7.1 Introduction
In 1973 Opelz published his clinical study that demonstrated the beneficial effects of pre-graft blood transfusions on graft survival. Blood transfusions have been used by several groups to achieve long term survival of organ allografts in rodent models. The immunosuppressive effects of blood transfusion have also been seen after bums and surgery where a higher incidence of infection is noted following blood transfusion (Galandiuk et a l 1990). It is now known that the formed elements of the blood are important (Fabre and Morris 1972), (El-Malik et a l 1984). The blood transfusion effect has been demonstrated in rodents using RBC (Wood et al 1985), platelets (Hibberd and Scott 1983) and leucocytes (Cranston et al 1987). However the blood transfusion effect appears to be strain specific (Soulillou et al 1984) and cannot be induced in every strain combination. The mechanisms of this blood transfusion effect are poorly understood, and have not previously been considered in terms of direct and indirect T cell recognition. In order to clarify the mechanisms behind the blood transfusion effect we wished to study the influence of priming to indirect allorecognition using M HC class I peptides. As we wished to study the effect of D A class I peptides on the blood transfusion effect it was necessary to use the DA-to-LEW strain combination as LEW rats have been shown to react to specific R T l.A class I peptides of the D A strain (Fangmann et al 1992a). Earlier studies giving a D A blood transfusion to LEW rats,
Freunds adjuvant, seven days prior to a (DAxLEW ) F I kidney graft resulted in no prolongation of survival even in the saline immunised group. This indicates that a single donor specific transfiision (DST) in this strain combination was insufficient to induce tolerance. This is in contrast to the studies by Wood et a l (1985) where long term survival was achieved and Fabre and Morris (1972) where marginal prolongation was achieved in this strain combination. However immunisation in adjuvant was not performed in these studies. Multiple blood transfusions i.e. repeated exposure to the donor blood given over a period of several weeks has been shown to be more effective (Fabre and Morris 1972). Therefore in this model multiple blood transfusions will be used. The timing of multiple blood transfusions is important as some investigators have found them to be ineffective (Bushell et al 1994).
In this study the influence of priming to indirect allorecognition on the blood transfusion effect prior to and during multiple blood transfusion was addressed.
LEW rats were immunised with either the D A class I peptide or saline in Freunds adjuvant either prior to or during the course of D A multiple blood transfusions. Twice weekly blood transfusions of 0.5ml D A blood in 0.5ml saline were administered via the tail vein for periods of up to 12 weeks. Antibody responses were studied to peptide 1 and intact D A class I molecules throughout the time course. T cell proliferation to the DA class I peptide was also studied.
7.2 Materials and Methods
7.2.1 Experimental animals
Inbred D A (RTl*''^) and LEW (RTl*) rats were purchased from Harlan UK Ltd (Oxen, UK). All rats were adult males.
7.2.2 Peptide immunisations
The RTl.A*''^ classical class I peptide termed peptide 1 described in section 5.4.0 was used. LEW rats received 50pg of unconjugated peptide emulsified in Freunds complete adjuvant subcutaneously into each hind footpad as described in section 5.4.1. Rats were boosted four weeks later with the same dose of peptide emulsifed in Freunds incomplete adjuvant. Control rats received an equal volume of saline emulsifed in Freunds adjuvant
7.2.3 Blood transfusions
LEW rats received twice weekly injections of 0.5ml of fresh heparinised D A blood in an equal volume of saline by i.v. injection into the tail vein as described in section 5.5.0. Control rats received 1ml of saline.
7.2.4 Assavs for alloantibodv response to intact class I molecules
Indirect radioactive binding assays (described in section 5.8.1) were performed on sera taken as stated in the experimental groups (7.3). D A fresh red blood cells were used as a source of intact DA class I molecules prepared as in section 5 .7.1.
7.2.5 Assavs for antibody response to peptide
Indirect radioactive binding assays using PVC plates coated with peptide 1 as described in section 5.8.3 were used on sera taken as stated in the experimental groups (7.3).
7.2.6 T cell proliferation assavs
T cell proliferation assays were performed 10 days following peptide boost as described in section 5.9.0. Cervical, popliteal and paraaortic lymph nodes were removed asceptically and dispersed into a single cell suspension plated out at 2x l0Vml
in culture medium. Peptide 1, 3 and an irrelevant peptide, peptide 32 from the p chain of mouse class II (H-2Ap‘*) were added at 20pg, lOpg and Ipg. Con A at lOpg/ml was used a positive control. 24 hours before harvesting IpCi in 20pl culture medium was added to the plates. Plates were harvested at day 4 and day 5. Cell bound radioactivity was measured using a rack beta counter.
7.3 Experimental Groups
7.3.1 Peptide immunisation during multiple blood transfusions
Group 1 5 LEW rats were injected i.v. twice weekly with 0.5ml D A blood in an equal