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Objective: To compare the suppressive effect of mesenchymal stem cells (MSC), derived from normal individuals or severe aplastic anemia patients (SAA), on T-cell activation., Patients and Methods: We studied bone marrow MSC from 19 healthy donors and 23 SAA patients in different phases of the disease: at diagnosis (n = 3), following immunosuppressive therapy (IS) (n = 16), or after a bone marrow transplant (BMT) (n = 4). MSC were tested for T-cell suppression in the following assays: mixed lymphocyte reaction (MLR), phytohemaglutinin (PHA)-primed cultures, activation surface markers, gamma-IFN production, hematopoietic colony formation (CFC), production of cyclic ADP-ribose (cADPR)., Results: The abnormalities of SAA MSC included: 1) significantly lower suppression of T-cell proliferation induced by alloantigens (p = 0.009) or PHA (p = 0.006); 2) impaired capacity to suppress CD38 expression on PHA-primed T cells (p = 0.001); 3) impaired ability to suppress gamma-IFN production in PHA cultures, resulting in an 11-fold higher gamma-IFN concentration; 4) no preventive effect on T cell-mediated inhibition of CFC; and 5) significantly reduced (p = 0.009) production of cADPR, a universal calcium mobilizer. MSC-mediated suppression of PHA-induced T-cell proliferation was restored to control levels in 3 of 4 patients post-BMT., Conclusion: The ability of MSC to downregulate T-cell priming, proliferation, and cytokine release is deficient in patients with SAA, persists indefinitely after immunosuppressive therapy, but seems to be restored after BMT. Whether these abnormalities are relevant to the pathogenesis of aplastic anemia remains to be determined.

This is a retrospective study of 97 patients who received either allogeneic bone marrow transplant (BMT) (n=52) or peripheral blood cell transplant (PBCT) (n=45) at our institution from human leukocyte antigen (HLA)-identical sibling donors between January 1994 and January 1997. The two groups were comparable with respect to diagnosis, age, sex, interval from diagnosis, and disease phase. They were prepared with cyclophosphamide (CY) and fractionated total-body irradiation (TBI) (n=51) or CY and thiotepa (n=46). Graft-vs.-host disease (GVHD) prophylaxis consisted of cyclosporin A and methotrexate. Patients who received PBCT exhibited faster neutrophil engraftment (day 14 vs. day 16, p = 0.002) than those in the BMT group, as well as higher platelet counts on day 20 (32x10(9)/kg vs. 21x10(9)/kg, p = 0.001), but graft function as assessed by platelet counts on days 50, 100, and thereafter was comparable. The number of days spent in the hospital, days on intravenous antibiotics, and days of fever were lower in the PBCT group, but not significantly. Acute GVHD, chronic GVHD, and cytomegalovirus infections were comparable between the two groups. The overall actuarial 3-year transplant-related mortality (TRM) rate for BMT vs. PBCT patients was 20 vs. 33% (p = 0.1), the survival rate was 53 vs. 48% (p = 0.3), and the relapse rate was 42 vs. 43% (p = 0.8). For patients in first complete remission, these figures were TRM 12 vs. 22% (p = 0.2), survival rate 75 vs. 70% (p = 0.4) and relapse rate 31 vs. 9% (p = 0.4), respectively, for the BMT and PBCT groups. These data suggest that the short-term outcome of allogeneic PBCT is not significantly different from that of allogeneic BMT in patients with hematologic malignancies. Long-term results are not available at present.

A total of 29 consecutive patients with leukemia or aplastic anemia who received an HLA-identical marrow graft were given cyclosporin A (CyA) to prevent graft-versus-host disease (GvHD). These patients were compared with an historic group of 25 similar patients with leukemia or AA given methotrexate (MTX) for GvHD prophylaxis at this institution. Engraftment was faster in patients given CyA when compared with MTX patients, with less days of granulocytopenia (P = 0.04), a shorter interval before reaching a platelet count of 70 X 10(9)/l (P = 0.04), fewer major infections (P = 0.01), and fewer days on intravenous antibiotics (P = 0.02). There were no graft failures in CyA patients compared with four of 25 in MTX patients (P = 0.01). Early mortality was lower in CyA patients but not significantly (P = 0.06). The incidence of pulmonary complications was comparable, five of 29 and seven of 25 in CyA and MTX patients, respectively, but the clinical features of such complications differed. Interstitial pneumonia developing after day 30 was seen in MTX patients, whereas an acute respiratory distress syndrome developing between day +8 and day +18 was seen in CyA patients. Acute GvHD was less severe in CyA patients (P = 0.04), but chronic GvHD was comparable (P = 0.3). The actual one-year survival is currently 72% and 52% in CyA and MTX patients, respectively (P = 0.1). Although our initial experience with CyA is encouraging with regard to engraftment and acute GvHD, optimization of CyA protocols will probably be needed for it to be proven as having a definite advantage over MTX.

