Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

1. Mononuclear Cell Isolation (day 0)

1. Insert spike of female luer adapter into outlet port of the 20% fraction of the cord blood unit, attach syringe and remove blood. Transfer thawed blood to 20 mL of warmed RPMI in 50 mL centrifuge tube. Rinse cord blood bag with 5 mL of RPMI and transfer to the same centrifuge tube.
2. Centrifuge cells for 10 minutes at 400 x g. Aspirate supernatant.
3. Resuspend cells in 20 mL of warm RPMI. Layer cells onto 15 mL of Lymphoprep in 50 mL centrifuge tube. Centrifuge 40 minutes @ 400 x g.
4. Harvest the interface containing the mononuclear cells and wash in 20 mL of RPMI. Centrifuge for 10 minutes @ 450 x g.
5. Aspirate supernatant and wash in 20 mL of RPMI. Count cells and spin for 5 minutes @ 400 x g.
6. Aspirate supernatant and resuspend cells at 5 x 10⁶ mononuclear cells/mL of Cellgenix DC media (serum free). Remove 1 mL for EBV-LCL generation and put into 15 mL centrifuge tube.

2. Dendritic Cell Generation (starting on day 0)

1. Plate 2 mL of cells (are already at 5 x 10⁶ cells/mL of DC media) per well into a 6-well plate. Leave in incubator for 1-2 hours.
2. After 1-2 hours, wash off the non-adherent cells by washing 3-4 times with PBS. Collect the non-adherent cells and freeze. Add 2 mL/well of DC media containing 1000 U/mL of IL-4 and 800 U/mL GM-CSF.
3. 3-4 days after DC initiation, replenish the IL-4 and GM-CSF by adding 100 μL/well of media containing a final concentration of 1000U/mL IL-4 and 800 U/mL GM-CSF.
4. On day 5-6, harvest the DC by scraping the well with a transfer pipette. Count the big cells and centrifuge for 5 minutes @ 400 x g. Aspirate media and resuspend DC at 2×10⁶ cells/mL of DC media. Add 0.5 mL/well of a 24-well plate.
5. Transduce the DC using the Ad5f35pp65 vector at an MOI per cell of 10 infectious units. Add vector to each well in a 24-well plate. Incubate cells for 1.5 hours. After 1.5 hours, add 1.5 mL/well of DC media containing: 1000 U/mL IL-4, 800 U/mL GM-CSF, 10 ng/mL TNF-alpha, 1 μg/mL PGE-1, 100 ng/mL IL-6, and 10 ng/mL IL-1beta. These cytokines mature the DCs.

3. Generation of EBV-LCL (starting on day 0)

1. Centrifuge the tube containing the 5 x 10⁶ mononuclear cells. Add 200 μL of concentrated EBV B95.8 supernatant and 1.8 mL of complete media (RPMI + 10% FBS + 2mM L-glutamine) containing cyclosporine (1 μg/mL).
2. Aliquot 100 μL of cells into 10 wells of a 96-well plate and 200 μL into 5 wells. Add 100 μL of complete media + cyclosporine to the wells containing only 100 μL of cells. Fill remaining wells with sterile water.
3. Feed the LCL weekly and expand from a 96-well plate to a 24-well plate after ~2 weeks. After another week, transfer to a T25 flask and in the subsequent week transfer to a T75 flask. At this stage it is recommended that you freeze aliquots of LCL. It typically takes 1 month to generate sufficient numbers of LCL.

