Isolation of cells from human tonsils.

Human tonsils were obtained immediately after routine tonsillectomy and kept at 4°C until further use. The protocol used was an adaptation of the method used by King et al (1990). The tonsils were washed in 70% ethanol to sterilise the external surface and then in Hanks balanced salt solution (HBSS). The tonsils were transferred into a sterile glass Petri-dish and cut into approximately 1mm pieces using a sterile scalpel blade and forceps and digested with collagenase type II at 1 mg/ml (in HBSS) for 90 minutes at 37° C. In order to stop further collagenase digestion, 5% FCS in HBSS was added to the digested tissue. A 20ml syringe plunger was then used to tease the digested tissue apart in the Petri-dish and then used to help force the resulting cell suspension through a sterile nylon mesh (125)iM pore size) stretched tightly over a sterile beaker. This separates the lympho-medulary cells from the surrounding connective tissue. The single cell suspension collected in the beaker was then washed 3 times (all washes were carried out at 600 xg for 5 minutes) with HBBS. After each wash, the pellet was resuspended in fresh HBSS and adhering fatty tissue was removed with a glass pasteur pipette until a clear suspension was obtained. The washed cells were resuspended at 5x10^ cells/ml in a solution composed of Percoll and lOx Hanks at a ratio of 9:1 (volume:volume) (base Percoll). The base percoll was then diluted to 70, 60, 50, 40, 30% in HBSS. The cell suspension in the base Percoll was aliquoted at 1.5 mis into 10 ml conical tubes and decreasing dilutions of the base Percoll (1.5 mis of 70, 50 and 30% and 1 ml of 60 and 40%) were layered successively on to it. This seven step, discontinuous Percoll gradient was centrifuged at 1000 xg for 30 minutes. Low density (LD) cells, comprising mainly activated lymphocytes, DC, macrophages and monocytes were collected from the 30- 40% and 40-50% interfaces. High density (HD) cells comprising mainly resting lymphocytes were collected from layers 50-60% and 60-70% interfaces. The HD cells and LD cells were then washed twice to remove the Percoll, resuspended at approximately 10^ cells/ml in culture medium (comprising Rosewell Park Memorial Institute 1640 medium {RPMI 1640} supplemented with 10% FCS; lOmM N-2

hydroxyethyl piperazine-N’-2-ethane sulphonic acid {HEPES}; 2mM L-glutamine; 50|iM 2-mercaptoethanol; lOOU/ml pennicillin; 100)ig/ml streptomycin; and 2.5|Xg/ml amphotericin B) and incubated overnight in 140mm tissue culture grade Petri-dishes at 10^ cells/dish, at 37°C. The following day the non-adherent cells were separated from the adherent cells such as monocytes, macrophages and fibroblasts.

The HD and LD cells were then further enriched for various T cell and APC subsets, respectively. The purification procedures employed for isolating these different cell populations are described below and is summarised in figure 2.1

2.21 Enrichment of T cells bv rosetting with sheep red blood cells (SRBCl.

The HD cells were harvested after overnight culture, washed and resuspended at 2x10^ cells/ml in 5% FCS (in HBSS). The HD cells were then incubated with SRBC (made up with 1 ml of packed SRBC and 9 ml of HBSS without phenol red) at 10^ HD cells/ml of SRBC for 10 minutes at RT, centrifuged at 1000 xg and then incubated at 4^ C for 1 hour. After this period, the SRBC rosetted cells were gently resuspended and allowed to reach RT before layering carefully over histopaque 1077 (at RT) and centrifuged at 750 xg for 30 minutes. The cells rosetted with SRBC sediment through the histopaque and form a pellet while the non rosetted cells separate at the histopaque/medium interface. The SRBS were removed from the rosetted cells by incubating with a lysis buffer (containing 0.17M Tris and 0.16M ammonium chloride, at pH 7.2) for 2-3 minutes to lyse the SRBC, washed at least 3 times before resuspending in culture medium. The cells were counted and left at 4°C until required for proliferation assays or phenotypic analysis. These cells will be subsequently referred to as E+ HD cells.

2.22 Enrichment of B cells bv rosetting with SRBC.

The LD cells were harvested after overnight culture and rosetted with SRBC as described above, but the non-rosetted cells from the histopaque/medium interface were collected on this occasion. These cells were washed twice, resuspended in culture

medium and counted. The cells were left at 4^C until required for proliferation assays or phenotypic analysis. These cells will be subsequently referred to as E- LD cells.

