The authors would like to acknowledge their funders: the Canadian Institutes of Health Research (CIHR) (grant #153082 to CC, MAJ, NC); the R&#233;seau SIDA et maladies infectieuses du Fonds de recherche du Qu&#233;bec-Sant&#233; (FRQ-S) who granted funding to CC and MAJ and the McGill University Faculty of Medicine who granted funding to CC. This study was also supported in part the Canadian Institutes of Health Research (CIHR)- funded Canadian HIV Cure Enterprise (CanCURE) Team Grant HB2 &#8211; 164064 to MAJ, CC and NC. MAJ holds the CIHR Canada Research Chair tier 2 in Immunovirology and CC and NC hold an FRQ-S Junior 1 and Junior 2 research salary award, respectively. ET holds an RI-MUHC Studentship MSc award.
In addition, the authors would like to acknowledge Jos&#233;e Girouard and all clinical staff involved in coordinating and obtaining the samples, as well as the respiratory therapists; Ekaterina Iourtchenko, H&#233;l&#232;ne Pag&#233;-Veillette, and Marie-H&#233;l&#232;ne Lacombe at the RI- MUHC Immunophenotyping platform; and Dr. Marianna Orlova for provision of the microscopy photos. Most importantly, the authors wish to thank the many volunteers without whom this research would not be possible.&#160;

<ol>
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The authors have nothing to disclose.

Herein we described a method for processing BAL fluid to obtain CD4 T cells and AMs, alongside matched PBMCs, which can be studied to investigate the HIV reservoir within the lungs. We recently reported on HIV DNA quantification in CD4 T cells from matched peripheral blood and BAL samples, and our group demonstrated that HIV is 13 times more abundant in pulmonary CD4 T cells than in those from peripheral blood15. However, the levels of HIV DNA in AMs are donor-dependent and so, thus far, there has not been a consistent correlation between HIV DNA levels in the lymphocytes compared to macrophages15. The access to these primary macrophage cell subsets, however, will be a vital tool to interrogating this question and gaining a better understanding of the viral load in the lung in the context of the HIV reservoir.
In the pre-ART era and in several other studies utilizing BAL fluid, participants underwent bronchoscopy in order to diagnose a suspected pathology or obtain a microbiological diagnosis for respiratory symptoms3. However, we were able to recruit participants without any active pulmonary symptoms or pathologies and all participants signed an ethical consent form15. We were able to recruit participants from our center who were participating in other studies, such as a spirometry screening study for obstructive lung disease24, as well as those undergoing other research procedures, such as leukapheresis and colonoscopy. Previous research amongst people living with HIV demonstrated that altruism is a key factor motivating participation in research studies25. Like with many human specimens, we noted a great deal of person-to-person variability. There was no way to &#34;predict&#34; from which participants we would obtain BAL with good versus poor cell yields. Unlike peripheral blood, which yields fairly consistent numbers of lymphocytes, the cell numbers in BAL fluid are very variable. Injecting a greater volume of normal saline into the lungs (with the hopes of obtaining a greater return of BAL fluid) is not always possible as larger volumes of normal saline are often associated with more coughing and a higher risk of fever postbronchoscopy. We noticed that using a smaller (rather than larger) diameter bronchoscope enabled the respirologist to reach deeper into the bronchi and obtain fluid containing greater quantities of cells. A consistent finding was that tobacco smokers had much larger proportions of AMs than lymphocytes within their BAL fluid, which is expected as AMs engulf debris and particulate matter. Furthermore, we observed that BAL fluid from smokers contained debris which may block the equipment used, such as PCR machines and flow cytometers. Similar issues may be observed in areas of high pollution or individuals exposed more frequently to poor air quality.
With regard to their role in the establishment of HIV reservoirs and viral persistence, the purity of CD4 T cells and AMs is a key consideration. For this reason, we opted to use fluorescence-activated cell sorting (FACS) to obtain highly pure cell populations. It is also possible that the collected BAL fluid may be contaminated with blood as some minor bleeding is expected during a bronchoscopy; the presence of naive B cells would indicate this, and cells can be washed in a red blood cell lysis buffer to circumvent this problem. Another challenge with studying BAL fluid relates to quantifying inflammatory markers and cytokines, which are important for understanding HIV persistence26. As the instilled saline dilutes the BAL fluid, levels of inflammatory mediators and cytokines may be difficult to measure. Although a urea correction factor has been proposed to account for dilution, there is relatively little literature describing its use27,28.
AMs are highly autofluorescent, which poses a problem during cell sorting and flow cytometry phenotypic analysis. In particular, the effect is more pronounced in smokers whose AMs may be completely black in color, significantly affecting their autofluorescence. When excited by a standard blue 488 nm laser, the AM autofluorescence is at its peak at approximately 540 nm, which overlaps with the fluorescence spectra of commonly used conjugates such as FITC and PE29,30. It is worth noting that two separate lasers can be used to excite FITC and PE (e.g., PE by the yellow/green and FITC by the blue 488 laser). To overcome the inherent autofluorescence with FITC, we used unstained AMs to determine the autofluorescence background. In addition, the use of fluorescence minus one (FMO) controls can be very useful to combat these technical issues. Larger beads (e.g., 7.5 &#181;m) can be used, which are closer in size for compensating macrophage populations, compared to smaller beads (e.g., 3.0 &#181;m), which can be used for compensating lymphocyte populations. An even more suitable approach would be to use a small fraction of cells as the single-stain controls, using a known, highly expressed marker on the subset, such as HLA-DR or CD45, conjugated to each of the desired fluorochromes, which would allow for a much more accurate compensation than can be achieved with beads. In the case of smokers&#39; samples, this tactic is particularly useful as the macrophages are much larger and more autofluorescent. In addition, from the preparation step, the whole BAL sample could be cultured in a plate before sorting as described in section 3 of the protocol, to allow a separation of the populations by adherence. In this way, the adherent macrophages can be isolated from other nonadherent cells such as lymphocytes. Compensation is far less challenging if the lymphocyte and AM populations are separated rather than examined together; however, relying on adherence will result in a loss of macrophages, which is an important consideration when cell numbers are already limiting. Also, an adherence step could result in the unwanted activation of adherent monocytes, which may affect downstream results generated using these cells. The value of efficiently sorting cells into purer populations must be weighed against the restriction of having fewer such cells for subsequent experiments.
Other models, most notably murine models, have been used to study macrophage immunological characteristics and biology. While these models are extremely useful and allow great insight into a cell type that is difficult to manipulate, they have limitations. Many of the cell surface markers vary between mice and humans such that the immunophenotype of human AMs is not completely understood. However, this model system requires the pooling of several mice for assays due to the low cell numbers available from each animal. In addition, the necessity to pool specimens precludes considerations of genetic predisposition and sex. Recently, it has been shown that sex plays a role in the infectivity of macrophages by HIV-1 due to the disparate expression of the restriction factor SAMHD-131. Nonhuman primates (NHP) represent the closest model to humans and facilitated the study of simian immunodeficiency virus (SIV) infection and its effect on the immune system, providing insight into the role of tissue-resident macrophages compared to monocyte-derived macrophages. In rhesus macaques, it has also been shown that lung macrophage isolates from BAL harbor a replication-competent virus; a viral outgrowth assay (VOA) was used to analyze the behavior of SIV in tissue-resident cells32. Such a finding is of significant research value but must still be validated in humans before it can be applied, and the high cost of using NHPs precludes the use of large sample populations. In addition, human AMs will be useful for many other applications such as in vitro viral/microbial infection assays and in studies of other pathogens such as Tuberculosis/HIV coinfection.

