We thank E. Massicotte and J. Lord for expert technical assistance during flow cytometry and cell sorting experiments. Also, we would like to thank the IRCM clinic staff and all donors for providing blood samples. Peripheral blood samples were obtained from healthy adult donors who gave written informed consent in accordance with the Declaration of Helsinki under research protocols approved by the research ethics review board of the Institut de Recherches Cliniques de Montr&#233;al (IRCM). The following reagents were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: MT4 cells from Dr. Douglas Richman; and Human rIL-2 from Dr. Maurice Gately, Hoffmann - La Roche Inc.
Dr. Cohen is the recipient of the Canada Research Chair in Human Retrovirology. This work was supported by grants from CIHR (CIHR 111226) to Dr. Cohen and from the Fonds de recherche du Qu&#233;bec-Sant&#233; (FRQ-S) AIDS network to Dr. Bego and Dr. Cohen.

General Notes
It is important to keep cells in culture without any antibiotic, since even controlled bacterial infections can be sensed by PBMCs. Such sensing of bacterial components will induce a background interferon production that cannot be distinguished from that triggered by specific HIV-1 sensing. Furthermore, all cells have to be routinely tested for the absence of mycoplasma contamination.
Always use endotoxin-free solutions whenever possible.
Flow cytometric characterization of the different cells to be used in the co-cultures (PBMCs, pDCs, MT4 and CD4+ T cells) is an optional step, but highly recommended since it can provide valuable information required for proper interpretation of a given experiment.
1. Preparation of Infected Target Cells
<ol>
	<li>Maintain human CD4+ T cell line MT4 in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS), referred from now on as RPMI-1640 complete medium.</li>
	<li>Isolation of primary CD4+ T cells.
	<ol>
		<li>Isolate mononuclear cells from human peripheral blood by density gradient centrifugation.</li>
		<li>Isolate CD4+ T lymphocytes using a CD4+ T cells negative selection kit, following manufacturer&#39;s recommendations.</li>
		<li>Activate CD4+ T lymphocytes with PHA-L (5 &#181;g/ml) for 48 hr and then maintain cells in RPMI-1640 complete medium supplemented with recombinant IL-2 (100 U/ml). Infect primary T cells 5 days post isolation.</li>
	</ol>
	</li>
	<li>Infection of target cells (MT4 T cell line or heterologous primary CD4+ T cells).
	<ol>
		<li>Two days prior to co-culture, infect T cells with HIV-1 virus. In the specific example described here pNL4.3-GFP_ires_Nef wild type (WT) or pNL4.3-GFP_ires_Nef delta Vpu (dVpu) viruses were used. These viral strains represent green fluorescent protein (GFP)-marked isogenic infectious molecular clones that differ only in their ability to express Vpu.</li>
		<li>Use different multiplicities of infection (MOIs) and select cultures with similar levels of infection for co-cultures. The day of the co-culture, determine the percentage of GFP+ cells by flow cytometry as a marker of infection. Use only cultures with a range of 20-50% infected cells for co-cultures.</li>
