2) Negative controis
2.9 Adoptive transfer of Thy1.2 cells Into Thy 1.1 mice
As described in chapter 5, these experiments allowed the CD45RA phenotype of a population of proliferating cells to be identified in vivo. 1 0^
A/Thyl .2 spleen cells from 4-6 week old female A/ST mice were injected i.v. into female congenic irradiated A/Thyl.1 mice. Either total spleen cells or nylon wool depleted spleen cells were transferred (see section 2.1 1). Spleen
cells from recipient mice were stained 1 , 2 , 6 , 1 3 , 1 6 and 60 weeks following transfer.
2 ,10 Purification o f CD4+ or CD8+ CD45RA+ and CD45RA’ ceiis
The general scheme for purification is outlined in figure 2.2. Each step is described below. For the C D 4 purifications for subsequent in vitro cultures, spleens from 60 mice were used for each purification. Fewer mice were needed for the in vivo adoptive transfer assays. For the C D 8 assays, spleens
from approximately 30 mice were used. Such a rigorous purification procedure was necessary in order to deplete of accessory cells as completely as possible since these are largely C D 45R A h rig h t and would have been greatly enriched in the C D 4 5 R A + fractions. All the following steps were
undertaken in sterile conditions.
2.10.1 Ficoll density centrifugation
To remove dead and red cells, spleen cells were resuspended at a concentration of 5 x 1 0 6 - 2 xlO^/ml in DMEM with 5%FCS and underlaid with
4 ml Ficoll Hypaque (1.09g/cm3). A stock of 14% Ficoll (Pharmacia, Sweden) was mixed in with metrizoic acid in a vol/vol ratio of 12/5, filter sterilized and kept at 4OC protected from light. The gradient was spun at 2000 xg for 20
minutes at 20°C and the interface collected and washed twice with DMEM. 2.10.2 Nylon wool columns
Passage over nylon wool removes 80-90% of B cells (Julius, et a!., 1973). For passage over nylon wool columns, cells were resuspended at 1-2
X 1 0 6/ml in warm DMEM containing 10%FCS and loaded on prewashed and
prewarmed nylon wool columns (O.Sg of teased, 5 x boiled, nylon wool for
1 0 6 cells). Columns were incubated at 37^0 in 5% CO2 for 45 minutes. Non
adherent T cells were washed off the columns with 15-40mls of prewarmed medium. Typically, 30% of the cells loaded would be recovered.
2.10.3 Complement killing of CD4+ or CD8+ cells.
Cells (5 X 10^/ml) were incubated with the concentrated supernatant of the anti CD4 mAB, RL172.4 or the anti CD8 mAb, 3.168, for 10 minutes at room temperature. The optimal concentration was determined by prior titration. Rabbit complement (Low Tox, Cedarlane, Canada) was then added at a final dilution of 1 in 10. The cells were incubated in a 37°C water bath for 50 minutes. Cold medium was then added and the cells washed twice.
Figure 2.2
Purification procedure used to isolate CD45RA+ and CD45RA- T cell subsets. Spleen Cells f Ficoll
t
Nylon wool CD4 or CDS killing Anti lgM+ Anti Class II panningT
Anti CD45RA Ab
t
Ficoll
T
Goat anti Rat Dynabeads
t
Magnet Positive^raction Trypsin CD45RA positive ceiis Negative fraction CD45RA negative ceils2.10.4 Panning to remove remaining igiVi+ and Class 11+ ceiis
Petri dishes (bacteriological grade, Sterilin, UK) were coated overnight at 4 0c with PBS containing rabbit anti-rat Ig (Dako, Denmark) at a
concentration of 20|ig/ml. The next day, non-specific binding sites were blocked by incubating the plates with DMEM + 5% PCS for 1 hour at room temperature followed by two washes with ice-cold medium. The cells to be panned were preincubated for 30 minutes at 4°C with anti IgM mAb, B7.6, with either of the anti lEk mAb's, M5 114.15.2 or 14.4.48, and 10-3.6.2 which bind lAk. 200pl of culture supernatant was used for 10^ cells. After one wash, the cells were resuspended at 10^/ml in ice cold DMEM. 4mls of cell suspension were loaded per plate followed by incubation for 1 hour at 4^0. Non-adherent cells were washed off the plates by gentle pipetting with cold medium.