One of the most significant challenges facing a cure to HIV infection is the presence of the latent HIV reservoir which causes a rebound of plasma viremia following the interruption of antiretroviral therapy (ART)1,2. While the HIV reservoir during long-term ART is well documented in several tissue compartments, including secondary lymphoid organs, gut-associated lymphoid tissue (GALT), and the central nervous system (CNS), the lungs have been overlooked as an area of study since the pre-ART era3. However, the lungs play a central role in the pathogenesis of HIV. Indeed, pulmonary symptoms were among the first indicators of AIDS-related opportunistic infections4. Even in the modern ART era, persons with HIV are at a greater risk of developing both infectious and noninfectious pulmonary diseases than persons without HIV. For example, persons with HIV infection are at elevated risk for invasive Streptococcal pneumoniae infection, as well as chronic obstructive pulmonary disease (COPD)5,6. Furthermore, coinfection of tuberculosis (TB) and HIV is a significant public health challenge in certain regions of the world, notably, sub-Saharan Africa, as HIV-infected individuals are 16 to 27 times more likely to have TB than persons without HIV7. Although some explanations for this susceptibility to pulmonary infection and chronic disease have been proposed8,9,10, the precise cellular mechanisms by which individuals with suppressed HIV plasma viral load remain at higher risk for pulmonary complications have not been fully elucidated. Importantly, HIV is a very strong risk factor for pulmonary infection and chronic disease, independent of smoking status6.
Analysis of the immune environment of the lung is, therefore, crucial in order to understand its role in health and disease. Although noninvasive, induced sputum samples tend to contain large amounts of epithelial cells and debris with rare pulmonary lymphocytes and no AMs, limiting their role to specific applications. Conversely, large biopsies of tissue cannot be obtained in the absence of suspected disease due to associated risks of significant bleeding and pneumothorax (collapse of the lung). Furthermore, the majority of pulmonary immune cells are mainly located at the mucosal level where the lungs are continuously stimulated by antigens during breathing. To that end, bronchoscopy to obtain BAL fluid has the advantage of providing relatively safe access to lymphocytes and AMs (see Figure 1). Macrophages constitute the largest proportion of cells within BAL fluid, followed by lymphocytes11. It is useful, therefore, to establish a method by which BAL fluid may be processed for use in subsequent applications, such as immunophenotyping, cell culture, transcriptomics, or any further applications. The protocol for processing the BAL fluid outlined here is adapted from general procedures previously described and optimized for the various downstream assays employed.This methodology allows for the isolation of both pulmonary lymphoid and myeloid mucosal immune cells for their phenotypical and functional characterization, as well as an assessment of the HIV reservoir in adults living with HIV.
To establish this protocol, we used the following criteria to recruit study participants15. For participants to be eligible to participate in this study, they had to be HIV-infected individuals who met the following criteria: (1) on ART for at least 3 years; (2) suppressed viral load (VL) for a minimum of 3 years; (3) CD4 T cell count of &#8805;200/mm3; (4) willing to undergo research spirometry and bronchoscopy. Patients with the following criteria were excluded from the study: (1) contraindication(s) to bronchoscopy; (2) high bleeding risk: coagulopathy or on warfarin or clopidogrel therapy; (3) thrombocytopenia (low platelets); (4) active pulmonary infection or another acute pulmonic process; (5) pregnant/trying to become pregnant.

