To establish the minimum number of marrow cells needed to achieve chimerism and tolerance, CBA mice were treated with three doses of 1 mg of each of the blocking mAbs to CD4, CD8 and CD40 L on alternate days 4 weeks prior to BMT, transplanted with different numbers of T cell-depleted B10 BM cells, and further treated with 1 mg of the same antibodies on day 0, 2 and 4 relative to BMT (Figure 1A). Peripheral blood samples were collected 50 days (not shown) and 120 days following BMT, and the level of chimerism was quantified by flow cytometry (Figure 1B). Chimerism was found to be stable since the levels detected at these two time points were always similar in any individual mouse. All animals transplanted with 1×10⁷, 2×10⁷ or 4×10⁷ T cell-depleted BM cells had detectable levels of chimerism, as did one mouse transplanted with 5×10⁶ BM cells. All mice transplanted with 1×10⁶ BM cells demonstrated no detectable chimerism.

To determine whether tolerance was dependent on the degree of chimerism, all mice were challenged with donor type skin grafts on day 50 following BMT. In addition, (BALB/c × B10)F₁ skin was transplanted on day 120 to determine whether any tolerance observed was dominant, in which linked suppression 16 would be the expected outcome. All animals that had detectable chimerism accepted donor-type skin indefinitely (Figure 1C). (BALB/c × B10)F₁ skin grafts were readily rejected by all mice (MST=13d) indicating that we could not elicit linked suppression and dominant tolerance.

These data demonstrate, therefore, that 1×10⁷ T cell-depleted BM cells together with this particular antibody protocol, is sufficient to achieve stable mixed chimerism, and a non-dominant form of transplantation tolerance. Remarkably, this is achieved in the absence of myeloablative or myelosuppressive conditioning.

We examined the requirements for a first-stage treatment in overcoming the immunologic resistance to BM engraftment. CBA or BALB/c mice were transplanted with different doses of donor BM and treated with 3 × 1 mg of CD4, CD8 and CD40L mAbs at the time of BMT, in the presence or absence of antibody treatment 4 weeks in advance of BMT. Only animals treated with the first-stage antibody-treatment in advance of BMT had detectable chimerism (Figure 2A and 2B). Mice transplanted with BM in the absence of first-stage treatment, readily rejected donor type skin grafts transplanted 50 days following BMT (Figure 2A and 2B).

The successful treatment regime used above was not, however, effective in B10 mice that had been transplanted with 2×10⁷ T cell-depleted BM from CBA donors. Chimerism was less than 1% and all mice rejected donor-type skin (not shown).

Although antibody-treatment was critical at the time of BMT (as described below), first-stage treatment did not, however, seem to be an absolute requirement in all experiments. In one experiment, we observed that omission of the CD40L mAb made no difference to the efficacy of the first-stage treatment (Figure 2C), while in another we actually achieved long term engraftment in the absence of any first-stage treatment whatsoever (Figure 2D). Clearly, there are variations between experiments in the need for first-stage treatment, but more importantly, treatment with CD4 and CD8 mAbs does seem to guarantee routine success in permissive strains. It should be noted that CD8 mAbs on their own could not be tested for the first-stage treatment because they lead to sensitization to rat antibodies, nullifying the efficacy of subsequent antibody administration 17. This does not happen when CD8 antibodies are combined with CD4 or CD40L antibodies.

To investigate the impact of the first-stage treatment on T cell populations, spleen cells from animals treated with CD4 and CD8 mAbs were collected 4 weeks following treatment (at the time donor BM is usually transplanted) and analysed by flow cytometry. Antibody-treated mice showed a marked reduction in CD8⁺ T cells (Table 1). Relative to untreated controls, antibody-treated mice had a significantly higher frequency of CD44⁺ cells amongst CD8⁺ cells. Small but significant changes within the CD4⁺ population were observed, including an increase of CD44⁺ and CD25⁺ cells in antibody-treated mice.

