Olle Ringdén

HLA expression and immunologic propertiesof differentiated and undifferentiated mesenchymal stem cells

OBJECTIVE Mesenchymal stem cells (MSC) do not elicit alloreactive lymphocyte responses due to immune modulations. We investigated the immunologic properties of MSC after differentiation along three lineages: bone, cartilage, and adipose. METHODS AND RESULTS Flow cytometry showed that undifferentiated MSC express HLA class I but not class II, although HLA class II was present intracellularly as detected by Western blot. Addition of interferon gamma (IFN-gamma) for 48 hours induced greater than 90% of cells to express HLA class II. No lymphocyte response was induced by allogeneic irradiated MSC as stimulators. Results were similar using MSC pretreated with IFN-gamma. After growth of cells in medium to induce differentiation to bone, cartilage, or adipose for 6 or 12 days, the expression of HLA class I increased but no class II was detected on the cell surface. The ability to upregulate HLA class II on the cell surface after exposure to IFN-gamma for 48 hours was clearly diminished after the cells had been cultured in differentiation medium for 6 or 12 days, with only 10% of cells expressing HLA class II. Using MSC grown in osteogenic, chondrogenic, or adipogenic medium as stimulator cells, no lymphocyte alloreactivity was seen, even if differentiated MSC had been pretreated with IFN-gamma. MSC inhibit mixed lymphocyte cultures, particularly after osteogenic differentiation. This suppression was further enhanced by IFN-gamma. CONCLUSIONS Undifferentiated and differentiated MSC do not elicit alloreactive lymphocyte proliferative responses and modulate immune responses. The findings support that MSC can be transplantable between HLA-incompatible individuals.

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.

Mesenchymal stem cells for treatment of therapy-resistant graft-versus-host disease.

Background. Mesenchymal stem cells (MSC) have immunomodulatory effects. The aim was to study the effect of MSC infusion on graft-versus-host disease (GVHD). Methods. We gave MSC to eight patients with steroid-refractory grades III-IV GVHD and one who had extensive chronic GVHD. The MSC dose was median 1.0 (range 0.7 to 9)×106/kg. No acute side-effects occurred after the MSC infusions. Six patients were treated once and three patients twice. Two patients received MSC from HLA-identical siblings, six from haplo-identical family donors and four from unrelated mismatched donors. Results. Acute GVHD disappeared completely in six of eight patients. One of these developed cytomegalovirus gastroenteritis. Complete resolution was seen in gut (6), liver (1) and skin (1). Two died soon after MSC treatment with no obvious response. One of them had MSC donor DNA in the colon and a lymph node. Five patients are still alive between 2 months and 3 years after the transplantation. Their survival rate was significantly better than that of 16 patients with steroid-resistant biopsy-proven gastrointestinal GVHD, not treated with MSC during the same period (P=0.03). One patient treated for extensive chronic GVHD showed a transient response in the liver, but not in the skin and he died of Epstein-Barr virus lymphoma. Conclusion. MSC is a very promising treatment for severe steroid-resistant acute GVHD.

Mesenchymal stem cells inhibit the formation of cytotoxic T lymphocytes, but not activated cytotoxic T lymphocytes or natural killer cells.

Background. Mesenchymal stem cells (MSCs) can reduce the incidence of graft-versus-host disease because of their ability to inhibit T-lymphocyte proliferation. There are no publications on the effect that MSCs have on cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, effector cells vital for the graft-versus-leukemia effect. Methods. Cytotoxic T cells were primed in mixed lymphocyte culture (MLC) against irradiated stimulator lymphocytes, and irradiated third-party MSCs were added at different time points. The CTLs were collected, and their cytotoxic potential was analyzed in a chromium-release assay against the same stimulator cells as in the MLC. Purified NK cells were mixed with irradiated MSCs, and the lysis was measured in chromium-release assay against K562 target cells. Results. We found that MSCs inhibited CTL-mediated lysis by 70% if added at the beginning of the 6-day MLC. The lysis was not affected on day 3 or in the cytotoxic phase. Furthermore, MSCs inhibited the formation of cytotoxic lymphocytes when the cells were separated in a transwell system, which indicates that the effect is mediated by a soluble factor. NK cell-mediated lysis of K562 cells was not inhibited by MSCs. MSCs did not induce proliferation of allogeneic lymphocytes, and they were not lysed by allogeneic CTLs or NK cells. Conclusion. Our findings indicate that MSCs escape recognition by CTLs and alloreactive NK cells, and inhibit the formation of cytotoxic T cells by secreting a soluble factor, but that they do not interfere with CTLs and NK cell lysis.

