GRANULOMETRÍA DEL AGREGADO

Freshly excised tissue was snap frozen in isopentane at -70®C for 5-7 minutes. The isopentane was contained in a glass beaker, placed in a metal kidney dish surrounded by dry ice pellets and acetone to cool the mixture. Frozen tissue was stored at -

70®C until required. Tissues were mounted in O. T. C. mounting medium (Raymond

Lamb, London, UK) and sections of 5|im thickness were cut in a cryostat (Bright Instrument Company Ltd, Huntingdon, UK) onto gelatinised slides, allowed to air dry for 2-20 hours and fixed in 100 % acetone at room temperature for 10 minutes. Sections were then allowed to air dry for 10 minutes and either stained or stored at -70®c until required.

4.4.2 Subbing solution- Gelatin coating of slides

Gelatin (5g) (Difco) was dissolved in 1 litre of distilled water, heated to 65®C, allowed to cool to room temperature and 0.5g of chromic potassium sulphate (KCr(S0^)2) (BDH Ltd) added. The mixture was then used to cover PTFE coated multispot microscope slides (Hendley (Essex) Ltd, UK). The slides were air dried in an oven and cooled before use. The subbing solution was kept at 4®c and remained fresh for 10 days.

4.4.3 Slide Mountant

2.5g of 1,4 Diazabicyclo (2,2,2) octane (Aldrich Chemical Company Ltd, Dorset, UK) was dissolved in 30 mis of Tris HCl buffer pH 8.0. The pH was then adjusted to 8.6 with concentrated HCl. 30mls of this solution was added to 70mls of glycerol (Analar, BDH Ltd, Poole, UK) mixed and stored at 4®C.

4.4.4 Antisera

Table 4.1 lists the monoclonal and polyclonal antibodies used in these studies and their specificity. Gratitude is extended to Prof. H. Waldmann (Dept. Pathology, Cambridge University) who supplied hybridomas and cloned supernatants of many rat anti-mouse T cell surface markers (CD4, CD8, Thy-1, Ly-1) and to Drs. Siamon Gordon , Paul Crocker and Hugh Rosen (Sir William Dunn School of Pathology, Oxford) who supplied me with supernatants of Mo. Abs to mouse macrophage cell surface markers (F4/80, Mac-1, SER- 4 and 7/4). A Mo.Ab against the mouse homologue of ICAM-1 (YNl/1. 7. 4.) was a kind gift from Dr. F Takei (Terry Fox Laboratory, BC Cancer Research Centre, Vancouver, Canada) and the anti-V611 monoclonal Ab supernatant was a kind gift of Dr. Tomonari (Clinical Research Centre, Harrow, London).

4.4.5 Immunoperoxidase.

Cryostat sections were removed from storage, allowed to thaw for 10 minutes, rehydrated in PBS for 10 minutes before staining commenced. Sections were incubated for the times indicated at 25®C in a humid staining tray (Ortho, High Wycombe, Bucks, UK). Following each antibody incubation slides were washed in three changes of PBS. Mouse T cell surface components were detected on frozen pancreatic tissue by a two layer peroxidase technique. Sections were blocked with 20% normal mouse serum (NMS) for 30 minutes, incubated with the rat anti-mouse monoclonal antibodies anti-L3T4 (YTS 191.1) 1/750 dilution, or anti-Ly-2 (YTS 169.4) 1/750 dilution for 30 minutes. Then a goat anti-rat IgG biotin conjugate 1/30 dilution (Seralab) was applied for 30 minutes, followed by avidin biotin horseradish peroxidase (Vectastain ABC kit,Vector Lab) for 30 minutes. The stain was developed for 5-10 minutes with 3.3-diaminobenzidine tetrahydrochloride (Sigma) at 0.6 |Xg/ml in PBS containing 0.01% hydrogen peroxide and lightly counter-stained in Harris' haematoxylin (Sigma) and mounted on DPX.

4.4.6 Immunofluorescence.

Macrophage cell surface antigens were detected by indirect immunofluorescence technique. Briefly fixed cryostat sections were preblocked as before then incubated for 30 minutes with the rat anti-mouse monoclonal antibodies. Mac-1 (Ml/70) IgG2b which binds to an epitope of the C3bi receptor (CR3) on neutrophils, macrophages and NK cells, F4/80 (specific for mature mouse macrophages), 7/4 (specific for neutrophils), and SER-4 (recognises the sheep erythrocyte receptor present on stromal macrophages from lymph node liver and spleen). This was followed by fluorescein conjugated goat anti-rat IgG (H-hL) (Jackson Immunoresearch Labs Inc. USA) in NMS. To facilitate double staining of macrophages, Mac-1 was detected as before followed by rabbit anti-mouse F4/80 which was detected by rhodaminated swine anti-rabbit Ig (Dako).

