly difficult to overcome when studying minority populations, or the cell population that lack exclusive markers. By combining "fluorescence activated cell sorting" (FACS) and "single-cell PCR gene-expression analysis" one can perform a high-throughput transcriptional analysis of the intraepithelial GD T cells in endocervix. This method exploits the capacity of modern flow cytometers to sort individual single cells with accuracy and precision, together with the use of microfluidic technologies to preform high sensitivity multiplexed PCR from minute amounts of mRNA, thereby allowing parallel analysis of the expression of up to 96 genes for each individual cell16.
The authors have nothing to disclose.
We thank the Miami WIHS study participants for their willingness to participate in this work. Data in this manuscript were collected by the Miami Women's Interagency HIV Study (WIHS). The contents of this publication are solely the responsibility of the authors and do not represent the official views of the National Institutes of Health (NIH). This work was supported by National Institute of Allergy and Infectious Diseases and Women's Interagency HIV Infection Study (WIHS) [grant number U01 AI103397], National Institute of Allergy and Infectious Diseases, National Institutes of Health [ grant number P30AI073961], National Center for Advancing Translational Sciences and the National Institute on Minority Health and Health Disparities, National Institute of Health [grant number UL1TR000460 and 1KL2TR000461], and National Institute of Child Health and Human Development, National Institute of Health [ grant number K23HD074489].
Cytobrush Plus GT | (CareFusion, San Diego, CA, USA | ||
IMDM,Iscove's Modified Dulbecco's Medium | ThermoFisher Scientific | 12440061 | |
Beckman Coulter automated cell counter (The Vi-CELL Series Cell Viability Analyzer | Beckman Coulter | 496178 | |
DMSO, Dimethyl sulfoxide | Sigma Aldrich | D2650 | |
FBS, Fetal Bovine Serum | Invitrogen, Gibco | 26140-079 | |
PBS | Invitrogen, Gibco | 10010023 | |
BSA | Sigma Aldrich | A-9647 | |
LIVE/DEAD Fixable Yellow Dead Cell Stain kit | Life Technologies, | L349S9 | |
1% paraformaldehyde (Sigma-Aldrich | Sigma Aldrich | D6148 | |
Fortessa flow cytometer | Becton Dickinson | ||
UltraComp eBeads | eBioscience | 01-2222-42 | |
ArC Amine Reactive Compensation Beads (Life Technologies, Grand Island, NY, USA) | Life Technologies | A10346 | |
anti-TCR gamma/delta MicroBead Kit | Milteny Miltenyi Biotec Inc. | 130-050-701 | |
MS column | 103-042-201 | ||
MACS separator | 4124 |