Immunomodulation by mesenchymal stem cells and clinical experience

Mesenchymal stem cells (MSCs) from adult marrow can differentiate in vitro and in vivo into various cell types, such as bone, fat and cartilage. MSCs preferentially home to damaged tissue and may have therapeutic potential. In vitro data suggest that MSCs have low inherent immunogenicity as they induce little, if any, proliferation of allogeneic lymphocytes. Instead, MSCs appear to be immunosuppressive in vitro. They inhibit T‐cell proliferation to alloantigens and mitogens and prevent the development of cytotoxic T‐cells. In vivo, MSCs prolong skin allograft survival and have several immunomodulatory effects, which are presented and discussed in the present study. Possible clinical applications include therapy‐resistant severe acute graft‐versus‐host disease, tissue repair, treatment of rejection of organ allografts and autoimmune disorders.

Transplantation of mesenchymal stem cells to enhance engraftment of hematopoietic stem cells.

Seven patients underwent treatment with mesenchymal stem cells (MSCs), together with allogeneic hematopoietic stem cell transplantation (HSCT). MSCs were given to three patients for graft failure and four patients were included in a pilot study. HSCT donors were three human leukocyte antigen (HLA)-identical siblings, three unrelated donors and one cord blood unit. The conditioning was myeloablative in four patients and reduced in three patients. MSC donors were HLA-identical siblings in three cases and haploidentical in four cases. Neutrophil counts >0.5 × 109/l was reached at a median of 12 (range 10–28) days. Platelet counts >30 × 109/l was achieved at a median of 12 (8–36) days. Acute graft-versus-host disease (GVHD) grade 0–I was seen in five patients. Two patients developed grade II, which in one patient evolved into chronic GVHD. One severe combined immunodeficiency (SCID) patient died of aspergillosis, the others are alive and well. One patient, diagnosed with aplastic anemia had graft failure after her first transplantation and severe Henoch–Schönlein Purpura (HSP). After retransplantation of MSCs and HSCs, she recovered from both the HSP and aplasia. Thus, co-transplantation of MSC resulted in fast engraftment of absolute neutrophil count (ANC) and platelets and 100% donor chimerism, even in three patients regrafted for graft failure/rejection.

Mesenchymal Stem Cells Inhibit the Expression of CD25 (Interleukin-2 Receptor) and CD38 on Phytohaemagglutinin-Activated Lymphocytes

Mesenchymal stem cells (MSC) are immunomodulatory and inhibit lymphocyte proliferation. We studied surface expression of lymphocyte activation markers and secreted cytokines, when lymphocytes were activated in the presence of MSC. MSC suppressed the proliferation of phytohaemagglutinin (PHA)‐stimulated CD3+, CD4+ and CD8+ lymphocytes. MSC significantly reduced the expression of activation markers CD25, CD38 and CD69 on PHA‐stimulated lymphocytes. Mixed lymphocyte culture (MLC) supernatants containing MSC suppressed proliferation of MLC and PHA‐stimulated lymphocytes dose‐dependently. MSC secrete osteoprotegerin (OPG), but not hepatocyte growth factor (HGF) or transforming growth factor‐β (TGF‐β). Stromal‐cell‐derived factor‐1 (SDF‐1) is not expressed on the cell surface. A recent report suggested that T‐cell suppression by MSC is mediated by HGF and TGF‐β. MSC suppression was not restored by the addition of neutralizing antibodies against SDF‐1, OPG, HGF or TGF‐β, alone or in combination. Addition of guanosine to PHA‐stimulated lymphocyte cultures containing MSC did not affect lymphocyte proliferation. The immunosuppressive effects of cyclosporine and MSC did not interfere, when present in the cultures of PHA‐activated lymphocytes. In summary, human MSC suppress proliferation of both CD4+ and CD8+ lymphocyte and decrease the expression of activation markers.

