Sofia Berglund

Improved Survival after Allogeneic Hematopoietic Stem Cell Transplantation in Recent Years. A Single-Center Study

We analyzed the outcome of allogeneic hematopoietic stem cell transplantation (HSCT) over the past 2 decades. Between 1992 and 2009, 953 patients were treated with HSCT, mainly for a hematologic malignancy. They were divided according to 4 different time periods of treatment: 1992 to 1995, 1996 to 2000, 2001 to 2005, and 2006 to 2009. Over the years, many factors have changed considerably regarding patient age, diagnosis, disease stage, type of donor, stem cell source, genomic HLA typing, cell dose, type of conditioning, treatment of infections, use of granulocyte-colony stimulating factor (G-CSF), use of mesenchymal stem cells, use of cytotoxic T cells, and home care. When we compared the last period (2006-2009) with earlier periods, we found slower neutrophil engraftment, a higher incidence of acute graft-versus-host disease (aGVHD) of grades II-IV, and less chronic GVHD (cGHVD). The incidence of relapse was unchanged over the 4 periods (22%-25%). Overall survival (OS) and transplant-related mortality (TRM) improved significantly in the more recent periods, with the best results during the last period (2006-2009) and a 100-day TRM of 5.5%. This improvement was also apparent in a multivariate analysis. When correcting for differences between the 4 groups, the hazard ratio for mortality in the last period was 0.59 (95% confidence interval [CI]: 0.44-0.79; P < .001) and for TRM it was 0.63 (CI: 0.43-0.92; P = .02). This study shows that the combined efforts to improve outcome after HSCT have been very effective. Even though we now treat older patients with more advanced disease and use more alternative HLA nonidentical donors, OS and TRM have improved. The problem of relapse still has to be remedied. Thus, several different developments together have resulted in significantly lower TRM and improved survival after HSCT over the last few years.

Mesenchymal stem cells inhibit thymic reconstitution after allogeneic cord blood transplantation.

Cord blood (CB) as a source of stem cells has been a successful addition to the field of allogeneic stem cell transplantation (ASCT). The increased human leukocyte antigen (HLA) permissiveness of CB grafts has made it possible for more patients to undergo treatment. The drawback is that patients suffer from a longer period of compromised immunity. We analyzed T-cell receptor excision circles (TRECs), immunoglobulin G (IgG), and immunoglobulin M (IgM) levels after cord blood transplantation (CBT) in 50 patients transplanted at our center. These immunological parameters were compared retrospectively with clinical factors and complications. We found that TREC levels after CBT were lower in adults, patients with myeloablative conditioning, and in patients with a lower nucleated cell dose in the graft. In addition mesenchymal stem cells (MSC) as co-infusion at the time of CBT had a negative effect on TREC reconstitution. This was found to be associated with decreased overall survival for this patient category. Reduced IgM and IgG levels post-CBT were associated with a major AB0 mismatch, and infusion of MSCs. Our results highlight the importance of close monitoring of the immune reconstitution after CBT. In addition it shows a potentially new suppressive effect of MSCs on the immune system.

Stable mixed donor–donor chimerism after double cord blood transplantation

Double cord blood transplantation (DCBT) has been used increasingly and has proven to be both safe and efficacious. In chimerism analysis, previous studies have indicated single unit predominance early after DCBT. In the present study, we evaluated the chimeric pattern in T-, B- and myeloid cells using PCR-based chimerism analysis in seven patients after DCBT: five patients had acute leukemia and two had lymphoma. Five patients received myeloablative conditioning and two patients were given reduced intensity conditioning. All patients received anti-thymocyte globulin (ATG) before DCBT. Three of the six evaluable patients showed donor–donor mixed chimerism in all cell lineages at 90 days after DCBT. Interestingly, two patients in long-term follow-up showed mixed donor chimerism in all cell lineages at 25 and 35 months after DCBT, respectively. Both patients are doing clinically well. Neither of the two developed GVHD after DCBT. In conclusion, in this study donor–donor mixed chimerism was common after high dose ATG and DCBT. Further studies are warranted concerning the immunological consequences of the phenomenon of donor–donor mixed chimerism after DCBT.

