Pediatric Virus-specific Bulk Stimulation Cytotoxic T Lympho

Wednesday, May 05, 2004

Description
This protocol is designed to increase the sensitivity of detection of HIV-1 specific CTL by the
antigen-stimulation of PBMC. Stimulator cells are prepared by infecting autologous B-lymphoblastoid
cell line (B-LCL) with a recombinant vaccinia vector (vv) expressing HIV-1 gene product (s),
followed by exposure to UV light while suspended in media containing psoralen. This inactivates viral
and cellular RNA and DNA while preserving antigenicity. Responder PBMC are mixed with
stimulation cells at a responder:stimulator (R:S) ratio of 10:1 and co-cultured for 10-14 days. This
protocol is written generically for use with any vaccinia vectors.

Procedure
EBV-Transformation (use the following method or one that has already been established in your lab)
1) To derive an autologus EBV-transformed B-lymphoblastoid cell line (B-LCL), incubate
10 x 106 fresh PBMC in 2 ml of EBV-containing cell-free supernatant from B-958
marmoset cells for 2 hrs in a 370C incubator (see current Protocol in Immunology for
further details). Add 8 ml of R-20 1 ug of cyclosporin A (final concentration cyclosporin
A is 0.1 ug/ml). Transfer to a T-25 tissue culture flask. Incubate in an upright position
undisturbed for 2-3 weeks at 370C in a humidified 5% CO2 incubator.
2) Maintenance of B-LCL Target cell lines- Cells should be split to maintain the
concentration between 0.5-1.0 x 106 B-LCL/ml in R-20. Lines that will be used for
assays should be split three times weekly for at least 1-2 weeks prior to use.
B. Antigen-specific in Vitro Stimulation Cultures
Preparation of stimulator cells
1) On the day (14-18 hours prior) before setting up the stimulation culture, infect autologus
B-LCL with the appropriate vaccinia vectors. A minimum of 1 x106 B-LCL will be
needed for each 4-5 x 106 responder PBMC. Pellet the B-LCL in a 15-ml conical
centrifuge tube. Pour off supernatant. Resuspend cells in the volume of the vaccinia
vector stock containing the appropriate MOI for that vector. Incubate for 90 minutes at
370C with occasional resuspension. After 90 minutes incubation, wash with 10 ml R-10
media. Resuspend in 1mL R-10.
2) Incubate for 14-18hr in a 5% CO2 incubator at 370C. After the incubation period remove
200-300ul of the supernatant for testing of protein expression if appropriate for the
stimulator vaccinia vector.
3) Wash 1x in cold culture media ( RPMI + 10 FBS, 100 units/ml penicillin, 100ug/ml
streptomycin)
4) Resuspend in 1-1.5 ml cold culture media, add 40 ul of Psoralen stock solution ( final
Psoralen concentration 10 ug/ml)
5) Cell suspensions are then transferred into T25 flasks and placed directly on top of a longwave
UV light Box. Cells are left there for 10-15 minutes with gentle rocking of flasks
every few minutes.
6) Cells are then transferred back into a new 15cc conicals and washed with R-10 culture
media 3x.
7) After the final wash, count and resuspend in culture media containing R-10-20U rIL-2 at
1x106/ml. B-LCL after infection should >80% viable.
C. Stimulation of PBMC
1) Ficoll blood as usual, count, and resuspend to a final conc. of 2.5x 106 PBMC/ml in
culture media containing R-10-20U rIL-2 . These cells will be the responder PBMC
2) In a 24 well tissue culture plate add 1.6 ml of your PBMC suspension with 0.4ml of your
stimulator cell suspension. Final conc. of PBMC 2x106/ml, stim cells 2x105/ml giving
you a ratio of 10/1 (Responder PBMC: Stimulator cells). Final volume 2 ml. Use as
many PBMC as are available but not to exceed 10 x 106.
3) The cultures are fed by 1ml media exchange with fresh culture media containing R-10-20
U rIL-2 every 3-4 days. Cells are cultured for 10-14 days.
D. Chromium Release Assay of Stimulated PBMC
After incubation period, stimulated PBMC are tested using a slightly modified standard
protocol. Due to high background activity that may have been induced during the stimulation
process cold target inhibition is required to optimally detect HIV-specific CTLs. The E/T
ratio's to be used are the same in a standard assay, 50/1, 25/1, and 12.5/1.
Day before the assay (Target set up)
1) Prior to the CTL assay (14-18 hours prior), pellet 1.0 x106 B-LCL in a 15 ml conical
centrifuge tube for each vaccinia vector to be tested. Pour off supernatant. Resuspend
