Whole Blood Assay with Dual Co-Stimulation for Antigen-Specific Analysis of Host Immunity to Fungal and Viral Pathogens

Our research focuses on the immune monitoring of patients in the intensive care unit. Some patients develop severe viral pneumonia during mechanical ventilation. We isolate the cause of the viruses, but also characterize the local and systemic immune response using the protocol presented in this video to identify immunological risk factors.

Traditional antigen-specific T-cell assays that are based on isolated mononuclear cells are highly susceptible to pre-analytical confounders, time-and resource-intensive, and require large amounts of blood. This poses severe limitations to the implementation of antigen-specific T-cell assays in the clinical research or clinical routine use as prognostic or diagnostic biomarkers. We present a versatile immunoassay protocol, which accommodates various downstream readouts from as little as 250 micros blood per stimulus.

This enables applications, even when that volume is limited or several antigens need to be screened. Furthermore, our assay is easy to perform and amenable to several pathogens. In addition to its versatility and the low blood volume required, our protocol used a stimulation environment that was thoroughly and specifically optimized for antigen-induced T-helper cell responses.

This was achieved through dual core stimulation, which also makes the protocol more robust towards confounders such as immunosuppressive pharmacotherapy or pre-analytical delays. We are using this protocol for immune monitoring of patients with ventilation-associated viral pneumonia. Our collaborators in Wuerzburg and Houston are working on a clinical translation of cytomegalovirus-specific immunoassays.

Furthermore, they're using this protocol for studies on fungal immunopathogenesis and preclinical immunotherapy studies in mouse models for opportunistic infections. To begin, prepare 2.7 milliliter blood collection tubes without anticoagulant, with antigens, and co-stimulatory antibodies under sterile conditions. Add anti-CD28 and anti-CD49d into each tube, including the negative control.

If frozen, fall the ready-to-use stimulation tubes at room temperature about 30 to 60 minutes before whole blood stimulation. Collect venous blood from the donor or patient using blood collection tubes with lithium heparin anticoagulant. Pipette the required amount of heparinized whole blood into the stimulation tubes under a sterile workbench.

Carefully invert the tubes about five to 10 times. Then place the tubes in an incubator at 37 degrees Celsius. After four hours of incubation, prepare one milligram per milliliter brefeldin A solution in RPMI medium.

In a sterile workbench, uncap the tubes used for flow cytometric analysis. Then transfer 1%of the sample volume of the brefeldin A solution into the stimulation tube. Incubate the cap tubes at 37 degrees Celsius for another 16 to 18 hours.

To begin sample preparation for flow cytometry, take out stimulation tubes containing whole blood samples with brefeldin A from the incubator. Add 500 microliters of 0.5 molar EDTA solution to each stimulation tube containing brefeldin A.Transfer the samples into new 15-milliliter centrifuge tubes. Then pipette one milliliter of erythrocyte lysis buffer into each stimulation tube to rinse it.

Transfer the buffer and cell suspension into the same centrifuge tube. Centrifuge the tubes at 600g for seven minutes and carefully pipette out the supernatant. Then add erythrocyte lysis buffer to the pellet and resuspend it.

Incubate samples at room temperature until the samples appear clear or for a maximum of six minutes. Centrifuge the tubes at 600g for seven minutes. After discarding the supernatant, add one milliliter of HBSS to the pellet.

Transfer the cell suspension into two-milliliter reaction tubes. Then centrifuge the tubes at 400g for five minutes. Discard the supernatant before performing flow cytometric staining.

Transfer the samples from the stimulation tubes that do not contain brefeldin A into 1.5-milliliter tubes. Centrifuge the tubes at 2000g for 20 minutes. Now, carefully pipette the supernatant into a fresh 1.5-milliliter tube.

If not analyzing the supernatant immediately, cryopreserve the sample at 80 degrees Celsius. When using frozen samples, centrifuge thawed supernatants again at a speed greater than 7000g for five minutes before analysis. Perform pre-dilution of the samples as required by the cytokine assay protocol.

Resuspend the cell pellet in one milliliter of RNA protection buffer. Cryopreserve it at 80 degrees Celsius for subsequent RNA isolation. Alternatively, resuspend the cell pellet in 700 microliters of lysis buffer for immediate RNA isolation.