The overall goal of the following procedure is to evaluate the innate sensing of HIV V one, infected CD four positive T cells by plasmacytoid dendritic cells, or PDCs. This is accomplished first by infecting CD four positive T cells with H HIV V one. In the second step, the infected T cells are co cultured with PDCs.
After 18 to 22 hours, the supernatants from the co cultures are recovered for quantification of PDC type one interferon production. Ultimately, the production of type one interferon can be used to assess the sensitivity of human blood, isolated PDC to CD four positive T-cell H HIV one infection. This method could be useful for studying viral and host factor governing the early innate immune response in recognition of pathogen, such as a recognition of HIV one infected CD four T-cell by plasmacytoid lytic cells.
Although these protocols are aimed at measuring type one interferon, they can be easily modified to detect other bioactive molecules released by PCs upon recognitions of infected T cells such as interfer gamma, TNF alpha, or kins For the infection of primary CD four positive T cells begin by activating human peripheral blood, isolated CD four positive T cells with PHAL for 48 hours. Maintain the cells in complete medium, supplemented with recombinant IL two for at least three days prior to infection. Then in a biosafety level three laboratory, infect the T cells with HIV one viruses using different multiplicities of infection, two days post infection.
Evaluate the cell cultures for their expression of GFP BST two and CD four. Use only cultures with 20 to 50%GFP positivity for the co-culture experiments in a biosafety level two facility. Next, isolate the mononuclear cells from a human peripheral blood sample by density gradient centrifugation within half an hour of the blood collection.
Then stain the cells with the appropriate antibodies to confirm that the desired cell lineages have been isolated at physiological ratios. Working quickly to keep the cells cold. Use A PDC negative selection kit to enrich for the PDCs within the isolated PBMC following the manufacturer's recommendations.
Then confirm the purity percentage of enrichment and relative amounts of PDC specific surface markers such as I LT seven and BDCA two by flu cytometry. Enriched samples should contain at least 40%PDCs now co-culture 220 microliters of the sensing cells with 30 microliters of infected T-cells per well in a 96 well U bottom plate Include control wells containing the sensing or target cells alone, adjusting these wells to a final volume of 250 microliters with complete media. To assess PDC responses to known toll-like receptor agonists, add a TLR seven or TLR nine agonist to the appropriate wells and incubate the plate at 37 degrees Celsius and 5%carbon dioxide for 18 to 22 hours.
At the end of the co-culture, transfer the cells to a 96 well V bottom plate and centrifuge the plate for five minutes at 400 Gs and room temperature. Then transfer the supernatants to a 96 well flat bottom plate. Resus suspend the cell pellets in 2%paraform aldehyde, and analyze them by flow cytometry using their size and granulation to distinguish the cells in a forward versus side scatter plot.
To measure bioactive type one interferon using hcbl interferon alpha beta reporter cells first plate the reporter cells at a density of 50, 000 cells per well in a 96 well flat bottom plate to a final volume of 180 microliters. Then add 20 microliters of the just harvested co-culture supernatant to each well in duplicate, include a set of internal standard controls and incubate the plate at 37 degrees Celsius and 5%carbon dioxide for 18 to 22 hours to determine the levels of alkaline phosphatase released from the reporter cells at 180 microliters of quantum blue solution to each well of a new flat bottom plate. Then add 20 microliters of the induced HEC PLU interferon alpha beta cells supernatants to each well and incubate the plate at 37 degrees Celsius until color develops in the standard interferon control wells.
The quantum blue solution changes from pink to purple blue in the presence of the enzyme. Finally, use a spectrophotometer to evaluate the levels of secreted alkaline phosphatase at 620 to 655 nanometers and determine the concentration of type one interferon by extrapolation from the linear part of the interferon standard curve. Due to the variable nature of primary cells, it is important that normal ratios of CD 14 positive myeloid and CD three positive T cells are observed as shown in these representative dot plots.
Furthermore, only a low number of activated T cells identified by their higher forward scatter and CD three levels are desirable in the freshly isolated PBMC cultures. Most important, the relative percentage of PDCs needs to be determined, 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 the PDCs using negative selection often yields 50 to 95%PDC purity as seen in these two representative dot plots from different donors.
A prototypical example of the overall experiment is demonstrated in the next few figures. In this representative study. MT four cells were infected with P and l 4.3 GFP, IRIS NF wild type, or delta VPU to achieve 30%infection at the time of co-culture.
As previously described, only the infections with wild type virus resulted in a significant downregulation of surface BST two. Due to their differences in size and granularity, MT four cells can easily be distinguished from p BMCs by flow cytometry after the co-culture only pbmc in the presence of known TLR seven or TLR nine agonists or pbmc in contact with hiv one infected cells released significant amounts of type one interferon. No interferon was detected in pbmc alone in Pbmc co-culture with mock infected MT four cells or an infected MT four cells alone in the example presented here, innate sensing of HIV one infected MT four cells by pbmc was found to be significantly reduced in the presence of VPU.
While attempting this procedure, it is important to remember to prevent all sources of contamination. Always use endotoxin free solutions. Remember to check routinely for mycoplasma contaminations and keep your cells in culture without any antibiotics as even controlled bacterial infections can be sensed by PBMs.
Don't forget that PCs are very fragile cells. They have a limited lifespan, and their responsiveness can be affected if the procedure is not performed in a timely manner.
