The overall goal of this experiment is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model. This method can help answer questions in immunology field, such as sufficiency and detection of antigen-specific cytolytic killing of target cells in an attacked immune system and how this could be modified by manipulation of the T-cells to enhance the function in vivo. The main advantage of this technique is that it allows the researcher to assess antigen-specific cytolytic potential directly and quickly, as the assay is not depend on tumor growth or infection.
Presenting this procedures will be Cara Haymaker, and Yared Hailemichael to instruct us from Department of Melanoma Medical Oncology. After anesthetizing mice, according to the text protocol, use 70%isopropyl alcohol to spray the tail base area for injection. Using a 27 gauge needle attached to a syringe, and with the bevel side facing up, penetrate four to five millimeters into the tail base region and inject 100 microliters of previously prepared peptide solution.
Then, lateral to the vaccine injection site, inject 100 microliters of anti-CD4D antibody. Carefully apply 50 milligrams per mouse of imiquimod cream on the vaccination sites. Rub the cream into the surface until the cream is no longer visible and is fully absorbed.
Then inject 100 microliters of 100, 000 IUs per milliliter of recombinant human rhIL-2 protein intraperitoneally, in the lower abdominal region. After isolating splenocytes from mice and lysing the red blood cells, according to the text protocol, carry out peptide-pulsing by first resuspending cells at 10 to the sixth cells per milliliter in complete medium. Divide the cells into two 15 milliliter conical tubes and label them as pulsed or unpulsed.
For OVA 257 to 264 pulsing, add one microgram per milliliter of peptide to the tube labeled pulsed. Then incubate pulsed and unpulsed cells at 37 degrees Celsius for one hour. Add 10 milliliters of complete medium to both the pulsed and unpulsed tubes to wash the target cells.
Then spin the remaining cells at 475 x g and room temperature, for five minutes, before aspirating the supernatent. Prepare a CSFE high-labeling medium by adding 1 microliter per milliliter of CSFE to RPMI 1640 with 2%FBS, for a final concentration of five micromolar per millimeter. Then prepare CSFE low-labeling medium by adding one microliter per milliliter of CSFE to RPMI 1640 with 2%FBS, for a final concentration of 0.5 micromolar per milliliter.
To label the target splenolytes, resuspend 10 to the sixth cells per milliliter of pulsed cells with prepared CSFE high-labeling medium, and 10 to the sixth cells per milliliter of unpulsed cells with CSFE low-labeling medium. Mix the cell suspensions by gentle inversion or swirling. Then incubate the cells at 37 degress Celsius for 15 minutes, remixing the cells every five minutes.
Next, add 10 milliliters of complete medium to each cell suspension, and centrifuge the cells at 475 x g and room temperature for five minutes. Then aspirate the supernatent and use 10 milliliters of cold PBS to resuspend the cells. Spin the cells at 475 x g and 4 degrees Celsius for five minutes, before repeating the PBS wash.
Count the cells and mix peptide-pulsed CSFE high-labeled with unpulsed CSFE low-labeled cells at a 1:1 ratio for injection into recipient mice. Keep an aliquot of one times 10 to the sixth mixed cells to use for a baseline flow cytometry assessment. After injecting and reisolating target cells, according to the text protocol, perform assessment of CTL activity using a standard flow cytometry protocol, acquiring cells using the FITC channel with a 488 nanometer laser for excitation.
Prior to injection of CSFE-labeled target cells, the 1:1 cell mixture was run on a flow cytometer to determine the baseline frequencies of both the CSFE high and CSFE low target cells. Shown here is the gating strategy to detect changes in the CSFE populations. An initial gate was made using FSC and SSC parameters.
The total CSFE positive cells were then subgated, prior to assessing changes in frequency, as this population is relatively small when compared to the unlabeled endogenous spenlocytes. An example of the relative frequency of the CSFE populations prior to injection is shown here and should be close to one to one. The necessity of the covex priming is seen in this panel, where no killing of the antigen-pulsed CSFE high-target cells was observed at 24 hours post-injection.
This figure demonstrates the effective killing of the antigen-pulsed CSFE high-labeled target cells, as the peak that was observed prior to injection was almost undetected, and the ratio was dramatically shifted from 50%to 1%detection. Finally, the figure also shows the kinetics of antigen-pulsed CSFE high-label target cell killing by assessing loss of this population at both 6 and 24 hours post-injection. Once mastered, this technique can be performed in two to three days, depending upon antigen and T-cell function.
While attempting this procedure, it is important to remember that CSFE labeling must be uniform, and the kinetics of CTL response must be assessed, prior to performing the assay. Following this procedure, other methods like ELISA, ELISPOT, and intracellular flow cytometry staining can also be performed to assess changes in effector function and answering other additional questions. After watching this video you should be able to have a good understanding of how to assess in vivo CTL function in the absence of an infection or tumor.
