Inbred female C57BL/6ByJ (B6, H-2b
), BALB/cByJ (BALB/c, H-2d
) mice, and C57BL/6-Rag1tm1/Mom
) were purchased from The Jackson Laboratory (Bar Harbor, Maine, USA). Female C57BL/6 I-Ab
-gene targeted MHC class II–/–
deficient (C2D, H-2b
) mice (19
) and immune-deficient C.B-17scid/scid
) female mice (20
) were obtained from Taconic Farms (Germantown, New York, USA). Animals were housed under pathogen-free conditions at the University of Colorado Barbara Davis Center Animal Facility, according to NIH guidelines.
Generation of C2D rag1–/– mice.
To generate immune-deficient rag1–/– mice that were also MHC class II deficient, C2D mice were crossed with B6 rag1–/– mice and then intercrossed to generate double-deficient mice. The rag1–/– phenotype was determined by the lack of detectable lymphocytes in peripheral blood leukocytes (PBLs), and the C2D genotype was assessed by PCR screening of genomic DNA for the disrupted IAβb allele. The C2D phenotype also was confirmed functionally by the inability of C2D stimulator cells to trigger in vitro proliferation of BALB/c CD4 T cells relative to MHC class II+/+ rag1–/– stimulator cells. Homozygous C2D rag1–/– mice then were interbred for experimental use.
Heterotopic heart transplantation.
Cardiac allografts from BALB/c mice were transplanted heterotopically into B6, B6 rag1–/–
, or C2D B6 rag1–/–
mice. Cardiac allografts from B6 or C2D mice were transplanted heterotopically into BALB/c mice or into SCID mice. Vascularized grafts were transplanted according to standard microsurgical techniques (21
). Briefly, the harvested donor heart was placed in 4°C saline until transplantation. Under avertin-induced anesthesia, a 2-cm midline vertical abdominal incision was made, and the abdominal cavity entered. The abdominal aorta and inferior vena cava (IVC) were isolated below the renal vessels. An end to side anastomosis of the donor aorta to the recipient aorta was made using running 10-0 nylon suture. An end to side anastomosis of the donor pulmonary artery to the recipient IVC was made in similar fashion. The abdominal wall was closed in two layers using 5-0 silk suture in a running fashion. A 1.0-ml bolus of sterile normal saline was administered into the abdomen as fluid resuscitation upon closing. No other supportive measures were required during the surgery. Heart graft survival was monitored by daily palpation with rejection defined as cessation of detectable beat.
CD4 T-cell depletion in vivo.
The rat anti-mouse CD4 mAb GK 1.5 (IgG2b) (22
) was produced for in vivo CD4 T-cell depletion. GK1.5 antibody was generated as ascites in SCID mice and quantitated by an isotype-specific ELISA. BALB/c recipients were either left untreated or were treated with GK1.5 antibody (10 mg/kg) administered intraperitoneally to the indicated BALB/c recipients on days –1, 0, 1, and 2 relative to transplant.
CD4 T-cell purification and adoptive transfer.
Cervical, axillary, and mesenteric lymph nodes (LNs) were harvested from BALB/c or B6 mice. Single-cell suspensions of LN cells were enriched for CD4 T cells by negative selection of CD8 T cells and B cells on an immunoaffinity column according to the manufacturer’s specifications (Cellect, Edmonton, Alberta, Canada). Where indicated, B6 CD4-enriched lymphoid populations were further depleted of residual MHC class II–bearing cells with anti-I-Ab antibody (25-9-3S; IgM) plus rabbit complement (LowTox-M; Accurate Chemical & Scientific Corp., Westbury, New York, USA) treatment for 1 hour at 37°C. Cellular phenotyping of freshly purified cells or of PBLs of adoptive transfer recipients was determined by flow cytometry assessing staining of FITC-labeled anti-CD4, anti-CD8, or anti-B220 mAb’s (PharMingen, San Diego, California, USA) detected by an EPICS ELITE ESP flow cytometer (Coulter Corp., Miami, Florida, USA). CD4-enriched T cells contained less than 0.5% contaminating CD8 T cells or B220+ cells. Ten million unseparated LNs or CD4-enriched T cells were injected intraperitoneally into the indicated adoptive transfer recipients on day 0 relative to cardiac transplant.
Mixed lymphocyte reaction of reconstituting lymphoid populations.
Mixed lymphocyte reaction (MLR) of adoptively transferred cell populations were also performed to demonstrate cell viability and function. Briefly, 2.0 × 105 responder cells were mixed with 3.0 × 105 irradiated (3000R) splenic stimulator cells in 96-well flat-bottom plates. Cells, cultured in EMEM supplemented with 10% FCS, 10–5 M 2-Me, and antibiotics, were incubated at 37°C in 10% CO2. Cultures then were pulsed with 1.0 μCi [H3]thymidine for 6 hours on the indicated day of cell culture. Plates were harvested and counted on a TopCount microplate scintillation counter (Packard Instrument Corp., Meriden, Connecticut, USA).
Tissue histological examination.
Transplanted and native hearts were removed and divided in half in long axis perpendicular to the intraventricular septum. One half of the transplant was fixed in 10% formalin, and the other was imbedded in OTC and snap frozen to –70°C. The formalin fixed tissue was paraffin embedded, and sections were cut and stained with hematoxylin and eosin (H&E) and trichrome stains. These were examined in a blinded fashion to determine the extent of myocardial damage, mononuclear and granulocyte cell infiltration, and vasculitis and/or vascular intimal proliferation.
Parallel sections were analyzed by immunohistochemistry on frozen tissue sections by air drying overnight and fixing with acetone. Sections were rehydrated in TBS, washed, and then blocked with 1:5 normal rabbit serum in TBS containing Vector avidin DH (Vector Laboratories Inc., Burlingame, California, USA). KT6 supernatant (rat anti-mouse CD4) or YTS105 supernatant (rat anti-mouse CD8) was applied and incubated for 45 minutes at room temperature. Biotinylated rabbit anti-rat Ig (1:200) was applied and incubated for 30 minutes. Vectastain Elite ABC Reagent (Vector Laboratories Inc.) was applied and then counter stained with Mayer’s hematoxylin. Tissue sections were examined for immunoperoxidase staining by light microscopy for the presence of CD4 and/or CD8 T cells.
Detection of alloantibody production.
Alloreactive antibody production was assessed by the ability of sera from grafted animals to fix complement to 51Cr-labeled Con A blast target cells. Briefly, 104 labeled blast target cells were mixed with serial twofold dilutions of test sera and rabbit complement (LowTox-M) in 200 μl and incubated in 96-well V-bottom tissue culture plates for 1 hour at 37°C. The 51Cr release was measured on a microplate scintillation counter, and end-point titers were determined as the reciprocal of the serum dilution producing 51Cr-release greater than 3 SD above replicate wells of target cells plus complement alone.
Mann-Whitney U and Fischer exact tests using commercially available software were used to determine significance of graft survival data in adoptive transfer studies.