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It is generally believed that homeostatic responses regulate T-cell recovery after peripheral stem cell transplantation (PSCT). We studied in detail immune recovery in relation to T-cell depletion and clinical events in a group of adult patients who underwent PSCT because of hematologic malignancies. Initially, significantly increased proportions of dividing naive, memory, and effector CD4+and CD8+ T cells were found that readily declined, despite still very low numbers of CD4+ and CD8+ T cells. After PSCT, increased T-cell division rates reflected immune activation because they were associated with episodes of infectious disease and graft-versus-host disease (GVHD). T-cell receptor excision circles (TRECs) were measured to monitor thymic output of naive T cells. Mean TREC content normalized rapidly after PSCT, long before naive T-cell numbers had significantly recovered. This is compatible with the continuous thymic production of TREC+ naive T cells and does not reflect homeostatic increases of thymic output. TREC content was decreased in patients with GVHD and infectious complications, which may be explained by the dilution of TRECs resulting from increased proliferation. Combining TREC and Ki67 analysis with repopulation kinetics led to the novel insight that recovery of TREC content and increased T-cell division during immune reconstitution after transplantation are related to clinical events rather than to homeostatic adaptation to T-cell depletion.

Haploidentical hematopoietic stem-cell transplantation (HSCT) is associated with an increased risk of graft failure. Adult bone marrow-derived mesenchymal stromal cells (MSCs) have been shown to support in vivo normal hematopoiesis and to display potent immune suppressive effects. We cotransplanted donor MSCs in 14 children undergoing transplantation of HLA-disparate CD34+ cells from a relative. While we observed a graft failure rate of 15% in 47 historic controls, all patients given MSCs showed sustained hematopoietic engraftment without any adverse reaction. In particular, children given MSCs did not experience more infections compared with controls. These data suggest that MSCs, possibly thanks to their potent immunosuppressive effect on alloreactive host T lymphocytes escaping the preparative regimen, reduce the risk of graft failure in haploidentical HSC transplant recipients. © 2007 by The American Society of Hematology.

Co-transplantation of CD34+ hematopoietic stem and progenitor cells (HSPC) and mesenchymal stromal cells (MSC) enhances HSPC engraftment. For these applications, MSC are mostly obtained from bone marrow. However, MSC can also be sourced from Wharton's jelly (WJ) of the human umbilical cord, which as a 'waste product', is cheaper to acquire and without significant burden to the donor. Here, we evaluated the ability of WJ MSC to enhance HSPC engraftment. First, we compared cultured human WJ MSC with human bone marrow-derived MSC (BM MSC) for in vitro marker expression, immunomodulatory capacity and differentiation into three mesenchymal lineages. Although we confirmed that WJ MSC have a more restricted differentiation capacity, both WJ MSC and BM MSC expressed similar levels of surface markers and exhibited similar immune inhibitory capacities. Co-transplantation of either WJ MSC or BM MSC with CB CD34+ cells into NOD-SCID mice showed faster recovery of human platelets and CD45+ cells in the peripheral blood and a 3-fold higher engraftment in the BM, blood and spleen six weeks after transplantation when compared to transplantation of CD34+ cells alone. Upon co-incubation, both MSC sources increased the expression of adhesion molecules on CD34+ cells, although SDF-1-induced migration of CD34+ cells remained unaltered. Interestingly, there was an increase in CFU-GEMM when CB CD34+ cells were cultured on monolayers of WJ MSC in the presence of exogenous thrombopoietin, and an increase in BFU-E when BM MSC replaced WJ MSC in such cultures. Our results suggest that WJ MSC is likely to be a practical alternative for BM MSC to enhance CB CD34+ cell engraftment. Disclosures No relevant conflicts of interest to declare.