Characterization of long-term mixed donor–donor chimerism after double cord blood transplantation

Double cord blood transplantation (DCBT) with two matched or partially matched cord blood units has been implemented successfully to circumvent the limitations of graft cell dose associated with single CBT. After DCBT, sustained haematopoiesis is derived almost exclusively from only one of the donated units. None the less, we previously observed two of six evaluable DCBT patients still having mixed donor–donor chimerism at 28 and 45 months post‐transplantation, respectively. In the present study we utilize flow cytometry techniques to perform the first thorough analysis of phenotype and functionality of cord blood units in patients with mixed donor–donor chimerism. Our results suggest that the two stable cord blood units are different phenotypically and functionally: one unit shows more naive T cells, lower T cell cytokine production and higher frequencies of natural killer cells, the other shows higher frequencies of well‐differentiated and functional lymphocytes. Additionally, in comparison with control patients having a single prevailing cord blood unit, the patients with donor–donor chimerism exhibit less overall T cell cytokine production and a smaller fraction of memory T cells. Furthermore, our results indicate that human leucocyte antigen‐C match of donor units may partly explain the development of a donor–donor mixed chimerism.

Cord blood T cells cultured with IL-7 in addition to IL-2 exhibit a higher degree of polyfunctionality and superior proliferation potential.

One disadvantage with umbilical cord blood transplantation is that donor lymphocyte infusion (DLI) for treatment of threatening rejection or relapse of underlying malignant disease is not available. We have previously expanded T cells from the cord blood graft in clinical setting using anti-CD3+/CD28+ paramagnetic beads and interleukin (IL)-2 for possible future DLI. Here we studied the effect of adding clinical-grade IL-7 to the expansion protocol. T cells were positively selected with anti-CD3+/CD28+ paramagnetic beads and cultured in increasing concentrations of IL-2 with and without IL-7 (20 ng/mL). After 7 days of expansion, the T cells were analyzed for proliferative capacity and investigated with flow cytometry and Luminex to determine phenotype, cytokine production, and responsiveness to mitogenic stimulus. Cultures with IL-7 had significantly greater proliferation rate, higher CD4+/CD8+ ratio, a lower percentage of central memory T cells (CD45RO+CCR7+), and a higher percentage of effector memory T cells (CD45RO+CCR7−). We assessed the production of IL-2, tumor necrosis factor-&agr;, interferon-&ggr;, and CD107a and found a higher percentage of polyfunctional T cells (positive for 3 to 4 factors) in cells cultured with IL-7. The addition of IL-7 gives a proliferative advantage, also in cultures with a lower dose of IL-2. This could prove advantageous in T-cell culture for adoptive transfer to decrease the risk of apoptosis and other negative effects of cytokine deprivation in vivo. Addition of IL-7 also had an effect on the differentiation status of the cord blood-derived T cells. T cells cultured in IL-7 had more polyfunctional traits, possibly increasing the activity of a putative future umbilical cord blood DLI.

Chimerism and use of mesenchymal stem cells in umbilical cord blood transplantation

We performed a retrospective single-center analysis of 50 umbilical cord blood transplantations (UCBTs), focusing on chimerism development. Complete donor chimerism (DC) was associated with acute graft-vs.-host disease (aGVHD) grades II–IV for the CD3+ cell lineage (p = 0.01) and, in multivariate analysis, with total body irradiation (TBI) for all lineages (p < 0.01). Overall survival (OS) was negatively associated with patient age, (p < 0.001); aGVHD grades III–IV, (p < 0.001); and treatment with mesenchymal stem cells (MSCs) (p = 0.027). In conclusion, though multiple factors may have contributed, the association of TBI and DC might be worthy of consideration in the treatment of patients with malignant disease in the UCBT setting. The negative influence of MSCs on OS may be a reason for more careful usage of this treatment modality in combination with UCBT.

