Combining Flow and Mass Cytometry in the Search for Biomarkers in Chronic Graft-versus-Host Disease.

Chronic graft-versus-host disease (cGVHD) is a debilitating complication arising in around half of all patients treated with an allogeneic hematopoietic stem cell transplantation. Even though treatment of severe cGVHD has improved during recent years, it remains one of the main causes of morbidity and mortality in affected patients. Biomarkers in blood that could aid in the diagnosis and classification of cGVHD severity are needed for the development of novel treatment strategies that can alleviate symptoms and reduce the need for painful and sometimes complicated tissue biopsies. Methods that comprehensively profile complex biological systems such as the immune system can reveal unanticipated markers when used with the appropriate methods of data analysis. Here, we used mass cytometry, flow cytometry, enzyme-linked immunosorbent assay, and multiplex assays to systematically profile immune cell populations in 68 patients with varying grades of cGVHD. We identified multiple subpopulations across T, B, and NK-cell lineages that distinguished patients with cGVHD from those without cGVHD and which were associated in varying ways with severity of cGVHD. Specifically, initial flow cytometry demonstrated that patients with more severe cGVHD had lower mucosal-associated T cell frequencies, with a concomitant higher level of CD38 expression on T cells. Mass cytometry could identify unique subpopulations specific for cGVHD severity albeit with some seemingly conflicting results. For instance, patients with severe cGVHD had an increased frequency of activated B cells compared to patients with moderate cGVHD while activated B cells were found at a reduced frequency in patients with mild cGVHD compared to patients without cGVHD. Moreover, results indicate it may be possible to validate mass cytometry results with clinically viable, smaller flow cytometry panels. Finally, no differences in levels of blood soluble markers could be identified, with the exception for the semi-soluble combined marker B-cell activating factor/B cell ratio, which was increased in patients with mild cGVHD compared to patients without cGVHD. These findings suggest that interdependencies between such perturbed subpopulations of cells play a role in cGVHD pathogenesis and can serve as future diagnostic and therapeutic targets.

Donor Cell Composition and Reactivity Predict Risk of Acute Graft-versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation.

Background. Graft-versus-host disease (GVHD) is a serious complication after allogeneic hematopoietic stem cell transplantation (HSCT). We designed a functional assay for assessment of individual risk for acute GVHD. Study Design and Methods. Blood samples were collected from patients and donors before HSCT. Two groups of seven patients each were selected, one in which individuals developed acute GVHD grades II-IV and one in which none showed any clinical signs of GVHD. Peripheral blood mononuclear cells (PBMCs) isolated from donors were incubated in mixed lymphocyte cultures (MLCs) with recipient PBMCs. The cells were characterized by flow cytometry before and after MLC. Results. Samples from donors in the GVHD group contained significantly lower frequencies of naïve γδ T-cells and T-cells expressing NK-cell markers CD56 and CD94. Donor samples in this group also exhibited lower frequencies of naïve CD95+ T-cells compared to controls. After MLC, there were dissimilarities in the CD4/CD8 T-cell ratio and frequency of CD69+ T-cells between the two patient groups, with the non-GVHD group showing higher frequencies of CD8+ and CD69+ T-cells. Conclusion. We conclude that a thorough flow cytometric analysis of donor cells for phenotype and allogeneic reactivity may be of value when assessing pretransplant risk for severe acute GVHD.

Long-Term Stable Mixed Chimerism after Hematopoietic Stem Cell Transplantation in Patients with Non-Malignant Disease, Shall We Be Tolerant?

Long-term stable mixed chimerism is a rare and poorly understood phenomenon post hematopoietic stem cell transplantation. This study aims to shed light on whether the two hematopoietic systems in patients with mixed chimerism remain functional. Additionally, we investigate possible immunologic differences in these individuals compared to patients with only donor derived immune cells. Patients with donor and mixed chimerism, at median 10 (5-16) years post-HSCT for non-malignant diseases, were assessed regarding clinical situation and immune system (phenotypical and functional). No difference in long-term outcome was seen in terms of general wellbeing, central phenotypic immune system features (e.g., differentiation status, CD4/CD8 ratio, B and NK-cell frequency) and antibody responses to immunizations. At a median of 10 years post transplantation, patients with mixed chimerism had significantly higher IgG3 and platelet levels. Additionally, these patients had higher NKT-cell levels (CD94+CD8+ and CD56+CD8+) than patients with donor chimerism. In depth phenotypic analysis of patients with mixed chimerism demonstrated recipient-derived fractions in most immune cell lineages (e.g., T-cell, B-cell and NK-cell subsets). Recipient cells were also capable of responding to mitogenic stimulation with production of several cytokines. In conclusion, long-term mixed chimerism did not negatively affect patient wellbeing and long-term outcome. Moreover, recipient-derived immunity may still be functional in these patients, suggesting an active state of tolerance and immunologic dependence on both hematopoietic systems.

Single-Cell Characterization of in vitro Migration and Interaction Dynamics of T Cells Expanded with IL-2 and IL-7.

