This protocol provides a long term, in vitro, functional method for the evaluation of chimeric antigen receptor, or CAR T cells, through repetitive tumor cell challenge. This technique is less time consuming, and less labor intensive than in vivo CAR T cell function evaluation while still achieving an accurate representation of the true anti-tumor efficacy. This in vitro technique enables the high throughput screening and differentiation of CAR T cell anti-tumor potency.
Which has the potential application for assessing clinical effectiveness of CAR T cell products. Begin by dissociating glioblastoma tumorspheres from a glioblastoma tumorsphere culture. With one milliliter of cold accutase, and 30 to 60 seconds of pipetting.
When the tumorspheres have been disrupted, stop the reaction with five milliliters of warm co-culture medium. And pellet the tumor cell suspensions by centrifugation. Re-suspend the glioblastoma cells to a 1.6 times 10 to the fifth tumor cells per milliliter of fresh coculture medium concentration, and dilute T cells harvested from a CAR T cell culture to the appropriate percentage of CAR positive T cells per milliliter of co-culture medium concentration.
Next, add 100 microliters of the diluted tumor cells to each well of a 96 well flat bottom tissue culture plate. And 100 microliters of CAR T cells into each well of tumor cells with gentle mixing. Then place the plate in a 37 degree Celsius, 5%carbon dioxide incubator.
On days two, four, and six of culture, carefully remove 50 microliters of medium from each well of the tumor cell, T cell co-culture plate slated for rechallenge according to the table. Then, add 50 microliters of fresh glioblastoma cell suspension, prepared as just demonstrated, but with twice the cell number of the initial co-culture to each well with gentle mixing. And return the plate to the cell culture incubator.
Days one, three, five, and seven of co-culture, transfer the supernatant from each well to be harvested into a new 96 well round bottom plate according to the table and add 50 microliters of pre-warmed 0.5%Trypsin EDTA into each medium depleted well. After five minutes at 37 degrees Celsius, confirm that the cells have detached from the bottom of each well by light microscopy, and gently pipette the enzyme solution around the bottom of each well to resuspend the cells. Before transferring the detached cell suspension into the appropriate corresponding wells of the new 96 wells plate.
Pellet the cells by centrifugation, and wash the cells with 200 microliters of fluorescents activated cell sorting standing solution, or FFS per well with a second centrifugation. Re-suspend the pellets in 100 microliters of the antibody cocktail of interest in 100 microliters of SSS per well for a 30 minute incubation at four degrees Celsius. A the end of the incubation remove any unbound antibody by sequential 100 and 200 microliter FSS washes and re-suspend the cells in 100 to 200 microliters of DAPI nuclear stain and FSS.
Then, analyze the cells according to standard flow cytometric analysis protocols. For functional and phenotypic analysis of the CAR T cells after tumor cell co-culture, retrieve the data files from the flow cytometer, and gate all of the live DAPI negative cells. To quantify the tumor cells, gate the CD45 negative population.
To quantify the CAR T cells gate the CD45 positive CAR positive population. Then plot the tumor and CAR T cell numbers over the time course of the experiment, identifying T cell activation by the co-expression of 41BB, and CD69. T cell exhaustion by the expression of PD1, LAG3, and TIM3, and the T cell memory status LAG3, and TIM3, and the T cell memory status by CD45RO, and CD62 ligand expression.
by CD45RO, and CD62 ligand expression. Both CD4 positive, and CD8 positive CAR T cells become equally activated against glioblastoma cells that express the target antigen as indicated by their CD107a and intracellular cytokine expression. However, as assessed by repetitive tumor challenge SA, CD4 positive, but not CD8 positive, CAR T cells are capable of multiple round killing.
CD4 positive CAR T cells also achieve a more robust expansion. When CD4 positive, and CD8 positive CAR T cells are mixed at a one to one ratio, this cell combination out performs CD8 positive, but not CD4 positive CAR T cells in terms of long term cytotoxicity. In addition, the expansion of CD8 positive CAR T cells is induced by the addition of CD4 positive T cells.
While CD4 positive CAR T cell expansion is inhibited in the presence of CDa positive cells. 24 hours after the initial co-culture, both CD4 positive and CD8 positive CAR T cells are similarly activated. During the repetitive tumor challenge, both CD4 positive and CD8 positive CAR T cells demonstrate a transition from a CD45RO positive, CD62 ligan positive central memory phenotype, to a CD45RO positive CD62 ligan negative effector memory phenotype.
CD8 positive CAR T cells are also more prone to exhaustion compared to CD4 positive CAR T cells as assessed by their PD1, LAG3, and TIM3 expression after three days of co-culture. Further, no differences observed in CDa positive CAR T cell exhaustion in the presence or absence of CD4 positive T cells, indicating that CD4 induced CD8 positive CAR T cell expansion is not associated with a better effector function. This essay can also be coupled with sorting the T cells form the co-culture to perform further analysis on transcriptomes, proteomics, and specific genes of interest.
This repetitive tumor challenge could also be used as a platform for investigating the biological events post CAR T cell tumor recognition.