Advances in umbilical cord blood cell therapy: the present and the future

ABSTRACT Introduction: Umbilical cord blood (UCB), previously seen as medical waste, is increasingly recognized as a valuable source of cells for therapeutic use. The best-known application is in hematopoietic stem cell transplantation (HSCT), where UCB has become an increasingly important graft source in the 28 years since the first umbilical cord blood transplantation (UCBT) was performed. Recently, UCB has been increasingly investigated as a putative source for adoptive cell therapy. Areas covered: This review covers the advances in umbilical cord blood transplantation (UCBT) to overcome the limitation regarding cellular dose, immunological naivety and additional cell doses such as DLI. It also provides an overview regarding the progress in adoptive cellular therapy using UCB. Expert opinion: UCB has been established as an important source of stem cells for HSCT. Successful strategies to overcome the limitations of UCBT, such as the limited cell numbers and naivety of the cells, are being developed, including novel methods to perform in vitro expansion of progenitor cells, and to improve their homing to the bone marrow. Promising early clinical trials of adoptive therapies with UCB cells, including non-immunological cells, are currently performed for viral infections, malignant diseases and in regenerative medicine.

Expansion of T-cells from the cord blood graft as a predictive tool for complications and outcome of cord blood transplantation

We have previously successfully expanded functional T-cells in vitro from cord blood grafts used for clinical transplantation, with the aim of creating donor lymphocyte infusions to treat e.g. malignant relapse. Here we show that the T-cell expansion in addition might work as a prognostic tool for complications after transplantation. We used multi-color flow cytometry to correlate in vitro phenotypical and functional data from 33 expansions to clinical outcome post-transplantation. Higher levels of CD69+ activated T-cells in the expansion were associated with prolonged survival of the patient. In addition, we found a correlation between T-cell expansions containing relatively high levels of effector memory T-cells and graft vs. host disease and relapse. Our data suggest that expansions of cord blood T-cells from the graft might not only be used as donor lymphocyte infusions, but also as in vitro indicators that could give essential information on how to manage cord blood transplanted patients.

Expansion of Gammadelta T Cells from Cord Blood : A Therapeutical Possibility

Gammadelta (γδ) T cells are found in both blood and tissues and have antiviral and antitumor properties. The frequency of γδ T cells in umbilical cord blood (UCB) is low, and the majority express δ1, in contrast to blood, whereas the main subset is δ2γ9 T cells. UCB γδ T cells are functionally immature, which together with their scarcity complicates the development of UCB γδ T cell therapies. We aimed to develop an effective expansion protocol for UCB γδ T cells based on zoledronate and IL-2. We found that culture with 5 μM zoledronate and 200 IU IL-2/ml medium for 14 days promoted extensive proliferation. The majority of the cultured cells were γ9δ2 T cells. The fold expansion of this, originally infrequent, subset was impressive (median and maximum fold change 253 and 1085, resp.). After culture, the cells had a polyclonal γδ T cell repertoire and the main memory subset was central memory (CD45RO+ CD27+). The cells produced cytokines such as IL-1B, IL-2, and IL-8 and displayed significant tumor-killing capacity. These results show that development of in vitro expanded UCB γδ T cell therapies is feasible. It could prove a valuable treatment modality for patients after umbilical cord blood transplantation.

Tissue-resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer

Tissue‐resident memory T (TRM) cells are CD8+ T lymphocytes that reside in the tissues, including tumours. This T cell subset possesses a magnitude of cytotoxicity, but its epigenetic regulation has not been studied. Here, we investigate the impact of perforin DNA methylation in TRM cells and correlate it with their functional potential. Fifty‐three urothelial urinary bladder cancer (UBC) patients were recruited prospectively. The DNA methylation status of the perforin gene (PRF1) locus in TRM cells was investigated by pyrosequencing. Flow cytometry with ViSNE analysis and in‐vitro stimulation were used to evaluate TRM cell phenotypes. We discovered that tumour TRM cells have low DNA methylation in the PRF1 locus (32·9% methylation), which corresponds to increased numbers of perforin‐expressing TRM cells. Surprisingly, programmed cell death 1 (PD‐1) expression is high in tumour TRM cells, suggesting exhaustion. Following interleukin‐15 and T cell receptor stimulation, perforin and T‐bet expressions are enhanced, indicating that TRM cells from tumours are not terminally exhausted. Moreover, a high number of TRM cells infiltrating the tumours corresponds to lower tumour stage in patients. In conclusion, TRM cells from UBC tumours are epigenetically cytotoxic with signs of exhaustion. This finding identifies TRM cells as potential new targets for cancer immunotherapy.