	</ol>
	</li>
</ol>
Optional steps: Perform flow cytometric staining at this point to evaluate surface expression of molecules/ligands of interest such as BST2, and CD4. For cell surface staining, resuspend 0.5 x 106 T cells in 100 &#181;l of wash solution, add 1 &#181;l of anti-CD4_PerCP/Cy5.5 and 1 &#181;l of anti-BST2_A660. Incubate tubes for 45 min at 4&#176;C, before washing with flow cytometry wash solution. If the studies are aimed at evaluating the role of Vpu-mediated BST2 antagonism, perform a HIV-1 particle release assay at this time as previously described 15.
2. Isolation and Enrichment of Sensing Cells (Whole PBMCs or Enriched pDCs)
Note: When PBMCs are used for sensing, start isolation within half hour after blood collection and conduct it in a timely manner. Use PBMCs immediately for co-cultures or pDC enrichment.
<ol>
	<li>Isolate mononuclear cells from human peripheral blood by density gradient centrifugation. 
	Optional step: Perform multi-parametric flow cytometry staining to confirm that freshly isolated PBMCs contain the different cell lineages at physiological ratios. For cell surface staining, resuspend 106 PBMCs in 100 &#181;l of Fc blocking solution, and incubate for 15 min at 4 &#176;C. After blocking, add 1 &#181;l of anti-CD3_Pacific Blue (for T cells), 1 &#181;l of anti-CD14-PE_Texas Red (for myeloid cells), 4 &#181;l of anti-BDCA2_APC and 1 &#181;l of anti-ILT7_PE (for pDCs). Incubate tubes for 45 min at 4 &#176;C, before washing with flow cytometry wash solution.</li>
	<li>Enrich pDCs using a pDC negative selection kit, following manufacturer&#39;s recommendations. It is recommended to work fast, keep cells cold, and use pre-cooled solutions. This will prevent the capping of antibodies on the cell surface and non-specific cell labeling as well as ensuring maximal pDC activity.
	<ol>
		<li>Perform multi-parametric flow cytometric analysis of the freshly isolated pDCs to confirm purity, percentage of enrichment, and relative amounts of pDC- specific surface markers (such as ILT7 and BDCA2). For cell surface staining, resuspend 104 pDCs in 100 &#181;l of Fc blocking solution, and incubate for 15 min at 4 &#176;C. After blocking, add 1 &#181;l of anti-CD3_Pacific Blue, 1 &#181;l of anti-CD14_PE_Texas Red, 4 &#181;l of anti-BDCA2_APC and 1 &#181;l of anti-ILT7_PE. Incubate tubes for 45 min at 4 &#176;C, before washing with flow cytometry wash solution (PBS, 5 mM EDTA, 5% FBS). A minimum of 40% pDC purity is recommended (if the starting material has 0.4% pDC, this would be equivalent to a 100-fold enrichment).</li>
	</ol>
	</li>
</ol>
3. Co-culture of Infected CD4+ T Cells and Sensing Cells (Whole PBMCs or Enriched pDCs)
<ol>
	<li>Mix 220 &#181;l of PBMCs (diluted at 850,000 cells/ml) or 220 &#181;l of pDCs (at a concentration ranging from 100,000 to 500,000 cells/ml) with 30 &#181;l of infected T cells (diluted at 106 cells/ml) per well in a U-bottom 96 well plate.
	<ol>
