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

HIV is incurable due to its persistence in cellular and anatomical reservoirs. This method allows an HIV reservoir assessment of immune cells isolated from the lungs. This technique allows the isolation of pulmonary mucosal T cells and alveolar macrophages from BAL fluid for further phenotypic or virologic assessment as cellular reservoirs of HIV.

Understanding the biology of alveolar macrophages and CD4-positive T cells and their contribution to the HIV reservoir could lead to important insights into the challenges facing an HIV cure. The isolation of primary macrophages from BAL can be applied to various other research areas, including inflammatory or infectious pulmonary diseases such as asthma and tuberculosis. After obtaining the BAL fluid from a human patient according to standard protocols, vortex the fluid in the original collection tube before transferring the sample to a 50-milliliter tube within a biosafety cabinet.

If the fluid appears very turbid or contaminated by filamentous tissue, filter the sample through a 70-micrometer nylon mesh filter into a new 50-milliliter tube. After centrifugation, transfer the supernatant to a new 50-milliliter tube and use a pipette tip to gently break up the pellet containing the whole BAL cells. Resuspend the pellet in one milliliter of RPMI 1640 medium and add one milliliter of the reserved supernatant of each of 10 1.5-milliliter microcentrifuge tubes.

Add 10-milliliter aliquots of the remaining supernatant into 15-milliliter conical tubes and place all of the supernatant samples in the minus 80-degrees Celsius storage. Dilute the pellet cell suspension in 10 milliliters of medium for every 25 milliliters of original sample and collect the BAL cells by centrifugation. Then resuspend the pellet in one milliliter of medium supplemented with 10%fetal bovine serum, or FBS, for counting.

For sorting of whole BAL and peripheral blood mononuclear cells, add five times 10 to the five cells to each of two five-milliliter round-bottomed polystyrene tubes per subset for the unstained and viability-stained compensation controls and add the rest of each subset sample into one five-milliliter tube per sample. Collect the cells by centrifugation and resuspend the control pellets in 100 microliters of PBS per tube on ice until they're used. Resuspend the pellets of the cells for sorting in 250 microliters of a one in 20 dilution of Fc receptor blocking buffer for one hour at four degrees Celsius to block any non-specific binding.

At the end of the blocking incubation, add the appropriate antibody cocktail of interest for an additional one-hour incubation at four degrees Celsius. At the end of the primary antibody incubation, add one milliliter of PBS to each tube for centrifugation and resuspend the pellets in 250 microliters of sorting buffer on ice protected from light until sorting. To prepare the compensation controls, add three drops each of the anti-mouse immunoglobulin kappa compensation beads and the negative control compensation beads per one milliliter of PBS to a microcentrifuge tube and transfer 100 microliters of the bead solution to each sample to be used for compensation.

Add one microliter of each antibody in the cocktail to each separate tube containing beads and add one microliter of viability stain to each viability stain compensation control tube. After 20 minutes at four degrees Celsius protected from light, wash the samples with one milliliter of PBS per tube and resuspend the pellets in 250 microliters of fresh PBS per tube. Load the whole-BAL cell unstained control sample onto the flow cytometer and set up the compensation matrix.

Next load the sample tube and sort the cells under low pressure, gating to exclude noise, to include live and CD45-positive cells, and to exclude the doublets. Within the larger myeloid population, sort the CD206 and CD169 double-positive cells as alveolar macrophages. Within the smaller lymphocyte population, isolate the CD3-positive cells and sort the CD4 and CD8 single-positive populations.

To sort the peripheral blood mononuclear cell sample, gate to exclude the noise and doublets and to include the live CD45-positive cells as demonstrated. After gating on CD3, gate the CD3-negative, CD14-positive cells for sorting the single-positive monocytes and gate the CD3-positive, CD4, and CD8-positive cells for sorting both of the single-positive populations. When counting the whole-BAL sample, different cell types can be visualized, including larger, round macrophages and smaller, round lymphocytes.

Macrophages are the most abundant cell type in the BAL fluid, accounting for approximately 85%of the cells in non-smoker lungs. These cells are enriched in smokers to the point of seeming almost exclusive and tend to be enlarged by about 40%compared to the macrophages observed in non-smokers. The markers used for sorting alveolar macrophages from BAL fluid samples were chosen based on previously described phenotypes of alveolar macrophages, such as the mannose receptor CD206 which is present on phagocytic cells and the sialoadhesin receptor CD169.

Following their isolation, alveolar macrophages and other cell types can be used for immunophenotyping by flow cytometry, immunological functional assays, or reservoir quantification by ultrasensitive PCR. This technique affords access to highly pure tissue-resident macrophages, which historically have been difficult to attain, allowing the previously unattainable examination of the significant immune cell population.