cells in the volume of the vaccinia vector stock containing the appropriate MOI for that
vector. Incubate for 90 minutes at 370C with occasional resuspension. After 90 minutes
incubation, wash with 10 ml R-10 media. Resuspend in 1mL R-10. If appropriate for the
vaccinia vector, remove 200ul of the cell suspension prior to labeling with chromium and
pipette into a 96 well tissue culture plate to test for expression of the vaccinia vector as
described in the ACTG protocol-specific appendix.
2) Add 40 uCi ( final concentration 50uCi/ml) of sodium chromate (51Cr). Incubate for
14-18 hr in a 5% CO2 at 370C. These will be HOT targets.
3) Along with the labeling of the standard hot targets, an additional 1.5 x107 of B-LCL
are infected with control vaccinia vector is prepared in the standard fashion,
however no 51Cr (Chromium) is added. These will be the COLD targets.
4) Cold and hot targets are incubated for 14-16 hours at 37oC.
Day of the assay
5) Simulated PBMC抯 (effectors) are removed from culture, washed 2 times in R-10 culture
media, counted and resuspended at 2.5 x 106 ml. This will be starting dilution of your
effectors.
6) Make 1:2 dilutions of the effectors to give you a minimum of 3 dilutions. This will give
you E/T ratios of 50/1, 25/1 and 12.5/1. Continue to E/T ratio of 6.25:1 and 3.125/1 if
there are sufficient cells.
7) Add 100 ul of effectors to triplicate wells of a 96 well round bottom tissue culture plate.
Set up one set of triplicates at each dilution for each target.
8) Wash the Hot and cold targets with R-10 culture media 2 times. Count and resuspend
COLD targets at 5x 106. This will give you a final ratio of cold:hot targets of 50:1
(A higher ratio of cold: hot targets may be used if you have high cytotoxicity against
the targets expressing the control vaccinia vector). Hold the hot targets on ice while
performing step D. 9.
9) Add 50 ul of cold targets (250,000 cold targets/well) to the wells containing the
effectors.
10) After the cold targets have been added to the effectors, wash the hot targets for a
third time with R-10 culture media. Count and resuspend 51Cr-labeled targets at
1x105. This delays the addition of the hot targets to effectors in order to increase the
time for cold targets to inhibit of non-specific lysis by responder cells. Note: The
final wash of the hot targets must be performed immediately before adding them to
the effectors to avoid high spontaneous release.
11) Add 50 ul of the appropriate 51Cr -labeled targets (5x103 hot B-LCL) to effectors
wells.
12) Make three spontaneous release wells for each target with 100 ul media, plus 50 ul cold
targets plus 50 ul hot targets. On a separate 96-well plate, make three maximum release
wells for each target with 100 ul of 5% triton X-100, plus 50 ul cold targets plus 50 ul hot
targets.
12) Incubate for 6 hr in a 5% CO2 at 37oC.
13) After incubation period, centrifuge plates for 5 minutes at 1000 RPM at 40C
14) Harvest 60-100ul supernatant; count chromium release on gamma counter per standard
protocol.
VI. Calculations:
For each well, calculate % cytotoxicity well=[(cpm effector well-cpm spontaneous
well)/(cpm maximum well-cpm spontaneous well)] x100. Calculate % mean for the
vector=[(% cytotoxicity well a) + (% cytotoxicity well b) + =(% cytotoxicity well c)]/3.
Calculate percent HIV-specific lysis by subtracting the % cytotoxicity of the control vector
from the % cytotoxicity of the experimental vector.

Recipes
Reagents and Supplies:
Psoralen (4' aminomethyl-4-5'-8 trimethylpsoralan hydrochloride)
Psoralen stock solution (.5mg/ml in sterile PBS/Ca2+Mg2+ Free)
24 or 48 well tissue culture plates
96-well round bottom tissue culture plates
15 ml conical tubes
T25 Culture flasks
sodium Chromate (51Cr) ?mCi/ml
EBV-transforming supernatant from B-958 marmoset cell line
Culture Medium (R-20):
RPMI Gibco #21870-050
2mM L-glutamine Gibco #35050-12
100U Penicillin + 100mg/ml Streptomycin Gibco #15070-014
20% Fetal Calf Serum
Culture Medium (R-10):
RPMI Gibco #21870-050
2mM L-glutamine Gibco #35050-12
100U Penicillin + 100mg/ml Streptomycin Gibco #15070-014
10% Fetal Calf Serum
Culture Medium (R-10-20U):
RPMI Gibco #21870-050
2mM L-glutamine Gibco #35050-12
100U Penicillin + 100mg/ml Streptomycin Gibco #15070-014
10% Fetal Calf Serum
rIL-2 20 U/ml

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Pediatric Virus-specific Bulk Stimulation Cytotoxic T Lympho