Part A: Comparison of the specificity of unresponsiveness induced in neonatal C3H mice by exposure to BALB-derived alloantigens using different assessments of tolerance
Experimental design: Within 24 h of birth, neonatal mice were injected with a cellular tolerizing inoculum as described below, derived from semi-allogeneic adult donors. No further treatment was administered. At approximately 8 weeks of age, treated mice and untreated control mice were assessed for tolerance using both in vivo and in vitro methods. No immunosuppressive therapy was administered.
Assessment of unresponsiveness: Treated and naïve control mice were transplanted with gender-matched, fully allogeneic donor-type or third-party heart grafts or skin grafts, or were sacrificed for harvesting of lymph node cells (LNC) for in vitro assessment of alloreactivity in proliferative and cytotoxicity assays.
Immunogenetic combinations: Neonatal recipients were all C3H (expressing the H-2k haplotype). Donors of tolerizing cells were adult (C3HxBALB)F1 mice of H-2k/d haplotype. Cardiac and skin allografts were from fully allogeneic adult BALB donors (H-2d) or unrelated third-party donors of other fully disparate H-2 haplotypes including B6 (H-2b), NZW(H-2z), SJL (H-2s) or adult outbred CD1 mice. Controls for transplant experiments were neonatally treated C3H mice transplanted with syngeneic heart or skin grafts and untreated C3H recipients transplanted with heart or grafts from all of the above donor strains. For in vitro experiments, responder cells were derived from neonatally treated and untreated C3H recipients. Stimulators and target cells were derived from BALB donors, B6 and NZW third-party strains and from syngeneic C3H mice.
General methods and materials
Animals: C3H/He (KkIAkIEkDk), C57BL/6 J (KbIAbIE–Db), BALB/c (KdIAdIEdDd), and CD1 mice were obtained from Charles River Laboratories (St. Consant, Quebec). NZW(KzIAzIEzDz), BALB.B (KbIAbIE–Db), B10.D2 (KdIAdIEdDd), DBA/2 (KdIAdIEdDd) and SJL (KsIAsIE–Ds) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All housing, treatment protocols and procedures were reviewed and approved by an independent Animal Care Committee in accordance with the current regulations and standards of The Canadian Council of Animal Care.
C3H breeding pairs were set up at staggered times to ensure continuous production of newborn pups to serve as neonatal recipients of tolerizing inocula, and subsequently as recipients of cardiac and skin allografts. Other breeding pairs were set up for production of various F1 donors of semi-allogeneic bone marrow and spleen cells.
Preparation of cellular inocula for neonatal injection: Bone marrow and spleen inocula were prepared from various adult F1 donor mice semi-allogeneic to the neonatal recipients. Bone marrow plugs were harvested from isolated tibia and humeri. Tissues were gently disrupted by mechanical prodding. Pooled single cell suspensions were prepared in PBS containing 2% fetal calf serum (Cansera). Splenocytes were layered over Histopaque-1083 (Sigma) and centrifuged for 20 min at 1800 r.p.m. and 20 °C; the interface containing lymphocytes was removed. Bone marrow cells and splenocytes were washed three times with PBS containing 2% fetal calf serum and counted; viability was assessed by trypan blue exclusion. Cells were resuspended (1 : 1 proportion) to a final concentration of 1.5 × 108/mL in 0.9% saline with 15 U/mL heparin (Leo Laboratories) for injection.
Induction of neonatal tolerance: Neonatal mice less than 24 h old were injected intravenously with 15 × 106 bone marrow and spleen cells into the anterior facial vein using a 30G needle. Injected mice were returned to the nest, received no further treatment and were weaned from mothers at 3 weeks of age. No immunosuppressive therapy was administered.
