Age-related factors in cyclosporine-induced syngeneic graft-versus-host disease: regulatory role of marrow-derived T lymphocytes

Mature Animals that Modify the Induction of SGVHD The studies described above demonstrated that marrow from mature animals modified the induction ofSGVHD when added to the marrow inoculum from animals 1 mo of age. A series of experiments was undertaken to identify and characterize the cell population responsible for this effect. Marrow from Lewis rats of 1 or 6 mo of age was separated on the basis of size and density by counterflow centrifugal elutriation, a technique routinely used to T cell deplete human bone marrow (16) and recently adapted for use in the rat, allowing excellent recovery of the cells (17) . Small lymphocytes are purged at a flow rate of 25 ml/min, larger lymphocytes and myeloid cells at 29 ml/min, and the clonogenic hemopoietic and blast cells are collected by stopping the rotor, thus named the R/O fraction . Initial studies compared the separation characteristics of marrow from 1-and 6-mo Lewis rats. There was a moderate increase in the number of cells isolated in fraction 25 of marrow from 1-mo-old animals (44%) compared with marrow from animals 6 mo of age (28%). This was reflected by differences in yield from the 29 and R/O fractions (fraction 29, 11% and 16%; R/O fraction, 43% vs. 53%, respectively) . Studies were performed to assess if a given fraction from the marrow of donors 6 mo of age was responsible for preventing the induction of SGVHD. Marrow from Lewis rats 6 mo of age was elutriated into 25, 29, and R/O fractions, and 4 x 107 cells from each fraction were transfused with 2 x 107 or 4 x 107 unfractionated bone marrow cells from 1-mo-old syngeneic donors into lethally irradiated recipients (8-9 wk of age). The incidence ofSGVHD in the recipients was recorded after 30 d of CsA therapy with clinical observations and sequential biopsies until the time of autopsy. The results are summarized in Table 2 Marrow was harvested from 6-mo-old Lewis rats and fractionated by elutriation . Unfractionated marrow or the respective 25, 29, R/O elutriation fractions (4 x 107 cells) were added to marrow derived from 1-mo-old syngeneic Lewis rats and infused into Lewis rats (8) (9) wk of age). As controls, the recipients received either marrow from rats 1 or 6 mo of age or the elutriation fractions separately. The recipients were treated with a 30-d course of CsA therapy (15 mg/kg/d) and observed for the onset of SGVHD. marrow from donors 6 mo of age resulted in five of eight rats developing SGVHD. However, SGVHD did not occur in recipients receiving either 2 or 4 x 107 bone marrow cells from the immature donors and 4 x 107 elutriated bone marrow cells isolated in fraction 25 from donors 6 mo of age. Thus, fraction 25 had the most potent inhibitory effect on the prevention ofsyngeneic GVHD. Addition ofthe R/O and/or fraction 29 from marrow of 6-mo-old animals to unfractionated marrow from donors 1 mo of age did not significantly influence the induction ofsyngeneic GVHD. Furthermore, animals grafted separately with the R/O fraction or the 29 fraction from marrow of animals 6 mo of age, engrafted and developed SGVHD after a course of CsA (15 mg/kg) therapy. Animals did not engraft when fraction 25 was used singly. The potent capability ofunfractionated marrow from donors 6 mo of age to inhibit the primary induction of syngeneic GVHD with marrow from immature animals appeared to be isolated in elutriation fraction 25. This fraction also accounted for the inability of marrow from animals 6 mo of age to allow the induction ofSG