		<li>As controls, plate PBMCs or pDCs and T cells alone. Adjust volume in well to 250 &#181;l with RPMI 1640 complete medium.</li>
		<li>Assess cellular responses to known agonist of the TLR7 and TLR9 pathways. To this end, plate 220 &#181;l of PBMCs (diluted at 850,000 cells/ml) or 200 &#181;l of pDCs (at a concentration ranging from 100,000 to 500,000 cells/ml) per well in a U-bottom 96 well plate and add TLR7 agonist (Imiquimod, final concentration: 2.5 &#181;g/ml) or TLR9 agonist (ODN 2216 CpG-A, final concentration: 5 &#181;M). Incubate cells at 37 &#176;C and 5% CO2 for 18-22 hr and process them in a manner similar to the co-cultures described below.</li>
	</ol>
	</li>
	<li>Incubate co-cultures at 37 &#176;C and 5% CO2 for 18-22 hr, and then transfer them to a V-bottom 96 well plate. Centrifuge for 5 min at 400 x g.
	<ol>
		<li>Transfer supernatants to a flat-bottom 96 well plate. Supernatants can be stored at -80 &#176;C or used for type-I IFN detection immediately.</li>
		<li>Resuspend co-cultured cells in 2% paraformaldehyde and analyze them using flow cytometry. Due to differences in size and granulation, MT4 cells are easily distinguished from PBMCs or pDCs based on their side-scatter (SS) versus forward-scatter (FS) profiles. If primary T cells are used as target infected cells, these can be stained with any cell tracker dye, such as CSFE, prior to co-culture.</li>
	</ol>
	</li>
	<li>Measurement of bioactive type-I IFN using HEK-Blue IFN-&#945;/&#946; reporter cells. 
	Note: These cells were generated by stable transfection of HEK293 cells with the human STAT2 and IRF9 genes to obtain a fully active type I IFN signaling pathway. They also contain the secreted alkaline phosphatase (SEAP) reporter gene under the control of the IFN-&#945;/&#946; inducible ISG54 promoter. Stimulation of these cells with type-I IFN activates the JAK/STAT/ISGF3 pathway and induces the production of SEAP.
	<ol>
		<li>Maintain HEK-Blue IFN-&#945;/&#946; cells in DMEM supplemented with 10% FBS. Plate them at a density of 50,000 cells per well in a flat-bottom 96 well plate, in a final volume of 180 &#181;l.</li>
		<li>Add 20 &#181;l of co-culture supernatant to each well in duplicate. Each plate also requires a set of internal standard controls (human IFN&#945;, final concentration from 100 U/ml to 2,500 U/ml). Incubate the plates at 37 &#176;C and 5% CO2 for 18-22 hr.</li>
		<li>Determine levels of alkaline phosphatase activity using QUANTI-Blue solution, which changes from pink to purple/blue in the presence of the enzyme. Prepare QUANTI-Blue following manufacturer&#39;s recommendations. Add 180 &#181;l of this solution to each well of a flat-bottom 96 well plate. To each well also add 20 &#181;l of the induced HEK-Blue IFN-&#945;/&#946; cells supernatants, and incubate the plate in a 37 &#176;C incubator until color develops in the standard IFN control wells.</li>
		<li>Evaluate levels of SEAP using a spectrophotometer at 620-655 nm and determine the concentration of type-I IFN by extrapolation from the linear part of the IFN standard curve.</li>
	</ol>
	</li>
</ol>