We investigated whether a 2-stage protocol consisting solely of co-receptor blockade with omission of co-stimulation blockade would still enable the engraftment of donor BM. All mice received the first-stage treatment consisting of co-receptor blockade alone initiated 4 weeks before transplantation. At the time of transfer of 2×10⁷ T cell-depleted B10 bone marrow cells, the mice were split into three groups. One group received CD4, CD8 and CD40L mAbs; a second received CD4 and CD8 mAbs and a third received no mAbs. Chimerism was only achieved in the group which received the triple cocktail at the time of BM transplantation.

Based on the failure of the 2-stage protocol to enable engraftment in B10 recipients of CBA bone marrow, we turned to a congenic system to provide a less stringent immunological barrier. In 2 experiments, no engraftment was observed in B6 recipients of congenic B6.CD45.1 bone marrow in the absence of antibody-treatment, while control CBA recipients which received the 2-stage protocol were chimeric (mean +/- SD = 6.4% +/- 1.5 and 4.3% +/- 1.3). B6 recipients of the 2-stage antibody protocol plus congenic B6.CD45.1 bone marrow showed low but significant levels of chimerism (mean +/- SD = 2.7% +/- 0.3 and 2.7% +/-0.6) (Figure 4A).

We investigated whether BM engraftment in congenic mice might be facilitated by targeting NK cells. The specific targeting of NK cells by treatment with anti-NK1.1 antibody five days prior to BMT did not, however, enable BM engraftment (Figure 4B).

CBA/Ca (CBA, H-2^k), BALB/c (H-2^d), C57BL/10 (B10, H-2^b), C57BL/6 (B6, H-2^b) and B6.SJL.CD45 (B6.CD45.1, H-2^b) mice were bred and maintained in specific pathogen free (SPF) facilities at the Sir William Dunn School of Pathology (Oxford, UK). The animals used in the experiments were sex-matched and between 8 and 10 weeks of age. Procedures were conducted in accordance with the Home Office Animals (Scientific Procedures) Act of 1986.

BM donors were depleted of T cells with an i.p. injection of 1 mg of the anti-CD8 mAbs YTS156 and YTS169, and the anti-CD4 mAbs YTS191 and YTA3.1 five days prior to BM collection 27. BM was collected by flushing the femurs and tibias with RPMI medium. The cells were counted, resuspended in PBS and injected intravenously into recipient mice.

Mice were anaesthetised with a mixture of 1 mg/ml Xylazine (Rompun^®, Bayer) and 10 mg/ml Ketamine (Ketaset^®, Fort Dodge). 0.2 ml per 20 g of body weight was injected i.p. Skin grafting was performed as described elsewhere 28. Briefly, skin grafting was conducted by grafting full thickness tail-skin (1 × 1 cm) on the lateral flank. Grafts were observed on alternate days after the removal of the bandage at day 7 and considered rejected when no viable donor skin was present.

BMT were performed under the cover of 1 mg YTS177.9, 1 mg YTS105.18 and 1 mg MR1 29 as described in the text 15. All mAbs were produced in our laboratory by culture in hollow fibre bioreactors, purified from culture supernatants by 50% ammonium sulphate precipitation, dialysed against PBS, and the purity checked by native and SDS gel electrophoresis (PhastGel, Pharmacia, St. Albans, UK).

Hematopoietic chimerism was quantified by staining peripheral blood with mAbs specific for H-2D^b, H-2K^k and H-2K^d (all from BD Biosciences). To distinguish cells from the congenic strains we used mAbs specific for CD45.1 and CD45.2 (BD Biosciences). To study the effect of first-stage antibody treatment only, splenocytes were subjected to osmotic lysis of red blood cells before staining with mAbs specific for CD3, CD4, CD8, CD25 and CD44 (all from BD Biosciences). Cells were analysed with a FACScalibur (BD Biosciences) and CellQuest software (BD Biosciences).

Statistical analysis of graft survival was made by the log rank method. P values in Table 1 were calculated by the student's T test (unpaired, 2-tailed).