Allogeneic bone marrow transplantation vs filgrastim-mobilised peripheral blood progenitor cell transplantation in patients with early leukaemia: first results of a randomised multicentre trial of the European Group for Blood and Marrow Transplantation

In a multicentre trial involving 20 transplant centres from 10 countries haematopoietic stem cells were obtained either from the bone marrow of 33 sibling donors or from the peripheral blood of 33 such donors after administration of filgrastim (10 μg/kg/day). The haematopoietic stem cells were infused into their HLA-identical recipients suffering from acute leukaemias in remission or chronic myeloid leukaemia in chronic phase. PBPC donors tolerated filgrastim administration and leukapheresis well with the most frequent side-effects being musculoskeletal pain, headache, and mild increases of LDH, AP, Gamma-GT or SGPT. Pain and haematoma at the harvest site and mild anaemia were the most frequent complaints of BM donors. Severe or life-threatening complications were not seen with any type of harvest procedure. Time to platelet recovery greater than 20 × 109/l was 15 days (95% confidence interval (CI) 13–16 days) in the PBPCT group and 19 days (CI 16–25) in the BMT group. Time to neutrophil recovery greater than 0.5 × 109/l was 14 days (CI 12–15 days) in the PBPCT group as compared to 15 days (CI 15–16 days) in the BMT group. The numbers of platelet transfusions administered to PBPCT and BMT patients were 12 (range: 1–28) and 10 (range: 3–39), respectively. Sixteen patients (48%) transplanted with bone marrow and 18 patients (54%) transplanted with PBPC developed acute GVHD of grades II–IV; acute GVHD of grades III or IV developed in six (18%) and seven (21%) patients, respectively. Kaplan–Meier plots for transplant-related mortality until day 100 and leukaemia-free survival at a median of 400 days after BMT or PBPCT showed no significant differences. Administration of filgrastim and leukapheresis in normal donors were feasible and well tolerated. The number of days with restricted activity and of nights spent in hospital was lower in donors of PBPC. Transplantation of PBPC to HLA-identical siblings with early leukaemia resulted in earlier platelet engraftment. The incidence of moderate to severe acute GVHD, transplant-related mortality, and leukaemia-free survival did not show striking differences. Further investigation of allogeneic PBPCT as a substitute for allogeneic BMT is warranted.