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S., 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=Endocytosis+of+HIV-1+activates+plasmacytoid+dendritic+cells+via+Toll-like+receptor-viral+RNA+interactions.">Endocytosis of HIV-1 activates plasmacytoid dendritic cells via Toll-like receptor-viral RNA interactions.</a> The Journal of clinical investigation. 115, 3265-3275 (2005).</li><li>Martinelli, E., 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=HIV-1+gp120+inhibits+TLR9-mediated+activation+and+IFN-{alpha}+secretion+in+plasmacytoid+dendritic+cells.">HIV-1 gp120 inhibits TLR9-mediated activation and IFN-{alpha} secretion in plasmacytoid dendritic cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 3396-3401 (2007).</li><li>Cao, W., 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=Regulation+of+TLR7/9+responses+in+plasmacytoid+dendritic+cells+by+BST2+and+ILT7+receptor+interaction.">Regulation of TLR7/9 responses in plasmacytoid dendritic cells by BST2 and ILT7 receptor interaction.</a> The Journal of experimental medicine. 206, 1603-1614 (2009).</li><li>Bego, M. 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The protocols described here measure sensing of T cells infected with GFP-tagged HIV-1 viruses that differ only in their ability to express Vpu, as described in our recent report 18. However, these methods could easily be modified to study sensing of untagged viruses as well as viruses lacking other viral genes that could potentially modulate innate sensing. If the viruses used do not express GFP, the percentage of infected target cells should be determined by another means prior to co-culture, such as p24 (capsid) intracellular staining and flow cytometry or by p24 ELISA. Furthermore, these protocols can be used with different target T cells, including a variety of available cell lines and primary cells.
The methods described in this manuscript have a number of advantages when compared to early methodologies used to study HIV-1 sensing. First and foremost it has been established by a number of groups that cell-free HIV-1 particles are poor inducers of type-I IFN 2,13,14. Furthermore, early studies relied on older IFN&#945; ELISA-based methods to quantify the amount of type-I IFN produced. A limitation of this detection technique is that most of the older IFN&#945; ELISA kits only measure one type of IFN&#945;, while overlooking the other types of IFN&#945; and IFN&#946;. The use of the described reporter cell lines overcomes this limitation as the concentration of all bioactive forms of human type-I IFNs can be measured at once. The technique is highly sensitive, less expensive than the new generation of IFN ELISA kit and for many donors large amounts of type-I IFN can easily be detected. Nevertheless, this protocol can be easily adapted for use with other type-I IFN read-out methods such as ELISAs.
Critical steps: It is important that the quality of all cellular components (both targets and effectors) be tightly controlled. Potential contamination, even when asymptomatic, needs to be monitored and controlled. As we previously mentioned, antibiotic-controlled asymptomatic bacterial contamination, and potentially other intracellular pathogens such as mycoplasma, can generate similar immune responses to the ones observed after HIV-1 infection, namely production of type-I IFN. Moreover, all reagents need to be free of LPS or other potential endotoxins. Similarly, monitoring of PBMCs and pDCs is also important since human samples can often be primed by underlying ongoing infections, which may affect normal sensing. We recommend always including the proposed negative controls, such absence of target cells as well as co-cultures with mock-infected cells. Viability of infected cells is also important for proper pDC sensing since type-I IFN is not produced following co-culture with apoptotic cells 2,14.
One important limitation of the technique is the need for freshly isolated primary cells. This procedure was not tested using PBMCs or pDCs that were either previously frozen or kept in culture. The isolation of PBMCs is labor intensive and these cells have a very limited lifespan. Furthermore, standard protocols for protecting against blood-borne pathogen should be used while working with human blood. There are often several sources of variability associated with work involving freshly isolated human cells. Among them are the different procedures implemented to isolate these cells, and the inherited variability encountered from donor-to-donor. While it is impossible to avoid variability among donor, the use of the suggested positive controls, such as measuring response to known agonists of TLR7 and TLR9 pathways, would provide an important internal control for these experiments. We observed that a robust response to agonists of the TLR9 pathway could be correlated with a healthy pDC response, while a responsive TLR7 pathway is required for a proper response against HIV-1 3.
Based on observations made by us and others 2, enrichment of pDCs from PBMCs is often not needed. However, if the experimental design requires it (for instance in the context where depletion of specific pDC surface markers is needed) negative selection is preferable over positive selection, as cells remain free of antibody after the selection process. Isolated pDCs undergo rapid apoptosis in culture. For experiments that require these cells to be cultured for extended period of time, culture media can be supplemented with IL-3. This cytokine induces pDC proliferation and inhibits their apoptosis 16. However, IL-3 usage needs to be tightly controlled since it may lead to pDC maturation or differentiation.
The method described here combines target HIV-1 infected cells with freshly isolated PBMCs or pDCs in order to recreate the initial steps involved during innate sensing. This method will undoubtedly be useful to explore early innate immune events associated with HIV infection. Although the associated protocols are aimed at measuring type-I IFN, they could easily be modified to measure other bioactive molecules released from pDCs upon recognition of infected cells. These could include: type-III IFN (IFN-&#955;), pro-inflammatory cytokines (such as TNF-&#945; and IL-6) and the chemokines CXCL10, CCL4, and CCL5 17.