<table><tbody><tr><td>70 &amp;micro;m Sterile Cell Strainer</td><td>Fisher Scientific</td><td>22363548</td><td>Nylon mesh filters with 70 &amp;micro;m pores to remove impurities from BAL sample before sorting</td></tr><tr><td>ACH-2 Cells</td><td>NIH</td><td>349</td><td>HIV-1 latent T cell clone with one integrated proviral copy which do not express CD4</td></tr><tr><td>BD FACSAria</td><td>BD Biosciences</td><td>N/A</td><td>Cell sorter (configured to detect 16 colours simultaneously)</td></tr><tr><td>BD LSRFortessa X-20</td><td>BD Biosciences</td><td>N/A&amp;nbsp;</td><td>Flow cytometer (configured to detect 14 colours simultaneously)</td></tr><tr><td>Bronchoscope</td><td>Olympus</td><td>BF-1TH190&amp;nbsp;</td><td>EEIII HD therapeutic bronchoscope; channel width 2.8 mm; outer diameter 6.0 mm</td></tr><tr><td>Cell Disassociation Solution</td><td>Sigma</td><td>C5914&amp;nbsp;</td><td>Non-enzymatic formulation for gently dislodging adherent cell types from plastic or glass surfaces.</td></tr><tr><td>CD169&amp;nbsp; BB515</td><td>BD Biosciences</td><td>565353</td><td>Sialic acid-binding molecule antibody used for flow cytometry</td></tr><tr><td>CD14&amp;nbsp; BV786</td><td>BD Biosciences</td><td>563698</td><td>Endotoxin receptor antibody used for flow cytometry</td></tr><tr><td>CD206&amp;nbsp; PE</td><td>BD Biosciences</td><td>555954</td><td>Mannose receptor antibody used for flow cytometry</td></tr><tr><td>CD3&amp;nbsp; Alexa700</td><td>BD Biosciences</td><td>557943</td><td>T cell co-receptor antibody used for flow cytometry</td></tr><tr><td>CD4&amp;nbsp; PE-Cy5</td><td>BD Biosciences</td><td>555348</td><td>T cell co-receptor antibody used for flow cytometry</td></tr><tr><td>CD45&amp;nbsp; PE Cy-7</td><td>BD Biosciences</td><td>557748</td><td>Receptor-linked protein tyrosine phosphatase antibody used for flow cytometry</td></tr><tr><td>CD8&amp;nbsp; BV605</td><td>BD Biosciences</td><td>564116</td><td>T cell co-receptor antibody used for flow cytometry</td></tr><tr><td>CompBead Plus</td><td>BD</td><td>560497</td><td>Anti-mouse&amp;nbsp; Ig, &amp;kappa;&amp;nbsp; and negative control polystyrene microparticles used to optimize fluorescence compensation in flow cytometry</td></tr><tr><td>DNase I</td><td>Invitrogen</td><td>18068015</td><td>Digests single- and double-stranded DNA to oligodexyribonuleotides containing a 5&#039; phosphate to remove contamination from RNA</td></tr><tr><td>dNTP Set 100 mM</td><td>Invitrogen</td><td>&amp;nbsp;10297-018</td><td>Consists of four deoxynucleotides (dATP, dCTP, dGTP, dTTP) for use in PCR</td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma</td><td>D8418</td><td>&amp;nbsp;Apolar, protic solvent used to make media for cryopreserving live cells</td></tr><tr><td>EDTA</td><td>Invitrogen</td><td>AM9912</td><td>Used to stop Dnase I enzyme activity</td></tr><tr><td>FBS</td><td>Wisent Bioproducts</td><td>080-150</td><td>Premium fetal bovine serum to supplement media</td></tr><tr><td>FcR Blocking Reagent, Human</td><td>Miltenyi</td><td>130-059-901</td><td>Binds to Fc receptor on the cell surface to prevent non-specific binding of flow antibodies</td></tr><tr><td>FlowJo v10</td><td>FlowJo LLC</td><td>N/A</td><td>Flow cytometry analysis software