Name: isolation and flow cytometric analysis of human endocervical gamma delta t cells
Uploaded: 2017-02-06T10:30:00
Description: Watch this Scientific Journal Video about Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells at JoVE.com

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&#39;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&#39;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].

Study activities took place at University of Miami HIV Research Unit in collaboration with the Miami Women&#39;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 ...

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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...

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.

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			<td height="42" style="height:42px;width:216px;">Cytobrush Plus GT&#160;</td>
			<td style="width:147px;">(CareFusion, San Diego, CA, USA</td>
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		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">IMDM,Iscove&#39;s Modified Dulbecco&#39;s Medium&#160;</td>
			<td style="width:147px;">ThermoFisher Scientific</td>
			<td align="right" style="width:119px;">12440061</td>
			<td></td>
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			<td height="63" style="height:63px;width:216px;">Beckman Coulter automated cell counter (The Vi-CELL Series Cell Viability Analyzer</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">496178</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO, Dimethyl sulfoxide&#160;</td>
			<td style="width:147px;">Sigma Aldrich&#160;</td>
			<td style="width:119px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS, Fetal Bovine Serum</td>
			<td style="width:147px;">Invitrogen, Gibco</td>
			<td style="width:119px;">26140-079</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:147px;">Invitrogen, Gibco</td>
			<td align="right" style="width:119px;">10010023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BSA</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">A-9647</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LIVE/DEAD Fixable Yellow Dead Cell Stain kit&#160;</td>
			<td style="width:147px;">Life Technologies,</td>
			<td style="width:119px;">L349S9</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1% paraformaldehyde (Sigma-Aldrich</td>
			<td style="width:147px;">Sigma Aldrich&#160;</td>
			<td style="width:119px;">D6148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fortessa flow cytometer&#160;</td>
			<td style="width:147px;">Becton Dickinson</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UltraComp eBeads</td>
			<td style="width:147px;">eBioscience</td>
			<td style="width:119px;">01-2222-42</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">ArC Amine Reactive Compensation Beads (Life Technologies, Grand Island, NY, USA)</td>
			<td style="width:147px;">Life Technologies</td>
			<td style="width:119px;">A10346</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-TCR gamma/delta MicroBead Kit&#160;</td>
			<td style="width:147px;">Milteny Miltenyi Biotec Inc.</td>
			<td style="width:119px;">130-050-701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MS column</td>
			<td style="width:147px;"></td>
			<td style="width:119px;">103-042-201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MACS separator</td>
			<td style="width:147px;"></td>
			<td align="right" style="width:119px;">4124</td>
			<td></td>
		</tr>
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isolation and flow cytometric analysis of human endocervical gamma delta t cells

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 &#39;unconventional&#39; 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&#39;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.

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.
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Watch this Scientific Journal Video about Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells at JoVE.com

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

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 ...

The overall goals of this procedure are to isolate and characterize an unconventional intraepithelial cell, gamma delta T cell population from a human endocervical brush sample. This method can help answer key questions in understanding the immune events within the endocervical intraepithelial compartment, to aid in the design of effective strategies for preventing infections. The main advantage of this technique is it provides a powerful and efficient tool for studying endocervical intraepithelial gamma delta T lymphocytes, in vitro.Demonstrating the procedure will be Laura Romero, a senior research associate from my laboratory. Immediately after collection, place the tube containing the cytobrush on ice. Within one hour of collection, vortex the tube containing the cytobrush for four pulses of 10 seconds.Then, centrifuge the tube, and carefully remove the brush without disturbing the palette. Next, add one milliliter of IMDM medium to the cells, and place the tube on ice. Place the cytobrush on top of a 100 millimeter cell strainer in a 50 milliliter conical tube, and wash the brush with 20 milliliters of fresh IMDM.Centrifuge the wash. Resuspending the palette in one milliliter of fresh IMDM, and pool with wash with the cells in the original cytobrush collection tube. To analyze the endocervical cells by flow cytometry, aliquot one to three times 10 to the fifth cells in 100 microliters of fax buffer per five milliliter tube, and label the cells with the antibodies and viability dye of interest for 30 minutes at four degrees celsius in the dark.At the end of the incubation, wash the cells in one milliliter of the kit buffer. After spinning down the cells, completely decant the supernatant. Use beads and the appropriate IgG control antibodies to set the compensation controls.Then, load the first tube, and set the gate on the total endocervical cell population in a forward by side scatterplot, followed by exclusion of the cell doublets. Exclude the dead cells according to their viability dye expression, gating on the live CD45 positive and CD3 positive cells. The gamma delta one and gamma delta two positive cells are defined as the subpopulations of CD3 positive cells that express gamma delta TCR.To isolate the gamma delta T cells by magnetic bead isolation, resuspend up to one times 10 to the seventh freshly harvested endocervical cells in 40 microliters of TCR gamma delta MicroBead kit buffer. Following the manufacturer's instructions for the two-step staining protocol, label the cells with anti-TCR gamma delta hapten antibody, followed by anti-hapten FITC conjugated MicroBeads for 15 minutes at four to eight degrees celsius. At the end of the incubation, wash the cells in one milliliter of the kit buffer, and completely decant the supernatant.Resuspend the cells in 500 microliters of kit buffer, and apply the cell suspension directly onto a magnetic bead column set in the appropriate separator magnet, collecting the flow-through in a new tube. When all of the cells have run through the column, wash the column three times with 500 microliters of fresh buffer, and pool the washes with the collected cells. To collect the gamma delta T cells, transfer the column into a conical tube, and use the column plunger to flush the T cells from the column with one milliliter of fresh buffer.To isolate the gamma delta T cells by fluorescence activated cell sorting, label the cells with a viability dye, in the antibody panel as just demonstrated. Then, sort the cells according to their viable dye, and CD45, CD3, and gamma delta positivity. A unique population of gamma delta V1 expressing T cells can be readily detected in human endocervical cell samples.Detailed phenotypic analysis of the gated CD3 positive gamma delta one positive cells reveals that the majority of these cells are CD4 and CD8 negative. Magnetic bead separation of the endocervical cells, as just demonstrated, results in the purification of a nearly 98%pure population of these TCR gamma delta cells. This level of purity can also be achieved via fluorescence activated cell sorting, validating the use of either technique for the isolation of endocervical gamma delta T cells.Interestingly, the analysis of endocervical intraepithelial cells, gamma delta T cells, harvested from female HIV infected patients and healthy donor controls, demonstrates a slight reduction in the numbers and viability of these important cells in HIV infected individuals. Once mastered, this technique can be completed in two to three hours if it is performed properly. While attempting this procedure, it's important to remember to quickly process the sample, and to keep the cells on ice throughout the entire procedure, to maintain a good cell viability.Following this procedure, other methods, like single cell sorting combined with a high-sensitivity throughput multiplex PCR, can be performed to answer additional questions about the inflammatory and signaling pathways within the endocervical intraepithelial compartment. After its development, this technique paved the way for the researchers in the field of HIV and infectious disease to explore novel cell markers of mucosal vulnerability. After watching this video, you should have a good understanding of how to prepare intraepithelial lymphocytes from a human endocervical brush sample.

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