2.10.5 Incubation with anti CD45RA antibodies
By this stage of the purification, the cells were generally more than 90% pure CD4+ or CD8+ cells. The remainder expressed neither class II nor IgM. The cells were incubated with purified anti CD45RA mAb, 14.8 (20pg for 10^ cells) for 30 minutes at 4 0C. Prior to incubation with Dynabeads (Dynal,
Norway), dead cells and excess antibody were removed by passage over Ficoll as described in section 2.10.1.
2.10.6 Magnetic separation of CD45RA+ and CD45RA- cells.
Cells at 5 X 10^/ml were incubated with goat anti-rat IgG coated Dynabeads at a bead to cell ratio of 10:1 for the CD8 purifications and 4:1 for the GD4 purifications. Prior to use the beads were washed three times with cold medium to remove the toxic preservative in which they were stored. Beads were washed by putting the tube next to the magnet for 2 minutes, the supernatant removed and fresh medium added off the magnet. The bead- antibody-cell mix was rotated at 4^0 for 30 minutes. Beads and cells were then put in contact with the magnet for 2 minutes and the magnet non-binding fraction which contained the CD45RA' cells, was collected. These cells were then washed and resuspended in the appropriate medium for subsequent culture or cell transfer. The bead-bound cells were washed twice and resuspended in warmed verse ne followed by an equal volume of warmed 0.125% trypsin to facilitate cell detachment. After incubation in a Petri dish for 5 minutes at 37^C, 2 ml of cold PCS was added to quench the trypsin and the bead/cell mixture was exposed to the magnet for 2 minutes. The supernatant
plus one wash were collected and used as the CD45RA+ fraction. The trypsin step was necessary because leaving the bead bound fraction to detach in culture overnight as recommended by the manufacturers led to almost 100% cell death.
2.10.7 Yields following purification
Typically, 60 spleens would yield between 2-5 x 10^ CD4+CD45RA+ cells whereas 20 spleens would yield between 4-10 x 10® CD8+CD45RA+ cells. The purity of the various starting populations is discussed in the relevant results sections in chapters 6 and 7. In each type of experiment where purified cells were used (proliferation assays, CTL assays and in vivo adoptive transfer assay) control experiments were undertaken to ensure that trypsinisation alone did not account for the findings. Thus, the CD45RA- subset was shown to behave similarly whether it was trypsinized or not.
2,11 Proliferation assays using purified CD4+ subsets. 2.11.1 Culture conditions
Flat bottomed 96 well plates were used for culture. 10^ T cells were added per well in a final volume of 200|il. Optimal concentrations of PHA (0.5-1 pg/ml final concentration, depending on batch) or purified biotinylated anti CD3 mAb, 2011, (4-6pg/ml final concentration, depending on batch) were added. The anti CD3 was cross linked with avidin which was used at a final concentration of 2-3pg/ml. The optimal ratio of avidin to biotinylated anti GD3 was determined by prior titration and was usually between 2-3:1. Feeder cells were added in some experiments and their preparation is described below. Control wells included medium, T cells alone, feeders alone, and feeders with either PHA or cross linked anti CD3. At relevant time points after the initiation of the cultures, the plates were pulsed for 16 hours with ImCi (= 37kBq/well) of ^H thymidine (Amersham international, UK) and then harvested for liquid scintillation counting. In some experiments, prior to addition of the ^h thymidine, 125|il of supernatant was removed from each
well. Supernatants from triplicate samples were pooled and frozen at -20^0 for subsequent IL2 and IL4 assays. Fresh medium was added to the wells which were then pulsed with ^H thymidine. Bulk cultures stimulated with PHA were set up simultaneously for subsequent staining.
2.11.2 Preparation of feeder cells
Spleen cells from syngeneic mice were mashed and resuspended as single cell suspension. They were irradiated with 3000 rads from a Cobalt source and washed well. 5 x 10$ feeders were added per well in a final volume of 200pl.
2.11.3 Harvesting of proliferation assays
After 16 hours pulsing with thymidine plates were either frozen prior to subsequent harvesting or harvested immediately. The cells were mashed and harvested on to glass fibre filters. After drying each filter corresponding to one well was placed in a tube and 2mls of scintillation fluid added (Optiscint, Dupont, UK). The incorporation of thymidine was measured in a liquid scintillation (3 counter (LKB, Pharmacia, Sweden) and results were expressed as the mean cpm from a triplet. Errors were usually less than 10%. The stimulation index was calculated thus:
mean com cells + stimulus mean cpm cells alone