used for all analyses</td></tr><tr><td>HLA-DR&amp;nbsp; BV650</td><td>BD Biosciences</td><td>564231</td><td>MHC class II cell surface receptor antibody used for flow cytometry</td></tr><tr><td>HyClone HEPES solution</td><td>Fisher Scientific</td><td>SH3023701</td><td>Buffer providing maintenance of physiological pH&amp;nbsp;</td></tr><tr><td>Live/Dead&amp;nbsp; APC-H7</td><td>Invitrogen</td><td>L34975</td><td>Viability marker used for flow cytometry</td></tr><tr><td>Lymphocyte Separation Medium (LSM)</td><td>Wisent Bioproducts</td><td>350-000-CL</td><td>Polysucrose for isolation of PBMC from whole blood</td></tr><tr><td>Mr. Frosty Freezing Container</td><td>ThermoFisher</td><td>5100-0001</td><td>Freezing container ensuring rate of cooling very close to -1 &amp;deg;C/min, the optimal rate for cell preservation</td></tr><tr><td>OneComp eBeads</td><td>Invitrogen</td><td>01-1111-41</td><td>Anti-mouse, rat and hamster antibodies for compensation of PBMC samples</td></tr><tr><td>PBS 1x</td><td>Wisent Bioproducts</td><td>311-010-CL</td><td>Phosphate buffered saline for cell washing and staining</td></tr><tr><td>PCR Tubes Corbett Rotor-Gene</td><td>Axygen</td><td>PCR-0104-C</td><td>4-strip PCR tubes with 0.1 mL capacity for use with Corbett Rotor-Gene</td></tr><tr><td>PerfeCTa qPCR ToughMix</td><td>Quantabio</td><td>95112</td><td>2x concentrated ready-to-use reaction cocktail for PCR amplification of DNA templates&amp;nbsp;</td></tr><tr><td>QiaAmp DNA Mini Kit</td><td>Qiagen</td><td>51304</td><td>Kit for isolation of genomic, mitochondrial, bacterial, parasite or viral DNA. Includes QIAamp Mini Spin Columns, QIAGEN Proteinase K, Reagents, Buffers, Collection Tubes</td></tr><tr><td>RNeasy Mini Kit</td><td>Qiagen</td><td>74104</td><td>Kit for purification of up to 100 &amp;micro;g total RNA from cells, tissues, and yeast. Includes RNeasy Mini Spin Columns, Collection Tubes, RNase-free Reagents and Buffers</td></tr><tr><td>Rotor-Gene Q</td><td>Qiagen</td><td>9001550</td><td>Real-time PCR cycler</td></tr><tr><td>RPMI 1640 1x</td><td>Wisent Bioproducts</td><td>350-000-CL</td><td>Cell culture media</td></tr><tr><td>Sterile Water</td><td>Wisent Bioproducts</td><td>809-115-CL</td><td>DNase, RNase &amp;amp; protease free</td></tr><tr><td>Superscript&amp;trade; III One-Step RT-PCR System&amp;nbsp;</td><td>Invitrogen</td><td>12574018</td><td>RT-PCR kit which performs both cDNA synthesis and PCR amplification in a single tube. Includes SuperScript III RT/Platinum Taq Mix, 2x Reaction Mix (containing 0.4 mM of each dNTP, 3.2 mM MgSO4), magnesium sulfate</td></tr><tr><td>Taq DNA Polymerase&amp;nbsp;</td><td>Invitrogen</td><td>18038-042</td><td>Thermostable enzyme that synthesizes DNA from single-stranded templates in the presence of dNTPs and a primer. Includes Taq DNA Polymerase, 10x PCR buffer, magnesium chloride</td></tr><tr><td>Transcription Factor Buffer Set</td><td>BD Biosciences</td><td>562725</td><td>Buffers for intracellular staining for flow cytometry. Includes fixation/permeabilization buffer, diluent buffer, perm/wash buffer</td></tr><tr><td>Trypan Blue</td><td>Sigma</td><td>T8154</td><td>Viability dye to count cells using haemacytometer&amp;nbsp;</td></tr></tbody></table>