Type-I IFN-producing pDCs represent the first line of defense against viral infections, and as such serve as a vital link between innate and adaptive immunity 1. While cell-free HIV-1 particles are poorly detected by the innate immune system, HIV-1-infected CD4+ T cells are effectively sensed by pDCs 2. The sensing mechanism requires an initial contact between the viral envelope glycoprotein (gp120) at the surface of the infected T cell and CD4 molecules on the pDC, which is then followed by virus capture and internalization by the pDC. Subsequent recognition of transferred HIV-1 RNA by TLR7 triggers the activation of the Myd88/IRF7 pathway and ultimately leads to type-I IFN production 3. Importantly, the second sensing pathway present in pDCs, which is mediated by TLR9, is inhibited by the interaction of the viral glycoprotein, gp120, with the pDC-specific surface marker BDCA2 4.
The TLR7 and TLR9 sensing pathways of pDC can potentially be negatively modulated by the engagement of BST2 (also designated Tetherin or CD317) with another pDC-specific surface inhibitory receptor called ILT7 5. BST2 is expressed at high levels at the surface of pDCs and some cancer cells, but at relatively lower levels in other cell types, such as B cells, macrophages, and T cells. Importantly, BST2 can be induced by type-I IFN in a number of transformed cell lines as well as in primary cell cultures of human and murine origins 6. Apart from its immune-regulatory function, BST2 was recently found to inhibit the release of HIV-1 and other enveloped viral particles by cross-linking nascent virus particles onto the infected cell surface 7. In the case of HIV-1, the virus-encoded accessory protein U (Vpu) was shown to act as a BST2 antagonist. It is believed that Vpu downregulates BST2 from the cell surface of HIV-1-infected cells, the site of its tethering activity and as a result enhances viral release and spread 7, although this notion has been challenged 8,9. In the absence of Vpu, a large number of fully formed and mature progeny HIV-1 particles are retained at the cell surface. Whether these tethered particles could represent effective targets for immune sensing still remains an open question. Additionally, it is unclear whether Vpu could dysregulate type-I IFN production from pDCs by modulating surface BST2 levels.
Early studies designed to assess HIV-1 sensing were conducted using cell-free HIV-1 particles, which are generally considered poor inducers of type-I IFN 3,10-12. Given that recent studies suggest that PBMCs and pDCs efficiently sense HIV-infected CD4+ T lymphocytes 2,13,14, we describe here a simple method to measure innate responses triggered by pDCs upon recognition of HIV-1-infected cells in vitro.

