Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex.

We aimed to study the effects of mesenchymal stem cells (MSCs) on alloreactivity and effects of T-cell activation on human peripheral blood lymphocytes (PBLs) in vitro. MSCs were expanded from the bone marrow of healthy subjects. MSCs isolated from second to third passage were positive for CD166, CD105, CD44, CD29, SH-3 and SH-4, but negative for CD34 and CD45. MSCs cultured in osteogenic, adipogenic or chondrogenic media differentiated, respectively, into osteocytes, adipocytes or chondrocytes. MSC added to PBL cultures had various effects, ranging from slight inhibition to stimulation of DNA synthesis. The stimulation index (SI = (PBL + MSC)/PBL) varied between 0.2 and 7.3. The SI was not affected by the MSC dose or by the addition of allogeneic or autologous MSCs to the lymphocytes. Suppression of proliferative activity was observed in all experiments after the addition of 10,000-40,000 MSCs to mixed lymphocyte cultures (MLCs). Lymphocyte proliferation was 10-90%, compared with a control MLC run in parallel without MSCs. In contrast, the addition of fewer MSCs (10-1000 cells) led to a less consistent suppression or a marked lymphocyte proliferation in several experiments, ranging from 40 to 190% of the maximal lymphocyte proliferation in control MLCs. The ability to inhibit or stimulate T-cell alloresponses appeared to be independent of the major histocompatibility complex, as results were similar using 'third party' MSCs or MSCs that were autologous to the responder or stimulating PBLs. The strongest inhibitory effect was seen if MSCs were added at the beginning of the 6 day culture, and the effect declined if MSCs were added on day 3 or 5. Marked inhibitory effects of allogeneic and autologous MSCs (15,000) were also noted after mitogenic lymphocyte stimulation by phytohaemagglutinin (median lymphocyte proliferation of 30% of controls), Concanavalin A (56%) and protein A (65%). Little, if any, inhibition occurred after stimulation with pokeweed mitogen. Low numbers of MSCs (150 cells) were unable to inhibit mitogen-induced T-cell responses. MSCs have significant immune modulatory effects on MLCs and after mitogenic stimulation of PBL. High numbers of MSCs suppress alloreactive T cells, whereas very low numbers clearly stimulated lymphocyte proliferation in some experiments. The effect of a larger number of MSCs on MLCs seems more dependent on cell dose than histocompatibility and could result from an 'overload' of a stimulatory mechanism.

Leukemia lineage-specific chimerism analysis is a sensitive predictor of relapse in patients with acute myeloid leukemia and myelodysplastic syndrome after allogeneic stem cell transplantation.

One of the major complications after allogeneic stem cell transplantation (SCT) in patients with malignant disease is a high frequency of relapse. We have prospectively analyzed the clinical impact of recipient-derived chimeric cells in 30 patients with acute myeloid leukemia and myelodysplastic syndrome after SCT. In order to improve sensitivity and specificity, all samples were cell-separated by using immunomagnetic beads according to the patient's leukemia phenotype, expressed at diagnosis or relapse before SCT. Twelve out of 30 patients experienced a clinical relapse after SCT. Median follow-up time for patients alive and without relapse (n = 15) was 30 (16-47) months. Mixed chimerism in peripheral blood (PB) and bone marrow (BM) > or =1 month post SCT, in the leukemia-affected cell lineage, was detected in 14/30 patients. Ten of these 14 patients relapsed as compared to 2/16 with donor chimerism (DC) (P <0.01). All eight patients with MC in peripheral blood > or =1 month after SCT relapsed vs 4/22 DC patients (P < 0.001). MC was detected a median of 66 (23-332) days before hematological relapse. No correlation was found between T cell MC and relapse. In this study, chimerism analysis showed a higher sensitivity and specificity vs morphological examination. In conclusion, this study may provide a rational basis for treatment with adoptive immunotherapy at an earlier time after SCT than at present, in patients with AML and MDS, in order to treat recurrences of malignant disease.

Poor immune reconstitution after four or five major HLA antigens mismatched T cell-depleted allogeneic and autologous stem cell transplantation.

Two adults with primary liver cancer underwent liver transplantation from 5/6 and 4/6 major HLA-antigen mismatched unrelated donors. They were then conditioned with 4 x 2 Gy of total lymphoid irradiation, 120 mg/kg cyclophosphamide, 7.5 Gy total body irradiation and anti-T cell antibodies. Thereafter, the patients received T cell-depleted autologous: unrelated mismatched bone marrow in a proportion of 0.5:3.0 and 0.35:1.1 x 10(6) CD34+ cells/kg, respectively. After allogeneic stem cell transplantation (ASCT), both became mixed chimeras, as determined with polymerase chain reaction amplification of variable number tandem repeats from DNA obtained from CD3+, CD19+ and CD45+ magnetic bead-separated cells. Due to a reduction in donor T cells, the first patient was given 10(5) donor T cells/kg and became a complete donor chimera within 3 months. The second patient rejected all donor cells within 1 month after ASCT. Leucocytes normalized in both patients within 1 month. CD8+ cells normalized after 4 and 2 months in the two patients, respectively. However, CD4+, CD56+ and CD19+ cells remained low, except for a transient increase in patient 2. Lymphocyte responses to mitogens were negative in patient 1 from 1 to 5 months after ASCT. This patient also showed an oligoclonal pattern of the B cell repertoire, performed by CDR3 spectratyping. Epstein-Barr virus DNA in lymphocytes increased by 4-5 log in both patients. Prior to ASCT, recipients and donors were mutually reactive in mixed lymphocyte cultures (MLC). In the first patient, who became a complete donor chimera, the chimera cells showed no response to recipient or donor, but a positive response to third party. In the other patient, recipient cells reacted vigorously against donor lymphocytes at the time of rejection. Both patients suffered from overwhelming bacterial, fungal and viral infections, and died of pneumonia 5 and 3 months after ASCT, respectively. To conclude, with a major HLA-mismatch barrier, stable mixed chimerism seems difficult to achieve. The first patient became a full donor chimera and the second one rejected the graft. Both suffered from immune incompetence.