In vitro colony formation (CFUC) was studied in 8 patients with severe aplastic anemia (SAA) in complete hematologic remission following high dose pulse methylprednisolone (P/6-MPr) and/or antilymphocyte globulin (ALG). All patients except one were off maintenance treatment at the time of study, and follow-up ranged from 271 to 630 days. All patients showed marked reduction of colonies (2 +/- 3/2.5 x 10(5) cells; our normal 61 +/- 14) and clusters (77 +/- 45; our normal 179 +/- 83), which persisted for as long as 600 days after initial therapy. Incubation of patients' marrow with ALG did not enhance significantly colony formation. Co-culture of bone marrow from 6 patients with SAA in remission and normal marrow produced a marked inhibition of the expected colonies (84 +/- 16%) and clusters (29 +/- 23%). Incubation of patients' marrow with ALG prior to the co-culture experiments did not prevent suppression of colonies and clusters. Co-culture of patients' peripheral blood lymphocytes with normal marrow had no effect on colony formation. Therefore remission marrow from patients with SAA exhibits severely impaired in vitro growth (CFUC) and marked myelosuppressive activity against normal marrow cells. Incubation of patients' marrow with ALG has no significant effect on either assay.

T derived colony inhibitory activity (Td/CIA) was obtained from unstimulated T cells from aplastic anemia patients (SAA), or from PWM primed normal T cells. Td/CIA suppressed CFUC growth of normal allogeneic marrow to less than 30% of expected growth. Td/CIA was then added to normal peripheral blood T and B cells, primed with PWM, to test whether it would interfere with in vitro immunoglobulin (Ig) production. When Td/CIA from normal T cells was added to cultures of T + B cells + PWM there was a 2-2.1-fold increase in Ig production. Similarly the addition of Td/CIA from SAA patients also resulted in a 1.4 up to 166-fold increase in Ig production. These results indicate that either (a) the targets for Td/CIA are expressed on hemopoietic but not on T and B cells, or (b) that Td/CIA inactivates an accessory cell which is essential for CFUC growth but not for the PWM driven in vitro B cell differentiation system.

T lymphocytes were derived by E-rosetting techniques from the peripheral blood and bone marrow of 12 healthy donors. Following incubation of 18 h with PWM, PHA, Con-A or culture medium (RPMI) alone, the lymphocytes were harvested by centrifugation and washed. Both lymphocytes and culture supernatants were tested for CFUC suppressor activity in semisolid bone marrow cultures. The results of this study indicate that (a) T cells and supernatants from unstimulated cultures contained no CFUC suppressor activity, (b) T cells and supernatants from cultures stimulated with PHA, PWM or Con-A significantly suppressed autologous and allogeneic bone marrow CFUC, (c) there was no significant difference between the ability of bone marrow or peripheral blood T cells to inhibit CFUC, and (d) mitogens alone had either no effect or a moderate enhancing activity on marrow CFUC. These findings suggest that generation of CFUC suppressor T cells occurs normally within a few hours of polyclonal T cell stimulation.

Forty-two patients with severe aplastic anemia (SAA) were treated with immunosuppressive therapy (IS) consisting of one or more courses of the following regimens: a) high dose bolus 6 methylprednisolone (BMPr); b) horse antilymphocytic globulin (HALG); c) rabbit ALG (RALG); d) ALG followed by the infusion of haploidentical marrow (ALG +/- BM); e) BMPr in combination with HALG; f) ALG in combination with androgens. All patients received one initial course of IS: 16 (38%) showed hematologic reconstitution and required no further transfusions, of 26 refractory patients 4 died, and 22 received a second course of IS 60 days after the first course. Of these 22, 6 (27%) responded, and of the 16 refractory patients, 9 died, and 7 received a third course of IS 60 days after the second course. Two of these responded (28%), 3 died and 2 are alive but pancytopenic. The overall response rate is 24/42 patients (57%): all of these patients are transfusion independent 6-60 months post-treatment, and 17 are off maintenance therapy with low dose steroids. The actuarial 5 year survival is currently 60%; 13/26 surviving patients have been followed for more than 3 years. The present study confirms that over 50% of patients with SAA can recover from their aplasia following IS treatment, and this is of relevance for patients who can not be offered a bone marrow transplant.