Mesenchymal stem cells (MSC's) can be used in the experimental transplant setting for treatment of life threatening GvHD or to improve engraftment. Presently, the European MSC Consortium has three ethically approved multi center clinical protocols in use. MSC's expanded ex vivo from donor bone marrow are mostly of parental origin. We reviewed donation data from two participating centers, to establish donor commitment, donation and procedural related complications. Parents of children undergoing haploidentical PBSCT or a cord blood transplant were asked to donate 50 cc of bone marrow 4–5 weeks prior to HSCT. Parents of children with non-steroid responsive aGvHD (grade III-IV) were asked at the time of treatment failure. Bone marrow was aspirated either under general or local anesthesia. Haploidentical PBSC donors received in addition 5 days of G-CSF immediately before stem cell harvest. Donors were asked to consider consent for additional MSC's to be donated for research or use as 3rd party donations for other patients. Donors were cleared for donation by an independent physician. Thirty three donors (14 mothers, 18 fathers, 1 brother) of 34 relatives asked, agreed to donate bone marrow for MSC expansion and all additional uses. One parent whose child died before receiving MSC's requested they be used for 3rd party donations. Fourty five patients (included 6 adults) were treated with MSC's (see table 1 for details). Thirty one patients (68%) received MSC's from a parent, the remaining treatments being 3rd party for acute GvHD. All donors were considered suitable for PBSC or BM donation, although excess weight, mild hypertension and depression were documented in a minority of donors. No adverse events were documented after bone marrow donation, but one parent suffered severe bone pain, requiring opiate analgesia, during G-CSF administration. At 6 weeks follow up no adverse effects were reported. MSC donation either alone or in combination with stem cell mobilization can be achieved without major problems. Parental donor commitment is definitely related to the needs of the child. However, donor altruism to include other unknown patients including adult recipients was uniformly evident in our studies. Donation of stem cells for parents is a stressful situation and requests for multiple donations of various types of cell products can raise questions about medical and ethical considerations. Our protocols show that well informed parents have a propensity to commit to others needs as well as their offspring's. It is therefore essential when introducing experimental treatments to establish a balance between donors commitment and patient needs. Table 1 . Patient and donor characteristics PATIENTS n=45 OUTCOME DONORS n=33 Male 29 21 alive 19 Female 16 12 alive 14 Age (range years) 7.0 (0.6–16.4) 38 (26–53.3) Hematological malignancy 35 Immune deficiency 3 Others 7 Haplo+ MSC 21 15 alive Own child donations Cord + MSC 9 1 rejection 1 death Own child donation aGvHD 15 CR in 11 patients 14/15 3rd party donations Complications 2 a GvHD (grade I) Severe pain 1 donor Infections 14 patients Platelet tx in 4 haplo-donors

Haploidentical CD34+ purified peripheral blood stem cell transplantation (PBSCT) is associated with an increased risk of graft failure/rejection. Animal models suggest that MSCs may promote engraftment after hematopoietic stem cell transplanation (HSCT) but there role in overcoming graft failure in human HSCT is still unclear. We recently reported our initial findings in 13 children receiving MSCs in conjunction with haplo-PBSCT. Here we report our extended series of 20 patients, now including those with MDS known to have an increased risk of rejection. MSCs were isolated from marrow of PBSCT donors and expanded under GMP conditions using same batch FCS containing medium and a standardized protocol, approximately 4–5 weeks before transplantation. MSCs (fresh or cryopreserved) were administered i.v. 4 hours before the allograft. Conditioning depended on underlying disease. No pharmacological GvHD prophylaxis was given after PBSCT. Characteristics of the patients and results are summarized in table 1. In comparison to historical controls (20% graft failure), all patients given MSCs showed successful and stable engraftment (p=0.03). Hematological recovery was accelerated (see table 2). Viral reactivations did not differ between patients and controls. MSC use was feasible and safe without infusion toxicities. In contrast to our initial findings, our extended analysis shows a statistically significant reduction in graft failure rates for patients cotransplanted with MSCs. Moreover, MSC cotransplantation may also prevent graft rejection in patients with MDS. In view of these findings, cotransplantation of MSCs could become standard practice for optimizing the outcome of children given this type of allograft. Table 1: Patient characteristics and summary of results PATIENT (n=20) CONTROL (n=52) P VALUE Male/female 14/6 (70/30%) 31/21 (60/40%) 0.6 Age (range) 8 years (2–16) 8 years (1–17) NS All hematological malignancies 16 (80%) 40 (70%) 1.0 MDS 2 (10%) 5 (10%) 1.0 Immune deficiencies 2 (10%) 2 (4%) NS Other 2 (10%) 10 (9%) NS Donor male/female 10/10 29/23 NS Conditioning TBI/Chemo based 12/8 30/22 1.0 N of CD34+cells infused (median, range) 21.3 (11.2–38.6) 23 (12.1–47.5) NS N. of CD3+ cells infused (mean, SD) 0.3 (0.30) 0.5 (0.7) NS aGvHD 0.2 I-II 2 12 III-IV 0 2 Relapse 4/16 (25%) 8/40 (20%) NS Infectious deaths 2 (10%) 7 (13%) NS GRAFT FAILURE 0 11 0.03 Tabel 2. Patient hematopoietic recovery compared to controls PATIENTS CONTROLS P value (student t-test Median (days, range) SD (days) Median (days, range) SD (days) P value equal or less than 0.05 is considered significant Leucocyte 〉1.0 × 109/L 11.5 (9–16) 2.0 14 (9–26) 4.2 0.01 Neutrophils 〉0.5 × 109/L 12 (10–17) 1.9 13 (9–27) 4.6 0.06 Platelets 〉20 × 109/L 11 (9–24) 3.8 13 (7–100) 22.3 0.11 Reticulocytes 〉20 × 109/L 12 (10–31) 5.0 23 (9–41) 10.2 0.01