<table border="0" cellpadding="0" cellspacing="0" width="652">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:203px;">MT4 cells</td>
			<td style="width:87px;">NIH AIDS Reagent Program</td>
			<td style="width:103px;">120</td>
			<td style="width:260px;">This reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: MT-4 from Dr. Douglas Richman.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEK-Blu IFN-&#945;/&#946; reporter cells </td>
			<td style="width:87px;">Invivogen</td>
			<td style="width:103px;">HKB-IFNAB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">RPMI</td>
			<td style="width:87px;">Wisent</td>
			<td style="width:103px;">350-000-CL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">DMEM</td>
			<td style="width:87px;">Wisent</td>
			<td style="width:103px;">319-005-CL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">FBS</td>
			<td style="width:87px;">Wisent</td>
			<td style="width:103px;">080-150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Ficoll-P&#226;que Plus</td>
			<td style="width:87px;">Myltenyi</td>
			<td style="width:103px;">17-1440-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">CD4+ T cell isolation kit II</td>
			<td style="width:87px;">Myltenyi</td>
			<td style="width:103px;">130-091-155</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">Diamond Plasmacytoid Dendritic Cell Isolation Kit II </td>
			<td style="width:87px;">Myltenyi</td>
			<td style="width:103px;">130-097-240</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">PHA-L</td>
			<td style="width:87px;">Sigma-Aldrich</td>
			<td style="width:103px;">O2769</td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:203px;">Human rIL-2</td>
			<td style="width:87px;">NIH AIDS Reagent Program</td>
			<td style="width:103px;">136</td>
			<td style="width:260px;">This reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: Human rIL-2 from Dr. Maurice Gately, Hoffmann - La Roche Inc.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Imiquimod</td>
			<td style="width:87px;">Invivogen</td>
			<td style="width:103px;">TLRL-IMQS</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">ODN 2216 CpG-A</td>
			<td style="width:87px;">Hycult Biotec</td>
			<td style="width:103px;">HC4037</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:203px;">Human IFN&#945;</td>
			<td style="width:87px;">PBL Interferon source</td>
			<td style="width:103px;">11100-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QUANTI-Blue </td>
			<td style="width:87px;">Cedarlane</td>
			<td style="width:103px;">REP-QB2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;"> </td>
			<td style="width:87px;"> </td>
			<td style="width:103px;"> </td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:203px;">Flow cytometry (Antibodies/reagents)</td>
			<td style="width:87px;"> </td>
			<td style="width:103px;"> </td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">Fc blocking solution (composition below):</td>
			<td style="width:87px;"> </td>
			<td style="width:103px;"> </td>
			<td style="width:260px;">Note: make in washing solution (PBS, 5mM EDTA, 5% FBS)</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:203px;">10% Goat serum</td>
			<td style="width:87px;">Sigma-Aldrich</td>
			<td style="width:103px;">G 9023</td>
			<td style="width:260px;"> </td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:203px;">10% Rabbit serum</td>
			<td style="width:87px;">Sigma-Aldrich</td>
			<td style="width:103px;">R 9133</td>
			<td style="width:260px;"> </td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:203px;">10% Mouse serum</td>
			<td style="width:87px;">Sigma-Aldrich</td>
			<td style="width:103px;">M 5909</td>
			<td style="width:260px;"> </td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:203px;">2.5 mg/ml human IgG</td>
			<td style="width:87px;">Sigma-Aldrich</td>
			<td style="width:103px;">I 4506</td>
			<td style="width:260px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">anti-CD3_Pacific Blue</td>
			<td style="width:87px;">Biolegend</td>
			<td style="width:103px;">300417</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">anti-CD14_PE-TxRed</td>
			<td style="width:87px;">Life Technologie</td>
			<td style="width:103px;">MHCD1417</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">anti-BDCA2_APC</td>
			<td style="width:87px;">Myltenyi</td>
			<td style="width:103px;">130-090-905</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">anti-ILT7_PE</td>
			<td style="width:87px;">Biolegend</td>
			<td style="width:103px;">326408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">anti-CD4_PerCP/Cy5.5</td>
			<td style="width:87px;">Biolegend</td>
			<td style="width:103px;">317427</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">anti-BST2_Alexa 660</td>
			<td style="width:87px;">eBioscience</td>
			<td style="width:103px;">50-3179</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:203px;">Paraformaldehyde </td>
			<td style="width:87px;">Sigma-Aldrich</td>
			<td style="width:103px;">P 6148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Cell strainer</td>
			<td style="width:87px;">BD Falcon </td>
			<td style="width:103px;">352340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;"> </td>
			<td style="width:87px;"> </td>
			<td style="width:103px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Equipment</td>
			<td style="width:87px;"> </td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Cyan ADP instrument </td>
			<td style="width:87px;">Beckman Coulter</td>
			<td style="width:103px;">CY20030</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing the innate sensing of hiv-1 infected cd4+ t cells by plasmacytoid dendritic cells using an ex vivo co-culture system.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

