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 ...

<ol>
	<li>Kaul, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genital+tract+immune+milieu:+an+important+determinant+of+HIV+susceptibility+and+secondary+transmission.">The genital tract immune milieu: an important determinant of HIV susceptibility and secondary transmission.</a> J Reprod Immunol. 77, 32-40 (2008).</li><li>Haase, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+early+infection+to+prevent+HIV-1+mucosal+transmission.">Targeting early infection to prevent HIV-1 mucosal transmission.</a> Nature. 464, 217-223 (2010).</li><li>Hayday, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gamma][delta]+cells:+a+right+time+and+a+right+place+for+a+conserved+third+way+of+protection.">gamma][delta] cells: a right time and a right place for a conserved third way of protection.</a> Ann Rev Immunol. 18, 975-1026 (2000).</li><li>Nakasone, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accumulation+of+gamma/delta+T+cells+in+the+lungs+and+their+roles+in+neutrophil-mediated+host+defense+against+pneumococcal+infection.">Accumulation of gamma/delta T cells in the lungs and their roles in neutrophil-mediated host defense against pneumococcal infection.</a> Microbes and infection / Institut Pasteur. 9, 251-258 (2007).</li><li>Autran, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor+gamma/delta++lymphocyte+subsets+during+HIV+infection.">T cell receptor gamma/delta+ lymphocyte subsets during HIV infection.</a> Clin Exp Immunol. 75, 206-210 (1989).</li><li>Pauza, C. D., Poonia, B., Li, H., Cairo, C., Chaudhry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gammadelta+T+Cells+in+HIV+Disease:+Past,+Present,+and+Future.">gammadelta T Cells in HIV Disease: Past, Present, and Future.</a> Front Immunol. 5, 687 (2014).</li><li>Hayday, A. C., Spencer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barrier+immunity.">Barrier immunity.</a> Sem Immunol. 21, 99-100 (2009).</li><li>Cheroutre, H., Lambolez, F., Mucida, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+light+and+dark+sides+of+intestinal+intraepithelial+lymphocytes.">The light and dark sides of intestinal intraepithelial lymphocytes.</a> Nature Rev Immunol. 11, 445-456 (2011).</li><li>Strbo, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+Intra-Epithelial+Endocervical+Gamma+Delta+(GD)+1+T+Cells+in+HIV-Infected+Women.">Loss of Intra-Epithelial Endocervical Gamma Delta (GD) 1 T Cells in HIV-Infected Women.</a> AJRI. 75, 134-145 (2016).</li><li>Szabo, S. E., Monroe, S. L., Fiorino, S., Bitzan, J., Loper, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+an+automated+instrument+for+viability+and+concentration+measurements+of+cryopreserved+hematopoietic+cells.">Evaluation of an automated instrument for viability and concentration measurements of cryopreserved hematopoietic cells.</a> Lab Hematol. 10, 109-111 (2004).</li><li>Alcaide, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+Vaginosis+Is+Associated+with+Loss+of+Gamma+Delta+T+Cells+in+the+Female+Reproductive+Tract+in+Women+in+the+Miami+Women+Interagency+HIV+Study+(WIHS):+A+Cross+Sectional+Study.">Bacterial Vaginosis Is Associated with Loss of Gamma Delta T Cells in the Female Reproductive Tract in Women in the Miami Women Interagency HIV Study (WIHS): A Cross Sectional Study.</a> PloS one. 11, e0153045 (2016).</li><li>Masson, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+genital+tract+cytokine+signatures+of+sexually+transmitted+infections+and+bacterial+vaginosis+in+women+at+high+risk+of+HIV+infection:+a+cross-sectional+study.">Defining genital tract cytokine signatures of sexually transmitted infections and bacterial vaginosis in women at high risk of HIV infection: a cross-sectional study.</a> Sex Transm Dis. 90, 580-587 (2014).</li><li>Fichorova, R. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin+(IL)-1,+IL-6,+and+IL-8+predict+mucosal+toxicity+of+vaginal+microbicidal+contraceptives.">Interleukin (IL)-1, IL-6, and IL-8 predict mucosal toxicity of vaginal microbicidal contraceptives.</a> Biol Reprod. 71, 761-769 (2004).</li><li>Juno, J. A., Boily-Larouche, G., Lajoie, J., Fowke, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collection,+isolation,+and+flow+cytometric+analysis+of+human+endocervical+samples.">Collection, isolation, and flow cytometric analysis of human endocervical samples.</a> JoVE. , (2014).</li><li>McKinnon, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+viable+leukocyte+sampling+from+the+female+genital+tract+for+clinical+trials:+an+international+multi-site+study.">Optimizing viable leukocyte sampling from the female genital tract for clinical trials: an international multi-site study.</a> PloS one. 9, e85675 (2014).</li><li>Dominguez, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+multiplexed+quantitation+of+gene+expression+on+single+cells.">Highly multiplexed quantitation of gene expression on single cells.</a> J Immunol Methods. 391, 133-145 (2013).</li></ol>

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.

<table border="0" cellpadding="0" cellspacing="0" width="648">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cytobrush Plus GT&#160;</td>
			<td style="width:147px;">(CareFusion, San Diego, CA, USA</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</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>
		</tr>
		<tr height="63">
			<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>
		</tr>
		<tr height="21">
			<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>
	</tbody>
</table>

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.
<p class="jove_content" fo:keep-togeth...

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 ...

Research

JoVE Journal

Immunology and Infection

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...