Heart transplantation: Mice were anesthetized with 3.6% chloral hydrate (Wiler). Primarily vascularized heterotopic heart transplantation into an abdominal position was performed by a modified technique as originally described by Corry et al. (33), in which the donor aortic root is anastomosed end-to-side to the recipient abdominal aorta and the donor pulmonary artery trunk to the inferior vena cava. Grafts were monitored by direct abdominal palpation of the pulsating graft in the awake animal. A qualitative score of 4 (strongest) to 0 (absence of pulsation) was assigned; rejection day was designated when the graft ceased functioning. Functioning grafts were monitored for 60 days; graft function beyond 60 days was considered ‘indefinite acceptance’. Results are given as median survival time (MST) and as percent of recipients with indefinite graft acceptance
Skin transplantation: Mice were anesthetized with 3.6% chloral hydrate (Wiler); a circumferential band was shaved around thorax and abdomen from the level of the shoulder joints to the level of the hip joints. Skin grafts were carried out using a modified technique originally described by Billingham et al. (34) by laying a full-thickness tail-skin graft into a prepared dermal bed in a dorsal thoracic position. Gender-matched allogeneic test grafts and syngeneic control grafts were placed on all animals on either side of the dorsal thorax with at least 2 cm of undisturbed recipient skin between grafts. Collodion (BDH) was applied at the junction of the recipient skin and donor skin to secure the grafts in place. Graft sites were first covered with vaseline gauze, then bandaged circumferentially with elastic bandage. Bandages were removed at 7 days and grafts inspected visually. Rejection day was assigned when no further viable skin was visible; grafts surviving longer than 60 days were considered to have achieved indefinite survival.
In vitro assessments: At 6–10 weeks of age, in vitro proliferation of lymph node cells (LNC) from treated and untreated mice as indicated by 3H-thymidine incorporation was assessed in mixed lymphocyte reaction (MLR), and effector function was assessed by chromium-release in cell-mediated lympholysis assay (CML) with stimulation by spleen cells from C3H, BALB, B6 and NZW mice, as described by Coligan (35).
Responder LNC (3 × 103−3 × 105 cells/well) were obtained from treated and naïve C3H mice. Stimulator cells(3 × 105 cells/well) were irradiated splenocytes(2000 rads) from naïve C3H and various test strain mice. The responder and stimulator cells were cultured in 96 roundbottom microtitre plates (Nunc) at 37 °C and 5% CO2 in DMEM (Gibco/BRL) with 10% fetal calf serum (Cansera), 10 mm hepes (Gibco/BRL), 5 × 10−6 M 2-mercapto-ethanol (Sigma) and penicillin/streptomycin (Gibco/BRL).
MLR assay: After 72 h culture, 3H-thymidine (Amersham) was added at a concentration of 1 μΧι/well and incubation continued for another 24 h. Cells were harvested with a cell harvester (Skatron), then counted in a beta-counter (Wallac). The stimulation index was calculated as the mean cpm of triplicate cultures of responder cells stimulated with allogeneic cells divided by the mean cpm of responder cells stimulated with syngeneic cells.
CML assay: On day 3, target cells were prepared from splenocytes of appropriate mice. Spleens were harvested and pressed though a sieve using sterile technique; samples were washed three times and counted. Splenocytes (1 × 107) were cultured in a vertical T-25 flask (Falcon) with 10 mL media and 2 μg/mL Concanavalin A (Con A) (Pharmacia) for 48–72 h. On day 5, the Con A stimulated blast target cells were washed and run through Lympholyte M (Cedarlane). The interface was removed, washed and counted. Con A lymphoblasts(1 × 106) were transferred into 15 mL conical tubes, centrifuged; the pellet was resuspended in 100 μΧι Na51CrO4(NEN) with fetal calf serum comprising 66% of the final volume. Targets were incubated in a lead case at 37 °C, 5% CO2 for 2 h with occasional shaking, then washed three times and resuspended in 5 mL media.
After 5 days of in vitro stimulation, 100 mL of the culture supernatant was removed from each well and 3 ×10351Cr-labeled target cells were added to each well. At the same time, spontaneous release (only stimulator and target cells) and maximum release (target cells only and 1% acetic acid) were set up. The plates were then centrifuged at 1000 r.p.m. for 1.5 min and incubated behind a lead shield at 37 °C for 4 h, then centrifuged at 1500 r.p.m. for 5 min. Subsequently, 100 mL of supernatant was removed from each well and counted with a gamma counter (Wallac). Specific lysis was calculated as [(experimental − spontaneous release)/(maximal − spontaneous release)]× 100%.