The overall goal of this procedure is to quantify bacterial CD4+T cell transinfection, using a mouse cell model. In vivo, some pathogenic bacteria such as Listeria monocytogenes and Salmonella enteritidis are able to infect T lymphocytes and modify their behavior. However, in vitro, T cells are not easy to infect.
Based on this observation, we investigated whether T cells can capture bacteria from infected dendritic cells during antigen presentation as occurs in viral infections. We found that T cells capture bacteria and then kill them. Actually, we found that T cells do this more efficiently than professional phagocytes.
These results contradict a central tenet of immunology, showing that cells from adaptive immunity can perform functions supposedly exclusive to innate immunity. Today we present the protocol used to study the bacteria transinfection process in a mouse model. Esteban Veiga and I will be demonstrating this procedure along with Guillermo Ramirez-Santiago, Raquelle Garcia-Ferreras, two graduate students, and Monica Torres-Torresano, a technician.
Note that all the procedures described in this video should be carried out in the hood, using only sterile media, instruments, pipette tips, and culture dishes. In a non-tissue culture treated sterile 15 centimeter Petri dish, add one microliter of one milligram per milliliter lipopolysaccharide to the bone marrow derived dendritic cells from a single mouse. Incubate the cells for 24 hours to increase major histocompatibility complex II, or MHC II, expression.
Following the incubation, perform flow cytometry to confirm MHC II expression and differentiation. After MHC II expression has been confirmed, as shown here, transfer the cells to a sterile 50 milliliter polypropylene tube. Add 10 milliliters of PBS containing five millimolar EDTA, then centrifuge the cells at 600 x g.
Once the spin is complete, perform a wash by resuspending the cells in RPMI with 10%FBS, then centrifuging again. Then, after the wash, add one milliliter of RPMI 10%FBS for every five times 10 to the sixth dendritic cells, and pipette up and down to resuspend them. Next, separate the resuspended dendritic cells into two tubes of equal volume.
To one tube, add 10 micrograms per milliliter OT-II OVAp. Leave the other tube untreated. It will serve as a negative control for antigen loading.
Incubate the tubes for at least one hour to load the MHC II molecules of dendritic cells with OVA peptide. Meanwhile, prepare the Salmonella enteritidis for infection by washing them with a PBS solution three times. Centrifuge between each wash at 1800 x g to eliminate the LB medium and any secreted toxins.
Then, resuspend the bacteria in RPMI. Following the incubation, infect each tube of dendritic cells at a multiplicity of infection, or MOI, of 10, and then incubate the cells for one hour. Once the cells have been infected, wash them three times with PBS, and then once with RPMI 10%FBS.
Centrifuge between washes at 450 x g to wash away most of the extracellular bacteria in the soluble OVAp. After the last centrifugation, resuspend the cells in RPMI 10%FBS at 20 times 10 to the sixth cells per milliliter. To a 24-well culture plate, add two million CD4+T cells in a volume of 0.5 milliliters to each of five wells.
Next, to control for physical contact, place polycarbonate trans-well barriers with a three micrometer pore size into the third and fourth wells. To the first well, add 0.1 milliliters of the infected overloaded dendritic cells. To the second well, add 0.1 milliliters of the infected non-loaded dendritic cells.
Add 0.1 milliliters of the infected overloaded dendritic cells to one well, and 0.1 milliliters of the infected non-loaded dendritic cells into the other well. Finally, as a negative control, directly infect the fifth well of CD4+T cells at an MOI of 10. Then, bring the total volume of the well up to 0.6 milliliters with RPMI.
Incubate the plate for 30 minutes to allow immune synapses to form. After the incubation, add 100 micrograms of Gentamicin per milliliter of medium to each well, and incubate for one hour. Gentamicin is an aminoglycoside antibiotic that is not able to penetrate eukaryotic cells.
Therefore, in this step, aminoglycoside will kill extracellular bacteria but not intracellular bacteria. Following the incubation, collect the non-adherent cells in 15 milliliter tubes, and resuspend them in 10 milliliters of PBS BSA EDTA buffer. Discard the plastic culture plate to which most of the dendritic cells remain attached.
Next, vortex the tubes gently to ensure cell desegregation. Centrifuge the tubes at 600 x g. Then, from any tube, aspirate 500 microliters of the cell supernatant and pipette it into a new tube.
This will be used as a control to show that the Gentamicin treatment was effective. Place all of the samples on ice. At this stage, there are six tubes:four tubes of test samples and two controls.
Although most of the dendritic cells were discarded with the plate, the samples that were not separated by the trans-well barriers could still contain some. To reduce contamination, use magnetic cell isolation or cell sorting to re-isolate the CD4+T cells from the samples that were not separated by trans-well barriers. The final isolate should contain less than 2%dendritic cells.
Keep the rest of the samples on ice. Once the CD4+T cells have been purified, it is time to save for bacteria class infection. This can be done by performing colony counts, or by flow cytometry.
Here, we will show you how to perform colony counts. Wash the transinfected CD4+T cells twice with PBS. Centrifuge at 600 x g between washes.
Then count the cells and resuspend them in PBS at a concentration of two times 10 to the sixth cells per milliliter. To 500 microliters of T cells, add 500 microliters of 0.1%Triton X-100 to lyse them. This releases the intracellular bacteria from the T cells.
For each sample of lysed cells, make three serial dilutions. Then, divide an LB agar plate into quarters and spread 50 microliters of each dilution, including the undiluted suspension, onto each section of the plate. On a separate LB agar plate, spread the stored cell supernatant as a control for Gentamicin efficacy.
Then, on another plate, spread the directly infected T cells as an additional control. Incubate overnight. The next day, count the colonies in each section of the plate and calculate the number of colony-forming units for each sample.
If low-level dendritic cell contamination occurs, which is defined as less than 2%subtract the colony-forming units corresponding to low dendritic cell contamination. T cell transinfection was performed using Salmonella enteritidis and quantified by Gentamicin survival assay as described in this video. When directly infected T cells were plated, as shown on the left, growth of colonies was not observed, indicated that T cells are refractory to direct bacterial infections.
When supernatant from transinfection assays was plated, as shown on the right, colonies were not observed, indicating that the Gentamicin worked properly. Results from transinfection assays performed in the presence of trans-well barriers are shown here. Only three colonies are observed in the undiluted suspension from both OVA-loaded and non-loaded samples.
In contrast, numerous colonies are seen when there is direct contact of T cells with infected dendritic cells. Moreover, when dendritic cells loaded with OVAp were incubated with T cells, the uptake of bacteria was increased by about five times. Taken together, these data indicate that direct contact of infected dendritic cells with T cells facilitates bacterial uptake and that cell transinfection is enhanced during antigen recognition.
After this video, you should have a good understanding of how to quantify transinfection using marine T cells. The advantage of quantifying transinfection with the Gentamicin survival is its great sensitivity. We are able to detect one single infecting bacterium.
Is it is important to remember that the purification step of this procedure is critical. The purity must be very high if colony counts will be used to determine the transinfection rate. Quantification by flow cytometry does not have this limitation, because you can easily distinguish dendritic cells and T cells, but the restriction is that the GFP expressing bacteria must be bright enough to detect the infected cells against the outer fluorescence background.
Don't forget that working with pathogens can be extremely hazardous and the appropriate safety precautions should always be used when performing this procedure.