Haploidentical peripheral blood stem cell transplantation from a parent is an accepted form of therapy in children lacking a suitable donor who require stem cell transplantation. The risk of graft failure/rejection increases with the intense T cell depletion resulting from positive CD34 selection. Mesenchymal stem cells (MSCs) isolated from bone marrow can be expanded to sufficient numbers for clinical use. MSCs can improve hematological recovery but their role in overcoming graft dysfunction post transplant is unknown. We present results of co-transplantation of MSCs in children undergoing haplo-identical PBSCT compared to 48 historical controls. MSCs were isolated from donor marrow and expanded under GMP conditions approximately 4–5 weeks before transplantation. MSCs (fresh or cryopreserved) were administered i.v. 4 hours before donor G-CSF mobilized PBSCs. To date, 8 children have been treated in two centers.Conditioning depended on underlying disease. No additional GvHD prophylaxis was given. Engraftment and immune recovery was compared to the control group. The local ethical committees approved the study. Characteristics and results are summarized in Table 1. There was no difference for age, gender, transplant indication, donor type or CD34+ or CD3+ cells infused. In comparison to historical controls (23% graft dysfunction) all patients had 100% documented and sustained engraftment. Hematological recovery of leucocytes was faster (p=0,008) with lymphocyte rather than neutrophil recovery accounting for this. Near significance in reticulocyte and platelet recovery (p=0.09; 0.08 resp) was evident. Viral reactivations were common in both patients and controls. Acute GvHD was 12.5% in the patient group compared to 39.5% of eligible controls. Two patients died, one relapse and one infection compared to 17 controls (9 relapse, 5 infection, 3 GvHD). Longer follow-up and immune recovery data are continuing. Our results suggest MSC co-transplantation overcomes graft dysfunction and stabilizes engraftment. MSC use is feasible and safe without infusional toxicities. Based on our findings, where graft dysfunction, delayed hematological and immune recovery compromise the success of stem cell transplant outcome, clinical randomized studies of MSCs are warranted. Table 1. PATIENT/CONTROL CHARACTERISTICS PATIENT CONTROL All patients received MSC between 1–2 x 106/kg MALE 4 (50%) 29 (60%) FEMALE 4 (50%) 19 (40%) AGE (range) 8y 2mo (2–15y 6m) 8y (6mo-17y 7mo) MALIGNANT DISEASES 5 (62%) 34 (70%) IMMUNE DEFICENCIES 2 (25%) 6 (13%) BENIGN CONDITIONS 1 (13%) 8 917%) DONOR MALE 5 24 DONOR FEMALE 3 24 CD34 INFUSED 31.8 (SD 3.1) 20 (SD 6.8) NS CD3 INFUSED 0.3 (SD 0.30) 0.68 (SD 0.7) NS aGvHD 1 13 OVERALL SURVIVAL 6 (75%: FU 3–48mo) 31(64%: FU 2–6Y) Table 2. HEMATOLOGICAL RECOVERY PATIENTS CONTROLS Average (days) SD (days) Average (days) SD (days) P value (T-test) SD: Standard deviation : NS: not significant LEUCOCYTES >1.0 X 109/L 12 1.9 16 4.15 0.008 NEUTROPHILS >0.5 X 109/L 13 1.5 15 3.6 0.14 (NS) PLATELETS >20 X 109/L 13 2.7 21 14.7 0.08 (near significance) RETICULOCYTES >20 16 4.9 27 13.7 0.09

