The overall goal of the following procedure is to rapidly generate donor derived virus specific cytotoxic T lymphocytes or CTLs that are suitable for adoptive transfer to allogeneic hematopoietic stem cell transplant or HSCT recipients. This is achieved by first nucle effecting dendritic cells or dcs with plasmids encoding viral antigens to prime the cells for antigen presentation. As a second step, the nucle affected dcs are used to stimulate antigen-specific T cells, which are then expanded in the presence of IL four and IL seven in a gas permeable culture device called a gre.
Next, after seven days of culture, the cells are split and receded and their media is replenished in order to sustain maximal in vitro CTL expansion. Finally, on day 12, cells are harvested and tested for purity, potency and safety and are cryopreserved for subsequent clinical use. Ultimately, CTL lines with multi virus specificity and minimal allo reactivity can be generated as evidenced by their intracellular cytokine staining, interferon gamma production and cytotoxicity.
The manufacturer of multi virus specific cytotoxic T-cell lines is time consuming and cumbersome. It requires the use of EP Epstein bio virus transform B-cell lines and adenoviral vectors and the procedure takes about three months. Our new method does not use viral vectors.
The cells grow exponentially in culture with minimal cell loss and therefore the procedure is rapid and cost effective. Demonstrating this procedure is Eureka Gerdeman, who's a clinical fellow in the laboratory Harvest monocyte derived dcs, which have been generated in Cellgenic media, supplemented with IL four and G-M-C-S-F and then matured for 24 hours in maturation cytokines by gentle Resus suspension with a three milliliter transfer pipette. Use trian blue to count the dcs and then transfer them into three 15 milliliter tubes with no fewer than 0.5 times 10 to the sixth and no more than two times 10 to the sixth cells per tube.
Next centrifuge the DCS for 10 minutes at 200 times G.During this time, prepare six milliliters of cell genix media supplemented with the same DC maturation cytokine cocktail as before and plate two milliliters in three wells of a 12. Well tissue culture treated plate fill unused wells with two milliliters of sterile water. Transfer the plate to an incubator at 37 degrees Celsius with 5%CO2 to prewarm.
Once the cells have finished spinning, aspirate the supernatants and add the relevant DNA plasmids to each tube at a final concentration of five micrograms of DNA per tube. Here the addition of the plasmid encoding IE one PP 65 is being added to tube number one heon penton to tube number two and E BNA one LMP two BZ F1 to tube number three reus. Suspend the DCS and DNA with 100 microliters of nuclear affection solution.
Mix well and transfer the mixture to NU nuclear affection Cuvettes. Place the cuvettes in the four D nucleo effector. Choose program CB one 50 and press start immediately after nuclear affection.
Add 500 microliters of the PREWARM CELLGENIC DC maturation media to each vete mix cells by gently pipetting up and down two to three times and transfer the nucle affected DCS to the prepared 12 well plate containing the remaining 1.5 milliliters of pre-war DC maturation media. Transfer the plate to the incubator for a further 12 to 18 hours harvest count and then irradiate the nucle affected. Dcs wash the cells once with 10 milliliters of CTL medium and then resuspend them at three times 10 to the fifth dcs per milliliter of media pool a minimum of 7.5 times 10 to the fifth or 2.5 milliliters and a maximum of 15 times 10 to the fifth or five milliliters of dcs from all three nucle infections and transfer the pooled plasmid transfected DC to the G Rex device.
Then place the device in the incubator for the preparation of the responder cells. Use the non-adherent mononuclear cells that remain after the DC selection. Transfer the responders to the pre-war culture solution.
Wash them once with CTL media reus. Suspend the cells in CTL media. Count the cells and bring them to a concentration of two times 10 to the six cells per milliliter.
Take 15 times 10 to the six cells or 7.5 milliliters and supplement with the cytokines IL four and IL seven. Transfer 7.5 milliliters of PBMC to the G Rex and top off the bioreactor with CTL media to a total volume of 30 milliliters. Finally, place the G REX in the incubator for six to seven days on day six to seven, aspirate 10 milliliters of the media.
Then mix the remaining 20 milliliters of media with a 10 milliliter pipette. Use trian blue to count the cells if there are fewer than 50 times 10 to the six cells. Replenish the culture with fresh media and cytokines.
If there are greater than 50 times 10 to the six cells, remove 10 milliliters of the cell suspension, transfer the cells to a new G rex and then feed both G rexes with fresh CTL media and cytokines culture the cells for an additional four to six days to expand the cells in sufficient number harvest and count the cells. Then cryopreserve any cells that are not used for phenotypic or functional analyses for future use. Representative results showing that multi cyonic DNA plasmids induce multi virus specific T cells as measured by interferon gamma ly spot and the frequency of reactive cells is superior to that of lines generated using conventional PHU plasmids expressing the same antigens.
The optimal ratio of DC to PBMC is important for potent T-cell stimulation as shown in this figure where a ratio of one to 50 produce suboptimal reactivation compared to a one to 20 stimulator to responder ratio CTL culture in the G rex as seen in this figure supports superior T-cell expansion compared with conventional 24 well plates. The addition of IL four and IL seven to cultures increases the repertoire and specificity of the expanded cells as shown here where the frequency of T cells reactive against the CMV PP 65 derived HLA A two restricted NLV peptide was assessing cultures generated in the presence or absence of IL four and or IL seven. This figure shows a representative sample of the phenotype of the rapidly expanded multi virus CTL, which are polyclonal with a mixture of CD four positive and CD eight positive T cells, of which the majority express the memory markers, CD 45 RO and CD 62 L.The expanded CTLs are specific for all the stimulating antigens and are poly functional as assessed here by intracellular cytokine staining to detect production of interferon gamma and TNF alpha after antigen stimulation as assessed by chromium release assay.
The expanded CTL are able to specifically kill viral targets, but shown no non-specific activity against non-viral HLA mismatch targets a crucial safety consideration. After watching this video, you should have a good understanding of how to rapidly manufacture multi virus specific cytotoxic T cell lines that are GNP compatible and suitable for the adoptive transfer into patients with virus associated diseases. The simplification of this procedure should facilitate the extension beyond specialized manufacturing facilities and allow the procedure to become standard of care for patients who are immunocompromised and highly susceptible to virus associated diseases.