Identical-twin bone marrow transplants for leukemia

Bone marrow transplants are increasingly used to treat leukemias including acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), and chronic myelogenous leukemia (CML). Approximately 4000 allogeneic transplants are done annually worldwide [1]. Most transplants are from HLA-identical siblings; few persons with leukemia receive transplants from identical twins. Consequently, analyses of results of twin transplants are limited by the small number of participants and diverse remission states. We analyzed the results of identical-twin transplants for leukemia in 103 persons reported to the International Bone Marrow Transplant Registry between 1978 and 1990. Transplant outcomes were compared with those of concurrent HLA-identical sibling transplants for leukemia. Methods The study population included 103 patients with ALL or AML in first remission or CML in first chronic phase receiving bone marrow transplants from identical twins between 1978 and 1990 and reported to the International Bone Marrow Transplant Registry by 66 centers. During the same period, 3214 recipients of non-T-cell-depleted HLA-identical sibling transplants for these indications were reported to the International Bone Marrow Transplant Registry by 163 centers: 581 for ALL in first remission, 1394 for AML in first remission, and 1240 for CML in first chronic phase. Assessing Comparability of Twin and HLA-identical Sibling Transplant Recipients For each disease, prognostic factors for transplant outcome were compared between twin and HLA-identical sibling transplant recipients using chi-square for categorical variables and the Mann-Whitney test [2] for continuous variables. Factors considered were those associated with HLA-identical sibling transplant outcome in previous International Bone Marrow Transplant Registry studies [3-7]. These included the following: for ALL, age, leukocytes at diagnosis, immune phenotype, and time to achieve first remission [3, 4]; for AML, age, leukocytes at diagnosis, FAB type, and Karnofsky performance score [5]; and for CML, age, previous splenectomy, interval between diagnosis and transplant, and previous treatment with busulfan [6, 7]. Conditioning regimens were also compared. For each twin transplant, 10 controls were selected from among 3214 HLA-identical sibling transplants matched for disease and any prognostic factors differing between the two cohorts with P < 0.1. For ALL, pairs were matched for age within 5 years; for AML, pairs were matched for FAB type and Karnofsky score; for CML, pairs were matched for age, interval between diagnosis and transplant, and use of busulfan before transplantation. Statistical Methods Actuarial probabilities of relapse, treatment-related mortality [8], and leukemia-free survival were calculated in the twins and matched HLA-identical sibling controls using life-table methods. Relapse was defined as hematologic recurrence in any site; patients in continuous complete remission were censored at death or, for survivors, at last contact. Treatment-related mortality was defined as death in continuous complete remission; patients were censored at time of relapse or at last follow-up. Leukemia-free survival was defined as survival in continuous complete remission. Relapse and death from causes other than leukemia were considered failures; patients alive and in remission were censored at time of last follow-up. Univariate comparisons of survival distributions were done using the matched log-rank test [9]. To compare relapse risks after adjusting for the effect of graft-versus-host disease, we used Cox proportional-hazards regression with acute and chronic graft-versus-host disease entered as time-dependent covariates [10, 11]. The International Bone Marrow Transplant Registry The Registry is a voluntary working group of more than 200 transplant teams worldwide that contribute detailed data on their allogeneic and identical-twin bone marrow transplants to a statistical center [12, 13]. The program is primarily funded by a program project grant from the U.S. National Institutes of Health. Participants are required to report all consecutive transplants; compliance is monitored by on-site audits. Approximately two thirds of all active allogeneic transplant centers report their data to the Registry. The Registry database includes 40% to 45% of all allogeneic transplant recipients since 1970. Patients are followed longitudinally. Computerized error checks, physician review of submitted data, and on-site audits of participating centers ensure data quality. Transplant outcomes estimated using Registry data are similar to those reported by large nonparticipating centers for comparable patients. Results Differences were found in patient-, disease- and treatment-related variables between the 103 recipients of identical-twin transplants and the 3214 recipients of HLA-identical sibling transplants reported to the International Bone Marrow Transplant Registry during the same interval. Some were intrinsic to the study design. For example, identical-twin donors and recipients were always of the same sex compared with only about one half of HLA-identical sibling donor-recipient pairs. Also, only eight (8%) twin recipients received immune suppression after transplantation compared with 100% of HLA-identical sibling transplant recipients. It was not possible to match or otherwise control for potential effects of these differences. Other differences were as follows: for ALL, younger median age in twins compared with HLA-identical siblings (17 compared with 22 years; P = 0.003); for AML, higher proportion of twins older than 40 years of age (22% compared with 8%; P = 0.002) and with Karnofsky performance scores at transplantation of less than 90% (15% compared with 10%; P < 0.001); and for CML in first chronic phase, shorter median interval from diagnosis to transplant in twins (6 compared with 13 months; P < 0.001) and fewer twins treated with busulfan before transplant (12% compared with 32%, P = 0.04). Ten HLA-identical sibling transplant controls were selected for each twin transplant, matching for diagnosis and for these variables. The resulting matched cohort was similar to the twin cohort for all variables considered, other than those intrinsic to twin transplants as indicated (Appendix Table 1). The median follow-up times of 6.3 years (range, 0.4 to 13.6 years) and 5.0 years (range, 0.3 to 14.8 years) were similar for twin and HLA-identical sibling transplant cohorts, respectively (P = 0.2). Appendix Table 1. Matched-Twin Compared with HLA-identical Sibling Cohorts* Transplant Outcome The two matched cohorts, twins and HLA-identical siblings, were compared for three transplant outcomes: relapse, treatment-related mortality, and leukemia-free survival. Cox regression analysis was used to compare relapse risk between the cohorts after adjusting for acute and chronic graft-versus-host disease. Acute Lymphoblastic Leukemia Ten of 24 twins had a relapse. The 3-year probability of relapse was 36% (CI, 17% to 55%) compared with 26% (CI, 20% to 32%) after 240 HLA-identical sibling transplants (P = 0.1; Figure 1). The 95% CI for the difference in relapse rates between twin and HLA-identical sibling transplants at 3 years was 10% to 30%. Regression analysis with adjustment for acute and chronic graft-versus-host disease showed no difference between identical-twin and HLA-identical sibling transplants in relapse risk (relative risk for twins compared with HLA-identical siblings, 1.4; CI, 0.6 to 2.8, P > 0.2). Figure 1. Outcome of transplants for acute lymphoblastic leukemia. top bottom Two recipients of twin transplants died in remission. The 3-year probability of treatment-related mortality in twins was 10% (CI, 3% to 30%) compared with 21% (CI, 16% to 26%) in HLA-identical sibling transplant recipients (P = 0.1). Twelve of 24 twins were alive in continuous complete remission between 1.2 and 9.5 years (median, 5.4 years) after transplantation. The 3-year probability of leukemia-free survival was 57% (CI, 37% to 77%) compared with 58% (CI, 52% to 64%) after HLA-identical sibling transplants (P > 0.2; Figure 1). Acute Myelogenous Leukemia Twenty-three of 45 twins had a relapse. The 3-year probability of relapse was 52% (CI, 37% to 67%) compared with 16% (CI, 12% to 20%) after 450 HLA-identical sibling transplants (P < 0.001; Figure 2). The 95% CI for the difference in relapse rates at 3 years was 20% to 52%. Regression analysis after adjustment for acute and chronic graft-versus-host disease also showed an increased relapse risk in twins (relative risk, 3.8; CI, 2.3 to 6.2, P < 0.001). Figure 2. Outcome of transplants for acute myelogenous leukemia. top bottom Five twins died of treatment-related causes. The 3-year probability of treatment-related mortality was 12% (CI, 1% to 23%) compared with 34% (CI, 29% to 39%) after HLA-identical sibling transplants (P = 0.004). Seventeen recipients of twin transplants were alive in continuous complete remission 5 months to 13.6 years (median, 6.3 years) after transplantation. The 3-year probability of leukemia-free survival was 42% (CI, 27% to 57%) compared with 55% (CI, 50% to 60%) after HLA-identical sibling transplants (P = 0.2; Figure 2). Chronic Myelogenous Leukemia Seventeen of 34 twins had a relapse. The 3-year probability of relapse was 40% (CI, 23% to 57%) compared with 7% (CI, 4% to 10%) after 340 HLA-identical sibling transplants (P < 0.001; Figure 3). The 95% CI for the difference in relapse rates at 3 years was 16% to 50%. Regression analysis after adjustment for acute and chronic graft-versus-host disease also showed increased relapse risk in twins (relative risk, 5.5; CI, 2.8 to 11.0, P < 0.001). Figure 3. Outcome of transplants for chronic myelogenous leukemia. top bottom One twin died of treatment-related causes. The 3-year probability of treatment-related mortality was 3% (CI, 0% to 16%) compared with 34% (CI, 29% to 39%) after HLA-ident