processing of bronchoalveolar lavage fluid and matched blood for alveolar macrophage and cd4+ t-cell immunophenotyping and hiv reservoir assessment

Bronchoscopy is a medical procedure whereby normal saline is injected into the lungs via a bronchoscope and then suction is applied, removing bronchoalveolar lavage (BAL) fluid. The BAL fluid is rich in cells and can thus provide a 'snapshot' of the pulmonary immune milieu. CD4 T cells are the best characterized HIV reservoirs, while there is strong evidence to suggest that tissue macrophages, including alveolar macrophages (AMs), also serve as viral reservoirs. However, much is still unknown about the role of AMs in the context of HIV reservoir establishment and maintenance. Therefore, developing a protocol for processing BAL fluid to obtain cells that may be used in virological and immunological assays to characterize and evaluate the cell populations and subsets within the lung is relevant for understanding the role of the lungs as HIV reservoirs. Herein, we describe such a protocol, employing standard techniques such as simple centrifugation and flow cytometry. The CD4 T cells and AMs may then be used for subsequent applications, including immunophenotyping and HIV DNA and RNA quantification.

In most nonsmokers, BAL fluid is received in a sterile container and is a slightly turbid yellow-orange-colored liquid. The fluid may be pinker in color if the donor underwent endobronchial biopsies during the bronchoscopy and some bleeding occurred. The fluid may be darker in color if the donor is a smoker. After centrifugation, the BAL supernatant will be almost clear and slightly orange, while the cell pellet can range in color from off-white to very dark brown, depending on the condition of the sample and whether the donor was a smoker or not.
When counting the whole BAL sample, different cell types can be visualized, including larger, round macrophages around 17 &#181;m in diameter and smaller round lymphocytes around 7.3 &#181;m in diameter18,19 (see Figure 2). Macrophages are enlarged in smokers by about 40%18. The distinction between the cell types allows for counting the macrophages and lymphocytes separately. There may also be some debris visible in the field, especially in samples from smokers. Macrophages are the most abundant cell type in the BAL, accounting for approximately 85% of cells in nonsmokers20, and they are enriched in smokers so they may seem almost exclusive.
The BAL cells have a tendency to aggregate, so they must be mixed well during all manipulations. The pellet may appear dark even after several wash steps. If filamentous debris is evident in the fraction after staining for cell sorting, pass the cells through a 70 &#181;m filter before running them through the cell sorter.
The sorting of BAL cells must be done at a low pressure to ensure droplet sizes large enough to accommodate the macrophages. The cells are first gated to include all CD45+21 cells, and then based&#160;on viability to ensure all dead cells are excluded (see Figure 3). Singlet cells are then chosen and within this, two populations are gated based on size and morphology, namely larger myeloid cells and smaller lymphocytes (see Figure 3). Within the larger cells, cells are gated on CD20622,23 and CD16922 and the double-positive cells are sorted as AMs, while within the smaller cells, CD3+ cells are chosen and gated on CD4 and CD8; CD4 single-positive and CD8 single-positive cells are sorted (see Figure 3). The markers used were chosen based on previously described phenotypes of AMs, such as the mannose receptor CD206, found on phagocytic cells23, and the sialoadhesin receptor CD16922.
When sorting the PBMCs, cells are first gated on forward and side scatter which should show a homogeneous lymphocyte population, all of which are taken, excluding noise close to the zero-axis (data not shown). The population is gated on viability and CD45, and live CD45+ cells are used. This population is then gated on CD3; to isolate monocytes, the CD3- population is subsequently gated on CD14 and all single positive cells are sorted. To isolate lymphocyte subsets, the CD3+ cells are gated on CD4 and CD8 and both single positive and cell populations are sorted.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59427/59427fig1.jpg" /> 