Mixed chimerism in the B cell lineage is a rapid and sensitive indicator of minimal residual disease in bone marrow transplant recipients with pre-B cell acute lymphoblastic leukemia.

One of the major problems after allogeneic bone marrow transplantation (BMT) is a high frequency of leukemia relapse. We have prospectively studied the presence of donor- and recipient-derived chimeric cells in bone marrow recipients with pre-B cell acute lymphoblastic leukemia (pre-B-ALL). The chimeric status of BMT recipients was compared to minimal residual disease (MRD) detection by analysis of immunoglobulin heavy chain (IgH) and T cell receptor (TcR) genes. Post-transplant blood and bone marrow samples from 12 patients with pre-B-ALL were studied. Five patients showed mixed chimerism (MC) in the CD19-positive cell fraction. Four of them have relapsed to date. The remaining patient with MC in the B cell lineage was also MRD positive in the same samples. All seven patients with donor chimerism in the B cell fraction remain in clinical remission (P = 0.01). In samples from all five patients having MC in the B cell lineage, the patient-specific IgH or TcR rearrangement was also detected. In three of four patients who relapsed, MC in the B cell lineage was seen more than 2.5 months prior to morphologically verified relapse. The results of this comparison suggest that routinely performed MC analysis of the affected cell lineage may facilitate post-BMT monitoring and rapid therapeutic decisions in transplanted patients with pre-B-ALL.

Faster neutrophil and platelet engraftment, but no differences in acute GVHD or survival, using peripheral blood stem cells from related and unrelated donors, compared to bone marrow.

In this retrospective study, 23 recipients of peripheral blood progenitor cells (PBPC) were compared to 23 recipients of bone marrow (BM). The donors were 12 HLA-A-B-DR identical siblings and 11 HLA-A-B-DR identical unrelated donors in the PBPC and BM groups, respectively. Diagnoses in the PBPC group were CML seven, AML, nine, ALL three, lymphoma one, myeloma two and aspartylglucosaminuria (AGU) one. The median age was 40 (5-55) years. The BM group was matched for diagnosis, age, conditioning therapy, GVHD prophylaxis and G-CSF treatment after BMT. A higher number of MNC (P<0.001), CD34+ (P = 0.05), CD3+ (P<0.001) and CD56+ (P<0.001) cells in the graft, a reduced number of platelet transfusions (P = 0.03) and a significant hastening of neutrophil and platelet recovery were seen in the PBPC group compared to the BM group. In logistic regression analysis, the following factors were important for engraftment of ANC >0.5 x 10(9)/l: peripheral blood progenitor cell transplantation (PBPCT) (P = 0.003) and mononuclear cells (MNC) > or =2.5 x 10(8)/kg recipient in the graft (above median) (P = 0.009) in univariate analysis. For recovery of platelets >30 x 10(9)/l: PBPCT (P = 0.03) and HLA-identical sibling donors (P = 0.05) were significant in multivariate analysis. A trend towards a lower incidence of bacteremia was seen in the PBPC group, ie 22 vs 48% (P = 0.06) in the BM group. GVHD, TRM and survival did not differ between the two groups.

Inhibition of immunoglobulin production in vitro by IgG and F(ab')2 fragments, but not by the Fc portion.

Antibody-secreting B cells were measured as plaque-forming cells (PFC) in a modified haemolysis-in-gel assay, using protein A coupled sheep erythrocytes as targets. Human lymphocytes from blood (PBL), bone marrow or spleen were stimulated in vitro by various polyclonal B-cell activators and incubated with intravenous immunoglobulin (IVIG) or peptide fragments of IVIG. IgG and IgM production from PBL and bone marrow cells, measured as PFC, was inhibited more than 50% by IVIG 2.5 mg/ml, compared to controls without IVIG. Inhibition of the IgG and IgM response of spleen cells by IVIG varied depending on the stimuli. Using Staphylococcus aureus protein A (SPA), inhibition was almost 90% (P < 0.001). The inhibition of the IgG and IgM responses to lipopolysaccharide from Escherichia coli (LPS) were 70% (P < 0.01) and 28% (P < 0.05), respectively. IgG stimulation by pokeweed mitogen (PWM) was inhibited by 57% (P < 0.01), but the IgM response was inhibited only by the higher IVIG concentration of 5.0 mg/ml. In mixed lymphocyte cultures of spleen cells, IgG and IgM production were inhibited by more than 60% (P < 0.05). The effect of IgG, IgG-F(ab')2 and IgG-Fc on LPS or PWM-stimulated spleen cells were compared, using equimolar concentrations of the various preparations. IgG- and IgM-producing PFC were significantly (P < 0.05) inhibited in a dose-dependent fashion by IgG and F(ab')2, but not by Fc. LPS-induced IgG and IgM production was inhibited also when IgG and F(ab')2 were added up to 48 h after the stimulator. A comparison of IgG, F(ab')2 and Fc products from different companies showed that all IgG and F(ab')2 preparations significantly inhibited IgG and IgM production of LPS-stimulated spleen cells. No significant inhibition was obtained with any of the purified Fc products.