2.23 Enrichment of T cells bv depletion using anti-mouse IgG coated dvnabeads.

The HD cells were harvested after overnight culture, washed, resuspended in 10% FCS (in HBSS without phenol red) and counted. mAbs specific for the cells to be depleted were added to the HD cells at 1/5 dilution o f the monoclonal culture supernatant and incubated at 4^C for 1 hour. Excess mAbs was removed by washing twice in 10% FCS (in HBSS without phenol red) and the cells resuspended at 10^ cells/ml in 10% FCS (in HBSS without phenol red). The sheep anti-mouse IgG coated magnetic beads were washed 3 times in 5% FCS (in HBSS) using a magnetic stand to separate the beads from the medium. The washed beads were added to the antibody-bound cells at 2 beads per targeted cell in 2ml conical tubes. Thus to enrich for CD3+ cells, either HLA-DR and/or CD 14 and CD 19 were targeted for removal. CD3+ cells comprise 50-60% o f the total population (Figure 3.5 and 3.6), 10^ beads/ml of HD cells were used. The beads/cell suspension was incubated at 4®C in a blood tube rotator (Horwell, Stuart, Sceintific) for 1 hour and the cells bound to the beads separated from the un-bound cells by placing the conical tube in a magnetic stand for 5 minutes. The un-bound cells were harvested and washed with HBSS, resuspended in culture medium, counted and left at 4^C until

required for proliferation assays or phenotypic analysis. Throughout the procedure the cells were maintained at 4®C.

2.24 Enrichment of CD4/CD8 T cells using anti-mouse coated IgG coated dvnabeads. The HD cells were harvested after overnight culture, washed, resuspended in 10% FCS (in HBSS) and counted. The cells were preincubated with either CD4 or CDS mAbs, and resuspended at 10^ cells/ml in 10% FCS (in HBSS). The same protocol for depletion of the antibody-bound cells as in section 2.23 was followed. The CD4 and CDS cells comprise approximately 40% and 20% of the total population, respectively

(data not shown). Thus 8x10^ and 4x10^ beads/ml of HD cells were used to enrich for CDS and CD4, respectively.

2.25 Enrichment of CD45RA/CD45RO T cells by depletion with anti-mouse IgG coated dvnabeads.

The HD cells were harvested after overnight culture, washed, resuspended in 10% FCS (in HBSS) and counted. The cells were preincubated with either CD45RO or CD45RA mAbs, and in addition, either HLA-DR and/or CD 14 and CD 19 at 1/5 dilution as in section 2.23. The antibody-bound cells were resuspended at 10^ cells/ml in 10% FCS (in HBSS). The protocol for depletion of the antibody-bound cells was followed as described in 2.23. The CD45RO+ and CD45RA+ cells comprise approximately 50% of the E+ HD cells (data not shown), and since CD3+ cells comprise .50-60% of the HD cells, this suggests that the CD45RA and CD45RO T cells each comprise approximately 25-30% of the HD cell population. Consequently, 1.5x10^ beads/ml of HD cells were used to enrich for either CD45RA or CD45RO T cells.

2.26. Enrichment of B cells bv depletion with anti-mouse IgG coated dvnabeads.

The LD cells were harvested after overnight culture, washed and resuspended in 10% FCS (in HBSS) and counted. The cells were preincubated vyith CD3 and CD14 mAbs, resuspended at 10^ cells/ml in 10% FCS (in HBSS) and the protocol for depletion of the antibody-bound cells using dynabeds described in section 2.23 was followed. Since CD 19+ cells comprise approximately 70% of the LD population (data not shown), 6x10* beads/ml o f LD cells were used to enrich for B cells.

2.27. Enrichment of DC bv depletion with anti-mouse IgG coated dvnabeads.

The LD cells were harvested after overnight culture, washed and resuspended in culture medium and incubated at 37°C for 2 hours after which time the non-adherent cells were removed from the adherent cells (DC enriched). Fresh culture medium was added to the adherent cells and reincubated at 37°C overnight (in experiments where the DC enriched

population was compared with a B cell enriched population, the later population was derived from the non-adherent cells after the 2 hour incubation and the enrichment for B cells as described above). The following day the non adherent cells from the DC enriched population were preincubated with CD 19, CD 14 and CD3 mAbs and resuspended at 10^ cells/ml in 10% FCS (in HBSS). The protocol for depletion of antibody-bound cells as described in 2.23 was followed. Since CD 19, CD 14, CD3 together comprise approximately 90-95% of the LD population (data not shown), 1.9x10^ beads/ml of LD cells were used to enrich for DC. In experiments comparing B cell enriched and B cell depleted populations, the 2 hour adherence step was omitted from the above protocol.