The co-culture system described in Figure 1 allows for a controlled study of HIV-1 innate sensing. Due to the variable nature of primary cells, it is important that normal ratios of myeloid and T cells (CD14+ and CD3+ respectively) are observed, such as in the example shown in Figure 2A. Furthermore, only a low number of activated T cells (higher FS and higher CD3 levels as compared to non-activated T cells) are desirable in the freshly isolated PBMCs cultures. PBMCs with abnormal ratios between the various cell-types are often incapable of mounting a proper innate immune response in vitro, likely due to an already activated phenotype as a consequence of an ongoing infection. Most importantly, the relative percentage of pDCs needs to be evaluated as shown in Figure 2B. Although this percentage can vary from 0.2 to 1.2%, an abnormal sensing phenotype is also observed with both extremes of this range. Enrichment of pDCs using negative selection often yields 50-95% pDC purity, as seen in the two examples from in Figure 2B (92% and 72%). A prototypical example for the overall experiment is shown in Figures 3 and 4. In this example, MT4 cells were infected with pNL4.3-GFP_ires_Nef WT or delta Vpu to achieve 30% infection at the time of co-culture (Figure 3A). As previously described, only the infections with WT virus resulted in a significant down-regulation of surface BST2 (Figure 3B). Due to their differences in size and granularity, MT4 cells can be easily distinguished from PBMCs by flow cytometry (Figure 4A). After the co-culture incubation, only PBMCs in contact with known TLR7 or TLR9 agonist and PBMCs in contact with HIV-1 infected cells released significant amounts of type-I IFN (Figure 4B). No IFN was detected in PBMCs alone, in PBMCs co-cultured with mock-infected MT4 cells, or in infected MT4 cells alone (Figure 4B). In the example presented here, innate sensing of HIV-1-infected MT4 cells by PBMCs was found to be significantly reduced in the presence of Vpu.
<img alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/51207/51207fig1highres.jpg" src="/files/ftp_upload/51207/51207fig1.jpg" width="500px" /> 
Figure 1: Schematic overview of the experimental design. SUP: supernatant <a href="https://www.jove.com/files/ftp_upload/51207/51207fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/51207/51207fig2highres.jpg" src="/files/ftp_upload/51207/51207fig2.jpg" width="500px" /> 
Figure 2: Phenotypic characterization of freshly isolated PBMCs and enriched pDCs. (A) Normal distribution of myeloid cells and T cells observed in PBMCs isolated from a healthy donor (Percentage of myeloid cells should range between 10-20% and T cells between 55-65%). PBMCs sample were stained using fluorophore-conjugated antibodies that recognize surface CD14 (myeloid lineage marker) and CD3 (T cell marker). (B) Phenotypic characterization of pDCs. Prior to enrichment pDCs represent between 0.2-1.2% of the total number of cells within the PBMCs (0.99% in the example shown). These cells can be identified based on expression of specific markers, such as BDCA2 and ILT7, present at the cell surface. Negative selection can be used to significantly enrich the number of pDCs and eliminate potentially detrimental contaminations, such as CD14+ myeloid cells (enrichment reaching 92% and 72% in the two examples shown). <a href="https://www.jove.com/files/ftp_upload/51207/51207fig2highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 3" fo:content-width="6in" fo:src="/files/ftp_upload/51207/51207fig3highres.jpg" src="/files/ftp_upload/51207/51207fig3.jpg" width="500px" /> 
Figure 3: Characterization of HIV-1-infected target MT4 cells. (A) Percentage of infected cells within the MT4 cultures infected with pNL4.3-GFP_ires_Nef WT or dVpu as determined by the percentage of GFP positive cells. (B) BST2 cell surface expression in the infected cells was evaluated after surface staining. The grey filled histograms represent mock-infected cells stained with the pre-immune rabbit serum (unstained control); the remaining histograms represent cells stained with anti-BST2 polyclonal rabbit serum. Histograms with solid lines represent control GFP negative (neg) cells; while histograms with dotted lines correspond to infected GFP positive (pos) cells. Mean fluorescence intensity (MFI) values are indicated for each sample. Flow cytometry was performed and analyzed as described in Figure 2. <a href="https://www.jove.com/files/ftp_upload/51207/51207fig3highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 4" fo:content-width="6in" fo:src="/files/ftp_upload/51207/51207fig4highres.jpg" src="/files/ftp_upload/51207/51207fig4.jpg" width="500px" /> 
Figure 4: Co culture of freshly isolated PBMCs and infected MT4 T cells. (A) Comparison of flow cytometry FS/SS profiles observed for MT4 T cells alone, PBMCs alone, or co-cultures between PBMCs and MT4 T cells. Due to differences in size and granularity, MT4 T cells can be easily distinguished from PBMCs in co-cultures using flow cytometry. (B) Representative example of the amount of type-I IFN released after stimulation of PBMCs with TLR7 or TLR9 agonist (ago), or after co-cultures with mock or the indicated MT4 cells infected with pNL4.3-GFP_ires_Nef WT or dVpu. Raw data shown as OD650 values and their corresponding converseion to type-I IFN concentration expressed in U/ml. <a href="https://www.jove.com/files/ftp_upload/51207/51207fig4highres.jpg" target="_blank">Click here to view larger image</a>.

