Mantas Okas

A novel haplo-identical adoptive CTL therapy as a treatment for EBV-associated lymphoma after stem cell transplantation

Epstein–Barr virus (EBV)-related malignancies such as post-transplant lymphoproliferative disease (PTLD) are severe complications after allogeneic stem cell transplantation and solid-organ transplantation. In immunosuppressed transplant recipients, the activity of EBV-specific CTLs are often decreased or absent which leads to an increased risk of developing PTLD. If primary treatment modalities of PTLD fail, the most efficient way of treating the malignancy is adopting EBV-specific CTLs from the donor or, more recently, third-party donors. However, both are time consuming and expensive and often it is too late to administer cells to the patient. We have for the first time, using a rapid isolation protocol of EBV-specific T cells, treated and cured a patient suffering from PTLD with multiple-associated tissue lesions, using her haplo-identical mother as a donor. This treatment approach paves way for a new possibility to within-days treat patients with life-threatening EBV-associated malignancies.

Unrelated cord blood and mismatched unrelated volunteer donor transplants, two alternatives in patients who lack an HLA-identical donor

The aim was to evaluate two transplant strategies for patients who lack HLA-identical donors, namely HLA-A, HLA-B or -DRβ1 mismatched unrelated donor (MM URD) transplants (n=14) and umbilical cord blood transplants (UCB, n=27). Diagnosis, disease stage and age were similar in the two groups. Cell dose was lower in the UCB group (P<0.001). Median time to ANC of >0.5 × 109/l was 30 days in the UCB group and 17 days in the MM URD group (P=0.002). Engraftment of plt was delayed in the UCB group (P=0.03). The UCB patients required fewer erythrocyte transfusions (P=0.001). At 100 days, complete donor chimerism for CD3 was 63 and 44% in the UCB and MM URD groups, respectively. Acute GVHD of grades II–IV were 30% in the UCB group and 21% in the MM URD group. The corresponding figures for chronic GVHD were 9 and 20%, respectively. TRM was 30% in the UCB patients and 50% in the MM URD patients. Three-year survival was 66% in the UCB group and 14% in the MM URD group (P=0.006). Although the material is small and heterogeneous, engraftment was delayed, leukocyte chimerism was not significantly different and survival was superior using UCB rather than MM URD transplants.

Clinical expansion of cord blood-derived T cells for use as donor lymphocyte infusion after cord blood transplantation.

Allogeneic stem cell transplantation (SCT) from cord blood (CB) as a stem cell source is a promising alternative when no human leukocyte antigen-matched donor is found. Donor lymphocyte infusion (DLI) is a possible treatment modality for threatening graft failure or relapse of an underlying malignancy after transplantation. Ethical and logistical reasons limit the possibility of DLI in the setting of CB SCT. To remedy this restriction, we performed expansion of donor T cells in vitro from CB grafts in a clinical setting for use as future DLI and characterized the expanded cells in comparison to T cells from CB acquired ex vivo and adult peripheral blood. T cells were expanded from grafts used for transplantation, upon CD3/CD28 crosslinking and culture in interleukin-2. Phenotype and function of T cells were assessed by flow cytometry and mixed lymphocyte culture assays. T-cell receptor repertoire distribution was evaluated with polymerase chain reaction-based spectratyping. We were able to amplify T cells to sufficient amounts for DLI in 13 out of 13 initiated expansions. Expanded T cells presented with an activated phenotype and could be induced to produce cytokines by a nonspecific stimulus. When exposed to allogeneic targets, expanded CB T cells proliferated at comparable levels to their ex vivo and adult blood counterparts. In summary, clinical expansion of CB T cells for DLI is feasible and may be a future modality for treatment of graft failure or relapse after SCT.

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.

Increased frequency and responsiveness of PSA-specific T cells after allogeneic hematopoetic stem-cell transplantation.

Background: Therapies for localized prostate cancer include curative surgery and radiotherapy while treatment of metastatic disease is often inefficient. Graft-versus-tumor effects of allogeneic stem-cell transplantation (ASCT) have been described for several types of solid tumors but have not been reported for prostate cancer. We, therefore, investigated the potential of ASCT as treatment for noncurable prostate cancer. Methods: A patient underwent ASCT from his human leukocyte antigen (HLA)-identical sister as treatment for his metastatic prostate adenocarcinoma. Frequencies of prostate-specific T cells in the peripheral blood of the patient, ASCT donor and a group of control individuals were determined by flow cytometry using pentameric HLA-A2 complexes containing peptides derived from the prostate-specific antigen (PSA). Cytotoxic activity of PSA-peptide-specific T cells against peptide-pulsed target cells was analyzed ex vivo by 51Cr-release assays. Results: Stable clinical and laboratory remission lasting more than 4 years was observed after ASCT. Using HLA-containing pentamers with PSA-derived peptides we could detect prostate-specific CD8+ T cells in this patient at high frequencies over several months. Furthermore, higher frequencies of PSA-specific T cells were revealed in the blood of the patient and female controls when compared with healthy males. Conclusions: Lymphocytes from the peripheral blood of the recipient, but not from donor or other tested control individuals, exhibited ex vivo cytotoxic activity against target cells pulsed with the relevant synthetic peptides and efficiently expanded in vitro following specific restimulations. Thus, the results of this study indicate that female to male ASCT can increase the frequency and enhance specific-responsiveness of PSA-specific T cells in transplant recipients.

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.