Figure 1: Protocol overview. A schematic showing the workflow of the protocol, including potential downstream uses of the generated samples. PBMC = peripheral blood mononuclear cells; BAL = bronchoalveolar lavage; LSM = lymphocyte separation medium. <a href="https://www.jove.com/files/ftp_upload/59427/59427fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59427/59427fig2.jpg" /> 
Figure 2: Microscope field view of whole BAL fluid. Microscope images from (A) a nonsmoker and (B) a smoker with visible lymphocytes (L), macrophages (M), and red blood cells (RBC). Magnification is 1,000x (10x ocular and 100x lens with oil immersion). <a href="https://www.jove.com/files/ftp_upload/59427/59427fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59427/59427fig3.jpg" /> 
Figure 3: Representative gating strategy for the cell sorting of whole BAL cells. Gating strategy used to sort alveolar macrophages (AM), CD4, and CD8 T cells from whole BAL cell samples. <a href="https://www.jove.com/files/ftp_upload/59427/59427fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample</td>
			<td>Antibody</td>
			<td>Fluorochrome</td>
			<td>Clone</td>
			<td>Volume per test (&#181;L)</td>
		</tr>
		<tr>
			<td rowspan="5">BAL and PBMC</td>
			<td>Live/Dead</td>
			<td>APC-H7</td>
			<td>-</td>
			<td>1</td>
		</tr>
		<tr>
			<td>CD45</td>
			<td>PE-Cy7</td>
			<td>HI30</td>
			<td>5</td>
		</tr>
		<tr>
			<td>CD3</td>
			<td>Alexa700</td>
			<td>UCHT1</td>
			<td>2</td>
		</tr>
		<tr>
			<td>CD4</td>
			<td>PE-cy5</td>
			<td>RPA-T4</td>
			<td>4</td>
		</tr>
		<tr>
			<td>CD8</td>
			<td>BV605</td>
			<td>SK1</td>
			<td>3</td>
		</tr>
		<tr>
			<td>BAL only</td>
			<td>CD206</td>
			<td>PE</td>
			<td>19.2</td>
			<td>10</td>
		</tr>
		<tr>
			<td></td>
			<td>CD169</td>
			<td>BB515</td>
			<td>7-239</td>
			<td>5</td>
		</tr>
		<tr>
			<td>PBMC only</td>
			<td>CD14</td>
			<td>BV786</td>
			<td>M5E2</td>
			<td>5</td>
		</tr>
	</tbody>
</table>
Table 1: Flow panel for the sorting of whole BAL cells and isolated PBMCs.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Target</td>
			<td>Step</td>
			<td>Primer Name</td>
			<td colspan="4">Primer Sequence</td>
		</tr>
		<tr>
			<td rowspan="2">HIV Total DNA or HIV LTR-Gag RNA</td>
			<td>Pre-amplification PCR</td>
			<td>UR1</td>
			<td colspan="4">5&#39;-CCA TCT CTC TCC TTC TAG C-3&#39;</td>
		</tr>
		<tr>
			<td></td>
			<td>ULF1</td>
			<td colspan="4">5&#39;-ATG CCA CGT AAG CGA AAC TCT GGG TCT CTC TGG TTA GAC-3&#39;</td>
		</tr>
		<tr>
			<td></td>
			<td>Real-time PCR</td>
			<td>UR2</td>
			<td colspan="4">5&#39;-CTG AGG GAT CTC TAG TTA CC-3&#39;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>LambdaT</td>
			<td colspan="4">5&#39;-ATG CCA CGT AAG CGA AAC T-3&#39;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>UHIV FamZen:&#160;</td>
			<td colspan="4">5&#39;-/56-FAM/CA CTC AAG G/ZEN/C AAG CTT TAT TGA GGC/3IABkFQ/-3&#39;</td>
		</tr>
		<tr>
			<td>CD3 DNA</td>
			<td>Pre-amplification PCR</td>
			<td>HCD3 out 5&#39;</td>
			<td colspan="4">5&#39;-ACT GAC ATG GAA CAG GGG AAG-3&#39;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>HCD3 out 3&#39;</td>
			<td colspan="4">5&#39;-CCA GCT CTG AAG TAG GGA ACA TAT-3&#39;</td>
		</tr>
		<tr>
			<td></td>
			<td>Real-time PCR</td>
			<td>HCD3 in 5&#39;</td>
			<td colspan="4">5&#39;-GGC TAT CAT TCT TCT TCA AGG T-3&#39;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>HCD 3 in 3&#39;</td>
			<td colspan="4">5&#39;-CCT CTC TTC AGC CAT TTA AGT A-3&#39;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>CD3 FamZen:</td>
			<td colspan="4">5&#39;-/56-FAM/AG CAG AGA A/ZEN/C AGT TAA GAG CCT CCA T/3IABkFQ/-3&#39;</td>
		</tr>
		<tr>
			<td>GUSB RNA</td>
			<td>Pre-amplification PCR</td>
			<td>GUSB Forward 1:&#160;</td>
			<td colspan="4">5&#8217;-ACC TAG AAT CTG CTG GCT ACT A-3&#8217;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>GUSB Reverse 1:&#160;</td>
			<td colspan="4">5&#8217;- GTT CAA ACA GAT CAC ATC CAC ATA C-3&#8217;</td>
		</tr>
		<tr>
			<td></td>
			<td>Real-time PCR</td>
			<td>GUSB Forward 2:&#160;</td>
			<td colspan="4">5&#39;-TGC TGG CTA CTA CTT GAA GAT G-3&#8217;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>GUSB Reverse 2:</td>
			<td colspan="4">&#160;5&#39;-&#160;CCT TGT CTG CTG CAT AGT TAG A-3&#39;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>GUSB-HEX:</td>
			<td colspan="4">5&#39;-/5HEX/TCGCTCACA/ZEN/CCAAATCCTTGGACC/3IABkFQ/-3&#39;</td>
		</tr>
	</tbody>
</table>
Table 2: Primer and probe sequences for HIV DNA and RNA quantification.