No alloantibodies against mesenchymal stromal cells, but presence of anti-fetal calf serum antibodies, after transplantation in allogeneic hematopoietic stem cell recipients

Background and Objectives Mesenchymal stromal cells (MSC) may be used in cellular therapy to treat graft-versus-host-disease and autoimmune disorders, and in regenerative medicine. Preliminary data suggest limited cellular allogeneic rejection, but less is known about humoral responses. The objective of this study was to investigate whether antibodies against MSC were present after hematopoietic stem cell transplantation (HSCT) including treatment with HLA matched or mismatched allogeneic MSC. Design and Methods Twelve patients were evaluated using flow cytometric cross matches (FCXM) and enzyme-linked immunosorbent assays. Expression of blood group antigens, regarded as alloantigens giving rise to humoral alloimmunity, on MSC were explored using flow cytometry and immunofluorescence. Results Three of 12 patients exhibited late positivity in the FCXM. In absorption studies, antibodies directed against fetal calf serum (FCS), a component of the MSC culture medium, were identified. Healthy individuals expressed varying levels of anti-FCS antibodies and the same pattern was seen in immunosuppressed HSCT patients. MSC did not express blood group antigens. The patients with positive FCXM are alive and well. Interpretation and Conclusions We have shown that immunosuppressed patients can exhibit anti-FCS antibodies, but no alloantibodies, which may bind to MSC. These antibodies seem clinically insignificant.