Watch this Scientific Journal Video about Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System. at JoVE.com

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

assessing-innate-sensing-hiv-1-infected-cd4-t-cells-plasmacytoid

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JoVE Journal

Immunology and Infection

8.6K 조회수.  Institut de Recherches Cliniques de Montréal (IRCM).  무 세포 HIV-1 입자와는 달리, 감염된 CD4 + T 세포를 형질 효과적으로 수지상 세포 (PDCS)에 의해 감지된다. 이 원고는 타입 I IFN의 방출에 의해 평가로서 말초 혈액 단핵 세포 (PBMC) 또는 격리 PDCS는 PDCS 의해 선천성 센싱을 평가하는 HIV-1 감염 T 세포와 공동 배양 방법을 설명한다. 

비디오: Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer&#039;s Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during inflammatory responses. Unfortunately, study of pDC function is hindered by their low frequency in lymphoid organs, and existing methods for in vitro DC generation predominantly favor the production of cDCs over pDCs. Here we present a unique approach to efficiently generate pDCs from common lymphoid progenitors (CLPs) in vitro. Specifically, the protocol described details how to purify CLPs from bone marrow and generate pDCs by coculturing with &#947;-irradiated AC-6 feeder cells in the presence of Flt3 ligand. A unique characteristic of this culture system is that the CLPs migrate underneath the AC-6 cells and become cobblestone area-forming cells, a critical step for expanding pDCs. Morphologically distinct DCs, namely pDCs and cDCs, were generated after approximately 2 weeks with a composition of 70-90% pDCs under optimal conditions. Typically, the number of pDCs generated by this method is roughly 100-fold of the number of CLPs seeded. Therefore, this is a novel system with which to robustly generate the large numbers of pDCs required to facilitate further studies into the development and function of these cells.

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand.

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during ...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&amp;#948;1+CD4+&amp;#947;&amp;#948; T Cells, an Extrathymic &amp;#945;&amp;#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can characterize thousands of proteins at a time. These powerful techniques allow us to have a systemic understanding of cellular changes, especially when cells are subjected to various stimuli, such as infections, stresses, and specific test conditions. Even with recent developments, analyzing the exosomal proteome is time-consuming and often involves complex methodologies. In addition, the resultant large dataset often needs robust and streamlined analysis in order for researchers to perform further downstream studies. Here, we describe a SILAC-based protocol for characterizing the exosomal proteome when cells are infected with HIV-1. The method is based on simple isotope labeling, isolation of exosomes from differentially labeled cells, and mass spectrometry analysis. This is followed by detailed data mining and bioinformatics analysis of the proteomic hits. The resultant datasets and candidates are easy to understand and often offer a wealth of information that is useful for downstream analysis. This protocol is applicable to other subcellular compartments and a wide range of test conditions.

Here, we describe a quantitative proteomics method using the technique of stable isotope labeling by amino acids in cell culture (SILAC) to analyze the effects of HIV-1 infection on host exosomal proteomes. This protocol can be easily adapted to cells under different stress or infection conditions.

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

&lt;em&gt;In Vitro&lt;/em&gt; Differentiation of Human CD4&lt;sup&gt;+&lt;/sup&gt;FOXP3&lt;sup&gt;+&lt;/sup&gt; Induced Regulatory T Cells (iTregs) from Na&amp;#239;ve CD4&lt;sup&gt;+&lt;/sup&gt; T Cells Using a TGF-&amp;#946;-containing Protocol

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...