T cells bearing particular V6 chains were detected by a similar indirect immunofluorescence techniques to that described for the rat anti-mouse monoclonals. Anti- V66 (44.22.1), anti-V88.1,2 (KJ16), and anti-V811 (K T ll) were used as tissue culture supernatants.

monoclonal Ml/42.3 ([rat IgG2a, Stallcup et al, 1981, kind gift from Dr G. Butcher, AFRC, Babraham, Cambridge) by the methodology previously described. Staining of class n of NOD was achieved using the mouse monoclonal OX-6 which was directly FITC conjugated (Serotec). This antibody reacts against a monomorphic determinant of the rat RTI-B antigen and cross reacts with I-A encoded molecules of certain mouse strains including NOD. The mouse equivalent of the human lymphocyte adhesion molecule ICAM-1, was observed by preblocking pancreatic sections as before with NMS followed by the rat anti-mouse M.Ab YN1/1.7.4 detected by goat anti-rat FITC. I1-2R staining was achieved using the rat anti-mouse M.Ab. 3C7 IgG2b (HPLC purified 5.19mg/ml, gift from Celltech, Slough, UK), (Ortega etal, 1984), again detected with rat anti-mouse Ig FITC.

Pancreatic beta ceUs were detected by preblocking sections with NMS followed by guinea pig anti-porcine insulin in NMS (ICN Immunobiologicals) and detected by rhodaminated goat anti-guinea pig IgG in NMS (Cappel).

Double immunolabelling was achieved by first staining sections with one set of primary and secondary reagents then incubating with a second set of primary and secondary antibodies. All reagents were added sequentially with three PBS washes in between as for single staining and were titrated before use. For dual immunofluorescence with rat M.Abs and rabbit polyclonal antibodies, species specific secondary reagents were used. After the final wash slides were mounted using glycerol based anti-fading mountant prepared as detailed above. Sections were examined using a Zeiss Axiophot photomicroscope (Carl Zeiss, West Germany) fitted with transmitted light and incident- light fluorescence with filter blocks for revealing fluorescein and rhodamine/Texas red fluorochromes. Photographs were taken on Kodak Ektachrome ASA 200/400 film with either automatic or manual exposure using the integrated camera system.

4 .5 In V ivo D epletions

4.5.1 In Vivo Depletions With Depleting Anti-CD4 and Anti-CD8.

Depleting monoclonal antibody to mouse cell surface antigens, L3T4 (YTS 191.1) and Lyt 2 (YTS 169.4) (anti-CD4 and anti-CDS respectively; Cobbold et aU 1984) and the non-depleting ant-CD4 monoclonal (YTS 177.9.6.1) were prepared as ascites in Lou x DA rats (Harlan OLAC Ltd) pretreated with piistane. The ascites was ammonium sulphate precipitated and analysed by SDS Page for Ig content. The depleting antibodies were of the IgG2b,k subclass and were diluted in phosphate buffered saline such that each mouse received 400|ig i.v. in a final volume of 200|xl on the first day of treatment and 400fxg intraperitoneally on the two following days. Control mice were either treated with YTH 3.2.6 a rat monoclonal antibody which recognises human CD7 (Benjamin etal, 1986A) or with PBS alone.

4.5.2 In Vivo Depletion With Non-Depleting Anti-CD4

Non-depleting anti-CD4 monoclonal (YTS 177.9.6.1) was prepared as detailed previously. The non-depleting anti-CD4 was of the IgG2a subclass and 2mg was administered i.v, i.p, i.p. on consecutive days prior to transfer of spleen cells from a diabetic donor. Recipient mice were then injected with YTS 177.9.6.1 three times weekly during the course of the experiment. The antibody does not lead to depletion of CD4+ T cells, but specifically binds to this cell population. Control mice were similarly treated with YTH 3. 2. 6.

4.5.3 In Vivo Depletion With The Monoclonal Antibody 5C6.

Groups of 4-7 male NOD/CRC mice of 2-3 months were injected on day-1 with 500|xg of 5C6 a monoclonal antibody specific for the myelomonocytic adhesion-promoting type-3 complement receptor (CR3 or CD 1 lb/CD 18), which does not bind to T cells (Rosen and Gordon, 1987). The initial injection was intravenous and all subsequent injections were intraperitoneal. On day 0, all mice were irradiated (650 rads from a cobalt source) and received 2x10^ spleen cells from diabetic donors or non-diabetic syngeneic age-matched males intravenously. On day 1 and then three times every week, 5C6 or isotype matched control YTH3.2.6 antibody was administered. Mice were killed at 4 weeks for histological

examination of the pancreata. A further group of mice were given 5C6 for 10 days only and another group was not started on this antibody treatment until day 10. Blood glucose was assessed using a glucometer and urine tested with Diastix.

4.5.4 In Vivo VB8 Depletion.

Mouse monoclonal F23.1 (Staerz et aU 1985) which recognises V68.1, 8.2, 8.3. was used to deplete the V68+ cells in the donor spleen ceU preparation and also in the recipient following diabetic spleen cell transfer. Donor animals were injected with 500|ig of

F23.1 i.p. three days before transfer to deplete V68'*' cells. 24 hours after transfer of the diabetogenic spleen cells into 650 rad irradiated recipients these animals were also injected with 500|ig F23.1 to ensure depletion. Control mice (donors and recipients) were injected with a comparable volume of phosphate buffered saline [PBS].

4.6 C vclonhosnham ide.

200mg/Kg of Cyclophosphamide (Faiitalia Carlo Erba Ltd. St. Albans, Herts), was administered i.p. twice 14 days apart to male or female NOD mice to induce the onset of diabetes.

4.7 Construction o f NOD E. NOD-ASP and NOD-PRO Transgenic