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.
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.
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 ≥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.
This research protocol was established directly based on the principles included in the Declaration of Helsinki and received approval from the Institutional Review Boards of the McGill University Health Centre (RI-MUHC, #15-031), the Université du Québec à Montréal (UQAM, #602) and the Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CR-CHUM, #15-180).
1. Bronchoalveolar Lavage
NOTE: This section describes bronchoscopy as performed by a licensed respirologist with assistance from a respiratory therapist16,17.
2. Isolation of BAL Cells
NOTE: The following procedure must be carried out under sterile conditions in a biological safety cabinet, class II (BSL2) or higher.
3. Adherence of BAL Cells (Optional)
NOTE: This alternative protocol can be performed prior to or instead of cell sorting. The following procedure must be carried out under sterile conditions in a BSL2 cabinet (or higher).
4. Isolation of Peripheral Blood Mononuclear Cells
NOTE: The following procedure must be carried out under sterile conditions in a BSL2 cabinet (or higher).
5. Sorting Whole BAL cells and PBMCs
NOTE: The following procedure must be carried out under sterile conditions in a BSL2 (or higher).
6. Immunophenotyping of AMs and PBMCs
NOTE: The following procedure must be carried out under sterile conditions in a BSL2 cabinet (or higher).
7. Remainder of BAL Cells
NOTE: The following procedure must be carried out under sterile conditions in a BSL2 cabinet (or higher).
8. HIV DNA and RNA Quantification
NOTE: The following procedure must be carried out under sterile conditions in a BSL2 cabinet (or higher).
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 µm in diameter and smaller round lymphocytes around 7.3 µm 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 µm 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 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.
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. Please click here to view a larger version of this figure.
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). Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Sample | Antibody | Fluorochrome | Clone | Volume per test (µL) |
BAL and PBMC | Live/Dead | APC-H7 | – | 1 |
CD45 | PE-Cy7 | HI30 | 5 | |
CD3 | Alexa700 | UCHT1 | 2 | |
CD4 | PE-cy5 | RPA-T4 | 4 | |
CD8 | BV605 | SK1 | 3 | |
BAL only | CD206 | PE | 19.2 | 10 |
CD169 | BB515 | 7-239 | 5 | |
PBMC only | CD14 | BV786 | M5E2 | 5 |
Table 1: Flow panel for the sorting of whole BAL cells and isolated PBMCs.
Target | Step | Primer Name | Primer Sequence | |||
HIV Total DNA or HIV LTR-Gag RNA | Pre-amplification PCR | UR1 | 5'-CCA TCT CTC TCC TTC TAG C-3' | |||
ULF1 | 5'-ATG CCA CGT AAG CGA AAC TCT GGG TCT CTC TGG TTA GAC-3' | |||||
Real-time PCR | UR2 | 5'-CTG AGG GAT CTC TAG TTA CC-3' | ||||
LambdaT | 5'-ATG CCA CGT AAG CGA AAC T-3' | |||||
UHIV FamZen: | 5'-/56-FAM/CA CTC AAG G/ZEN/C AAG CTT TAT TGA GGC/3IABkFQ/-3' | |||||
CD3 DNA | Pre-amplification PCR | HCD3 out 5' | 5'-ACT GAC ATG GAA CAG GGG AAG-3' | |||
HCD3 out 3' | 5'-CCA GCT CTG AAG TAG GGA ACA TAT-3' | |||||
Real-time PCR | HCD3 in 5' | 5'-GGC TAT CAT TCT TCT TCA AGG T-3' | ||||
HCD 3 in 3' | 5'-CCT CTC TTC AGC CAT TTA AGT A-3' | |||||
CD3 FamZen: | 5'-/56-FAM/AG CAG AGA A/ZEN/C AGT TAA GAG CCT CCA T/3IABkFQ/-3' | |||||
GUSB RNA | Pre-amplification PCR | GUSB Forward 1: | 5’-ACC TAG AAT CTG CTG GCT ACT A-3’ | |||
GUSB Reverse 1: | 5’- GTT CAA ACA GAT CAC ATC CAC ATA C-3’ | |||||
Real-time PCR | GUSB Forward 2: | 5'-TGC TGG CTA CTA CTT GAA GAT G-3’ | ||||
GUSB Reverse 2: | 5'- CCT TGT CTG CTG CAT AGT TAG A-3' | |||||
GUSB-HEX: | 5'-/5HEX/TCGCTCACA/ZEN/CCAAATCCTTGGACC/3IABkFQ/-3' |
Table 2: Primer and probe sequences for HIV DNA and RNA quantification.
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 "predict" 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 µm) can be used, which are closer in size for compensating macrophage populations, compared to smaller beads (e.g., 3.0 µm), 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' 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.
The authors have nothing to disclose.
The authors would like to acknowledge their funders: the Canadian Institutes of Health Research (CIHR) (grant #153082 to CC, MAJ, NC); the Réseau SIDA et maladies infectieuses du Fonds de recherche du Québec-Santé (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 – 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 Josée Girouard and all clinical staff involved in coordinating and obtaining the samples, as well as the respiratory therapists; Ekaterina Iourtchenko, Hélène Pagé-Veillette, and Marie-Hélène 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.