Mesenchymal stem cells (MSC) from adult bone marrow have the capacity to differentiate into several mesenchymal tissues and inhibit T-cell alloreactivity in vitro. Within the EBMT MSC expansion consortium we have used MSC to treat grades III–IV acute graft-versus-host disease (GvHD) in 40 patients. The MSC dose was median 1.0 (range 0.4–9) 10^6 cells/kg body weight of the recipient. No side-effects were seen after MSC infusions. Nineteen patients received one dose, 19 patients received two doses, two patients received three and five doses respectively. MSC donors were in five cases HLA-identical sibling donors, 19 haploidentical donors and 41 third-party HLA-mismatched donors. Among the 40 patients treated for severe acute GvHD, 19 had complete responses, nine showed improvement, seven patients did not respond, four had stable disease and one patient was not evaluated due to short follow-up. Twenty-one patients are alive between six weeks up to 3.5 years after transplantation. Nine of these patients have extensive chronic GvHD. One patient with ALL has recurrent leukaemia and one patient has denovo AML of recipient origin. We conclude that MSC have immunomodulatory and tissue repairing effects and should be further explored as treatment of severe acute GvHD in prospective randomized trials.

The immune system of patients with severe combined immunodeficiency (SCID) reconstitutes to a large extent during the first years after hematopoietic stem cell transplantation (HSCT). It was suggested, however, that accelerated loss of thymus output may cause impaired immune function at the long term. To address this issue, we studied patients with SCID who underwent allogeneic HSCT 5 to 32 years earlier and identified early determinants of long-term T-cell reconstitution. A variety of immune parameters were analyzed both early (1-4 years) and late (5-32 years) after HSCT. Late after HSCT, a clear distinction could be made between a group of 8 patients with impaired T-cell reconstitution and 11 patients with good immune reconstitution. Importantly, in patients with decreased long-term T-cell reconstitution, T-cell recovery was already poor early after HSCT, demonstrating that long-term immune failure was not caused by accelerated loss of thymus output or long-term graft failure, but resulted from poor early grafting. The number of T-cell receptor excision circles (TRECs) early after HSCT was most predictive for long-term T-cell reconstitution. Frequent monitoring of T-cell immunity and TREC numbers early after HSCT may thus serve to timely identify patients who will fail to reconstitute properly and who may need additional treatment.

Graft failure or rejection is an identified problem in haplo-identical or second attempt transplantation. In the bone marrow, mesenchymal stem cells (MSCs) have been identified and shown in animal models to enhance hematopoietic stem cell (HSC) engraftment. Advances in techniques and higher quality culture components have allowed the development of an MSC expansion procedure resulting in sufficient MSCs for clinical application. Adult studies have suggested that co-transplantation of MSCs and HSCs in the HLA-identical setting, is feasible and safe. Here we present the first combined clinical experience within the EBMT MSC expansion consortium with respect to co-transplantation of haplo-identical MSCs in the pediatric allogeneic transplantation setting. Six weeks before the SCT, 50 cc of bone marrow are sampled under sterile conditions. Density gradient-separated MNCs are plated into tissue culture flasks in low-glucose DMEM supplemented with 10% fetal calf serum and incubated at 370C with 5% CO2. At 70% confluence, the cells are trypsinized and re-plated at 4x103 cells/ cm2 until the target dose of 1–2 x106/kg recipient body weight is obtained. Enrichment and expansion of MSCs is performed under GMP conditions in nationally accredited laboratories. MSCs (either fresh or cryopreserved) are administered i.v. 4 hours before infusion of donor HSCs. To date 4 children have undergone co-transplantation. Patient characteristics are summarized in the table. All toxicities associated with the procedure were documented, as were the engraftment kinetics and immune recovery. The study was carried out with approval of the respective local ethical committees. The data indicate that expansion and co-transplantation of MSCs is feasible and well tolerated. While the study is ongoing initial engraftment and immune recovery data (compared to historical data) is encouraging and to date there have been no episodes of graft rejection or severe adverse events related to this treatment. Patient characteristics Patient demographics HSC characteristics MSC characteristics UPN Gender Age Diagnosis Donor CD34 dose (106/kg) Donor Cell dose (106/kg) 1 M 15 ANLL mother-haplo 16 mother 1.5 2 M 2 XLPD father-haplo 20 father 1.9 3 M 2 XLPD father-haplo 18.7 father 1.56 4 M 8 SAA URD (2 loci mismatched) 2.65 mother 1.0