Watch this Scientific Journal Video about Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment at JoVE.co

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment

We describe a method for processing bronchoalveolar lavage fluid and matched peripheral blood from chronically HIV-infected individuals on antiretroviral therapy to assess pulmonary HIV reservoirs. These methods result in the acquisition of highly pure CD4 T cells and alveolar macrophages that may subsequently be used for immunophenotyping and HIV DNA/RNA quantifications by ultrasensitive polymerase chain reaction.

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment

Bronchoscopy is a medical procedure whereby normal saline is injected into the lungs via a bronchoscope and then suction is applied, removing ...

processing-bronchoalveolar-lavage-fluid-matched-blood-for-alveolar

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JoVE Journal

Immunology and Infection

20.7K Views.  Research Institute McGill University Health Centre. HIV is ongeneeslijk door zijn persistentie in cellulaire en anatomische reservoirs. Deze methode maakt een HIV-reservoir beoordeling van immuuncellen geïsoleerd uit de longen. Deze techniek maakt het mogelijk om longslijmvlies T-cellen en alveolaire macrofagen van BAL-vloeistof te isoleren voor verdere fenotypische of virologic-beoordeling als cellulaire reservoirs van HIV.Inzicht in de biologie van alveolaire macrofagen en CD4-positieve T-cellen en hun bijdrage aan het HIV-reservoir ...

Video: Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

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CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

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We describe a research technique for fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) using manual hand held suction in order to remove nonadherent cells and lung lining fluid from the mucosal surface. In research environments, BAL allows sampling of innate (lung macrophage), cellular (B- and T- cells), and humoral (immunoglobulin) responses within the lung.
BAL is internationally accepted for research purposes and since 1999 the technique has been performed in &#62; 1,000 subjects in the UK and Malawi by our group.
Our technique uses gentle hand-held suction of instilled fluid; this is designed to maximize BAL volume returned and apply minimum shear force on ciliated epithelia in order to preserve the structure and function of cells within the BAL fluid and to preserve viability to facilitate the growth of cells in ex vivo culture. The research technique therefore uses a larger volume instillate (typically in the order of 200 ml) and employs manual suction to reduce cell damage.
Patients are given local anesthetic, offered conscious sedation (midazolam), and tolerate the procedure well with minimal side effects. Verbal and written subject information improves tolerance and written informed consent is mandatory. Safety of the subject is paramount. Subjects are carefully selected using clear inclusion and exclusion criteria.
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Bronchoalveolar Lavage BAL for Research; Obtaining Adequate Sample Yield

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Medicine

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

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T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.

The host immune response to pathogen infection is a tightly regulated process. Utilizing a lipopolysaccharide lung exposure model in mice, it is possible to conduct high resolution evaluations of the complex mechanisms associated with disease pathogenesis.

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain insight into an ongoing disease state.
Here, a simple and efficient method is described to perform BAL on murine lungs without the need of special tools or equipment. BAL fluid is isolated by inserting a catheter in the trachea of terminally anesthetized mice, through which a saline solution is instilled into the bronchioles. The instilled fluid is gently retracted to maximize BAL fluid retrieval and to minimize shearing forces. This technique allows the viability, function, and structure of cells within the airways and BAL fluid to be preserved.
Numerous techniques may be applied to gain further understanding of the disease state of the lung. Here, a commonly used technique for the identification and enumeration of different types of immune cells is described, where&#160;flow cytometry is combined with a select panel of fluorescently labeled cell surface-specific markers. The BAL procedure presented here can also be used to analyze infectious agents, fluid constituents, or inhaled particles within murine lungs.