70 µm Sterile Cell Strainer | Fisher Scientific | 22363548 | Nylon mesh filters with 70 µm pores to remove impurities from BAL sample before sorting |
ACH-2 Cells | NIH | 349 | HIV-1 latent T cell clone with one integrated proviral copy which do not express CD4 |
BD FACSAria | BD Biosciences | N/A | Cell sorter (configured to detect 16 colours simultaneously) |
BD LSRFortessa X-20 | BD Biosciences | N/A | Flow cytometer (configured to detect 14 colours simultaneously) |
Bronchoscope | Olympus | BF-1TH190 | EEIII HD therapeutic bronchoscope; channel width 2.8 mm; outer diameter 6.0 mm |
Cell Disassociation Solution | Sigma | C5914 | Non-enzymatic formulation for gently dislodging adherent cell types from plastic or glass surfaces. |
CD169 BB515 | BD Biosciences | 565353 | Sialic acid-binding molecule antibody used for flow cytometry |
CD14 BV786 | BD Biosciences | 563698 | Endotoxin receptor antibody used for flow cytometry |
CD206 PE | BD Biosciences | 555954 | Mannose receptor antibody used for flow cytometry |
CD3 Alexa700 | BD Biosciences | 557943 | T cell co-receptor antibody used for flow cytometry |
CD4 PE-Cy5 | BD Biosciences | 555348 | T cell co-receptor antibody used for flow cytometry |
CD45 PE Cy-7 | BD Biosciences | 557748 | Receptor-linked protein tyrosine phosphatase antibody used for flow cytometry |
CD8 BV605 | BD Biosciences | 564116 | T cell co-receptor antibody used for flow cytometry |
CompBead Plus | BD | 560497 | Anti-mouse Ig, κ and negative control polystyrene microparticles used to optimize fluorescence compensation in flow cytometry |
DNase I | Invitrogen | 18068015 | Digests single- and double-stranded DNA to oligodexyribonuleotides containing a 5' phosphate to remove contamination from RNA |
dNTP Set 100 mM | Invitrogen | 10297-018 | Consists of four deoxynucleotides (dATP, dCTP, dGTP, dTTP) for use in PCR |
Dimethyl Sulfoxide (DMSO) | Sigma | D8418 | Apolar, protic solvent used to make media for cryopreserving live cells |
EDTA | Invitrogen | AM9912 | Used to stop Dnase I enzyme activity |
FBS | Wisent Bioproducts | 080-150 | Premium fetal bovine serum to supplement media |
FcR Blocking Reagent, Human | Miltenyi | 130-059-901 | Binds to Fc receptor on the cell surface to prevent non-specific binding of flow antibodies |
FlowJo v10 | FlowJo LLC | N/A | Flow cytometry analysis software used for all analyses |
HLA-DR BV650 | BD Biosciences | 564231 | MHC class II cell surface receptor antibody used for flow cytometry |
HyClone HEPES solution | Fisher Scientific | SH3023701 | Buffer providing maintenance of physiological pH |
Live/Dead APC-H7 | Invitrogen | L34975 | Viability marker used for flow cytometry |
Lymphocyte Separation Medium (LSM) | Wisent Bioproducts | 350-000-CL | Polysucrose for isolation of PBMC from whole blood |
Mr. Frosty Freezing Container | ThermoFisher | 5100-0001 | Freezing container ensuring rate of cooling very close to -1°C/minute, the optimal rate for cell preservation |
OneComp eBeads | Invitrogen | 01-1111-41 | Anti-mouse, rat and hamster antibodies for compensation of PBMC samples |
PBS 1X | Wisent Bioproducts | 311-010-CL | Phosphate buffered saline for cell washing and staining |
PCR Tubes Corbett Rotor-Gene | Axygen | PCR-0104-C | 4-strip PCR tubes with 0.1 mL capacity for use with Corbett Rotor-Gene |
PerfeCTa qPCR ToughMix | Quantabio | 95112 | 2X concentrated ready-to-use reaction cocktail for PCR amplification of DNA templates |
QiaAmp DNA Mini Kit | Qiagen | 51304 | Kit for isolation of genomic, mitochondrial, bacterial, parasite or viral DNA. Includes QIAamp Mini Spin Columns, QIAGEN Proteinase K, Reagents, Buffers, Collection Tubes |
RNeasy Mini Kit | Qiagen | 74104 | Kit for purification of up to 100 µg total RNA from cells, tissues, and yeast. Includes RNeasy Mini Spin Columns, Collection Tubes, RNase-free Reagents and Buffers |
Rotor-Gene Q | Qiagen | 9001550 | Real-time PCR cycler |
RPMI 1640 1X | Wisent Bioproducts | 350-000-CL | Cell culture media |
Sterile Water | Wisent Bioproducts | 809-115-CL | DNase, RNase & protease free |
Superscript™ III One-Step RT-PCR System | Invitrogen | 12574018 | 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 |
Taq DNA Polymerase | Invitrogen | 18038-042 | 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 |
Transcription Factor Buffer Set | BD Biosciences | 562725 | Buffers for intracellular staining for flow cytometry. Includes fixation/permeabilization buffer, diluent buffer, perm/wash buffer |
Trypan Blue | Sigma | T8154 | Viability dye to count cells using haemacytometer |