4. CTL Initiation – 7 days After Initiation of DCs

1. Harvest DCs and irradiate at 30 Gy. Wash 4 x and count. Resuspend DC in T cell media containing human serum (45% RPMI, 45% CLICK’s, 10% human serum, 2mM L-glutamine) @ 1 x 10⁵ DCs/mL.
2. Thaw non-adherent cells from step 2.2. Wash cells, and count. Resuspend at 2 x 10⁶ non-adherent cells/mL in T cell media containing human serum. Add 10 ng/mL IL-7 and IL-12, and 5 ng/mL IL-15. Add 1 mL of cells per well in a 24-well plate. Add 1 mL of DC per well. Fill empty wells of 24-well plate with sterile water.
3. 7 days after T cell initiation, feed and/or split the cells with T cell media containing human serum.
4. 9-12 days after T cell initiation, freeze the T cells until the LCL are ready.
5. Once LCL have expanded and are passaged in T75 flasks, transduce the LCL by centrifuging for 5 minutes @ 400 x g. Add the vector to the cell pellet at an MOI of 100 infectious units per cell. Incubate for 1.5 hours. After 1.5 hours, resuspend in complete media at 5 x 10⁵ LCL/mL and add 2 mL per well in a 24-well plate.¹²
6. After 2 days, harvest the LCL and irradiate at 40 Gy. Wash 4 x and count. Resuspend the LCL in T cell media containing human serum at 2.5 x 10⁵ cells/mL. Add 1 mL of LCL per well of a 24-well plate. Also, add 5 x 10⁶ LCL to a Grex40 culture device as well as 5 ng/mL IL-15.¹³
7. Harvest the T cells. Centrifuge for 5 minutes @ 400 x g , aspirate the supernatant, and resuspend in T cell media containing human serum at 1 x 10⁶ T cells/mL. Add 5 ng/mL IL-15 and add 1 mL/well of T cells (total of 1 x 10⁶ T cells) to the LCL that are already in the 24-well plate. In the Grex40, bring the total volume of media up to 30 mL.
8. On day 3-4 post-stimulation, if the cells are confluent then split them (in the plate) 1:1 and add fresh media containing 50 U/mL IL-2. If they are not confluent then simply aspirate ½ the media and replace with media containing 50 U/mL IL-2. In the Grex40, aspirate half the media, replace with fresh media, and add 50 U/mL IL-2.
9. On day 7, repeat as in step 4.6 but use IL-2 on the day of the stimulation, not IL-15.

5. Representative Results

A schematic of the GMP-compliant FDA-approved manufacturing protocol is depicted in Figure 1. The process takes about 50 days. Typical methods of generating virus-specific T cells expand pre-existing memory T cells; however, cord blood lacks virus-experienced T cells and therefore we need to prime naïve T cells ex vivo. To do so, we use dendritic cells as well as the cytokines IL-7, IL-12, and IL-15, necessary for generating viral specificity.

After 3 stimulations, the yield should be over 100×10⁶ cells. If sufficient cells are not available, additional stimulations can be performed until the desired number of CTLs are available. The majority of these cells should be CD3+ with a mixture of CD4+ and CD8+ T cells. There should be less than 15% NK cells (CD3-/CD56+) and less than 1% CD19+ B cells (Figure 2).

The expanded CTL should recognize the antigen pp65 from CMV, hexon and penton from adenovirus, as well as numerous EBV antigens that are expressed on EBV-LCL. When tested in an ELISPOT assay, CTL should secrete more IFN-? in response to these antigens than irrelevant antigens (Figure 3).

The CTL should also lyse viral peptide-pulsed targets such PHA blasts. In a ⁵¹Cr release assay, CTL should lyse LCL, CMVpp65-pulsed and Adenovirus hexon- and/or penton-pulsed targets but not targets pulsed with irrelevant peptides (Figure 4).

Figure 1. Generation of multi-virus-specific CTL from cord blood. Schematic showing the entire process of CTL manufacture from cord blood. First the cord blood mononuclear cells are isolated from the 20% fraction of the fractionated umbilical cord blood unit. With the exception of 5×10⁶ cells that are saved for LCL generation, all the cells are then plated in dendritic cell media for 1-2 hours, at which point the non-adherent cells are harvested and frozen. The DC are then fed DC media containing IL-4 and GM-CSF. After 5 days of culture, the DC are matured and transduced with an adenoviral vector containing the immunodominant CMV antigen pp65. At initiation, these DC are combined with the non-adherent cells as well as the cytokines IL-7, IL-12, and IL-15. At subsequent stimulations, the same adenoviral vector is used to transduce EBV-LCL, which are used as the antigen presenting cells. IL-15 is used at the second stimulation and IL-2 thereafter.

Figure 2. Phenotype of resulting T cells. Shown is the percentage of live cells included in the lymphocyte gate. CTL are CD3+ and CD4+ or CD8+ but largely CD3-/CD56- and CD19-.

Figure 3. T cell functionality. T cell specificity was tested by IFN- γELISPOT. T cells were pulsed with overlapping peptides spanning the entire protein of hexon, penton, pp65, and the irrelevant antigen IE-1. CTL alone indicates media alone. Autologous LCL were irradiated and added at 1×10⁵/well. Shown is the mean spot forming cell count of triplicate wells.

Figure 4. Cytolytic activity of CTL. The ability of the resulting CTL to lyse targets expressing viral antigens was tested in a ⁵¹Cr assay. ⁵¹Cr-labelled Autologous PHA blasts were pulsed with overlapping peptides spanning the entire antigen or ⁵¹Cr-labelled Autologous LCL were cocultured with CTL. After 4 hours, gamma release was counted on a gamma counter.