The health status of the lung is reflected by the type and number of immune cells that are present in the bronchioles of the lung. We describe a bronchoalveolar lavage technique that allows the isolation and study of nonadherent cells and soluble factors from the lower respiratory tract of mice.

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain ...

Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer metastasis. The 4T1 cells expressing GFP in combination with imaging and confocal microscopy are used to monitor tumor growth, track metastasizing tumor cells, and quantify the metastatic burden. These approaches are supplemented by digital histopathology that allows the automated and unbiased quantification of metastases. In this method the routinely prepared histological lung sections, which are stained with hematoxylin and eosin, are scanned and converted to the digital slides that are then analyzed by the self-trained pattern recognition software. In addition, we describe the flow cytometry approaches with the use of multiple cell surface markers to identify alveolar macrophages in the lungs. To determine impact of alveolar macrophages on metastases and antitumor immunity these cells are depleted with the clodronate-containing liposomes administrated intranasally to tumor-bearing mice. This approach leads to the specific and efficient depletion of this cell population as confirmed by flow cytometry. Tumor volumes and lung metastases are evaluated in mice depleted of alveolar macrophages, to determine the role of these cells in the metastatic progression of breast cancer.

Here we describe the model and approach to study functions of pulmonary alveolar macrophages in cancer metastasis. To demonstrate the role of these cells in metastasis, the syngeneic (4T1) model of breast cancer in conjunction with the depletion of alveolar macrophage with clodronate liposomes was used.

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer ...

Isolation of Leukocytes from Human Breast Milk for Use in an Antibody-dependent Cellular Phagocytosis Assay of HIV Targets

Even in the absence of antiretroviral drugs, only ~15% of infants breastfed by HIV-infected mothers become infected, suggesting a strong protective effect of breast milk (BM). Unless access to clean water and appropriate infant formula is reliable, the WHO does not recommend cessation of breastfeeding for HIV-infected mothers. Numerous factors likely work in tandem to reduce BM transmission. Breastfed infants ingest ~105&#8722;108 maternal leukocytes daily, though what remains largely unclear is the contribution of these cells to the antiviral qualities of BM. Presently we aimed to isolate cells from human BM in order to measure antibody-dependent cellular phagocytosis (ADCP), one of the most essential and pervasive innate immune responses, by BM phagocytes against HIV targets. Cells were isolated from 5 human BM samples obtained at various stages of lactation. Isolation was carried out via gentle centrifugation followed by careful removal of milk fat and repeated washing of the cell pellet. Fluorescent beads coated with HIV envelope (Env) epitope were used as targets for analysis of ADCP. Cells were stained with the CD45 surface marker to identify leukocytes. It was found that ADCP activity was significant above control experiments and reproducibly measurable using an HIV-specific antibody 830A.

Breast milk transmits human immunodeficiency virus (HIV), though only ~15% of infants breastfed by HIV-infected mothers become infected. Breastfed infants ingest ~105&#8722;108 maternal leukocytes daily, though these cells are understudied. Here we describe the isolation of breast milk leukocytes and an analysis of their phagocytic capacity.

Even in the absence of antiretroviral drugs, only ~15% of infants breastfed by HIV-infected mothers become infected, suggesting a strong protective effect ...

Visualization of Extracellular Traps in Macrophages from Human Bronchoalveolar Lavage Fluids

After obtaining lung macrophages from humans and mice by bronchoalveolar lavage, or BAL, according to the text protocol, use trypan blue and a hemocytometer to perform a viable cell count on the BAL solution. Pipette 1 to 4 x 105 macrophages in 500 microliters of culture medium onto coverslips in the wells of 24-well plates. Then, incubate the cells at 37 degrees Celsius overnight for the cells to adhere.
Remove the culture medium and use PBS to wash the cells once. Then, add 2% paraformaldehyde/periodate/lysine or PLP fixative, and incubate the samples for 10 minutes. After using PBS to briefly wash the cells, add 0.2% Tween-20 in PBS, and incubate the cells for 20 minutes.
Next, to block the cells, add 10% chicken serum in 5% BSA diluted in PBS, and incubate the samples at room temperature for 30 minutes. Then, replace the block solution with a 1 to 100 concentration of primary and isotope control antibodies, and incubate the cells at room temperature for one hour. Following the incubation, use PBS to wash the cells before adding the corresponding secondary antibodies. Then, incubate the samples for 40 minutes at room temperature.
After washing the cells in PBS, mount the samples with DAPI-based mounting medium. Then, visualize the samples using a confocal microscope.

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4⁺ T-cell Immunophenotyping and HIV Reservoir Assessment

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