T cells are pivotal in the immune defense against cancers and infectious agents. To mount an effector response against cancer cells, T cells need to migrate to the cancer-site, engage in contacts with cancer cells, and perform their effector functions. Adoptive T cell therapy is an effective strategy as treatment of complications such as relapse or opportunistic infections after hematopoietic stem cell transplantations. This requires a sufficient amount of cells that are able to expand and respond to tumor or viral antigens. The cytokines interleukin (IL)-2 and IL-7 drive T cell differentiation, proliferation, and survival and are commonly used to expand T cells ex vivo. Here, we have used microchip-based live-cell imaging to follow the migration of individual T cells, their interactions with allogeneic monocytes, cell division, and apoptosis for extended periods of time; something that cannot be achieved by commonly used methods. Our data indicate that cells grown in IL-7 + IL-2 had similar migration and contact dynamics as cells grown in IL-2 alone. However, the addition of IL-7 decreased cell death creating a more viable cell population, which should be beneficial when preparing cells for immunotherapy.

Expanded umbilical cord blood T cells used as donor lymphocyte infusions after umbilical cord blood transplantation.

BACKGROUND: Umbilical cord blood (UCB) is an alternative graft source for hematopoietic stem cell transplantation and has been shown to give results comparable to transplantation with other stem cell sources. Donor lymphocyte infusion (DLI) is an effective treatment for relapsed malignancies after hematopoietic stem cell transplantation. However, DLI is not available after UCB transplantation. METHODS: In this study, in vitro-cultured T cells from the UCB graft were explored as an alternative to conventional DLI. The main aim was to study the safety of the cultured UCB T cells used as DLI because such cell preparations have not been used in this context previously. We also assessed potential benefits of the treatment. RESULTS: The cultured UCB T cells (UCB DLI) were given to 4 patients with mixed chimerism (n = 2), minimal residual disease (n = 1) and graft failure (n = 1). No adverse reactions were seen at transfusion. Three of the patients did not show any signs of graft-versus-host disease (GVHD) after UCB DLI, but GVHD could not be excluded in the last patient. In the patient with minimal residual disease treated with UCB DLI, the malignant cell clone was detectable shortly before infusion but undetectable at treatment and for 3 months after infusion. In 1 patient with mixed chimerism, the percentage of recipient cells decreased in temporal association with UCB DLI treatment. CONCLUSIONS: We saw no certain adverse effects of treatment with UCB DLI. Events that could indicate possible benefits were seen but with no certain causal association with the treatment.

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 (CD45ROCCR7), and a higher percentage of effector memory T cells (CD45ROCCR7). We assessed the production of IL-2, tumor necrosis factor-α, interferon-γ, 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 patterns of long-term stable mixed chimeras posthematopoietic stem cell transplantation in patients with nonmalignant diseases: follow-up of long-term stable mixed chimerism patients.

Long-term stable mixed chimerism (MC) is a rare phenomenon after hematopoietic stem cell transplantation (HSCT) characterized by 5% to 95% residual recipient hematopoietic cells. The underlying mechanisms of MC are largely unknown. In this study we compared full donor chimerism with long-lasting stable MC for a median of 9.5 years (range, 5 to 16.5) post-HSCT in patients with nonmalignant diseases. Several factors significantly associated with the likelihood of stable MC development were identified by univariate analysis, eg, younger donor age, sibling donor, and conditioning regimen. Despite a limited patient cohort, our multivariate analysis could confirm that a sibling donor was associated with stable MC development. Furthermore, development of acute-graft-versus-host disease and blood stream infection was significantly more prevalent in the full donor chimerism patient group. Additionally, significant fluctuations in the recipient-to-donor chimerism ratio decreased over time after HSCT in MC patients.

Factors with an impact on chimerism development and long-term survival after umbilical cord blood transplantation.

BACKGROUND: Umbilical cord blood transplantation (UCBT) is increasingly used and produces similar results to matched unrelated donor transplantation. METHODS: We performed a retrospective single-center analysis of 50 umbilical cord blood transplantations UCBTs performed from 2001 to 2010, including 37 single and 13 double umbilical cord blood transplantations UCBTs. RESULTS: The rate of engraftment of neutrophils was 88% at a median time of 29 days (range, 3-79). Complete donor chimerism (DC) within the CD19, CD3, and CD33 cell lineages was seen in 74%, 72%, and 76% of the patients, respectively. DC was associated with acute graft-versus-host disease (GVHD) grades II to IV for the CD3 cell lineage (P=0.01) and, in multivariate analysis, with total body irradiation for all lineages (P<0.01). Overall survival (OS) at 1 and 5 years was 55% and 43%. Nonmalignant diseases were associated with better 5-year OS (72%) than malignancies (28%; P=0.026). In multivariate analysis, a negative correlation was seen between OS and age (hazard ratio [HR], 1.04; 95% confidence interval [95% CI], 1.02-1.06; P<0.001), acute GVHD grades III and IV (HR, 3.43; 95% CI, 1.95-6.02; P<0.001), and mesenchymal stem cell treatment (HR, 2.66; 95% CI, 1.11-6.35; P=0.027). Transplant-related mortality at 100 days and 1 year was 16% and 30%. The incidence of acute GVHD grades II to IV was 34%. Acute GVHD grades III and IV was associated with ABO incompatibility (HR, 2.61; P=0.05) and myeloablative conditioning (HR, 4.17; P=0.047). CONCLUSIONS: The outcome in patients with nonmalignant diseases was acceptable, but transplant-related mortality in the whole group remains high. A significantly higher rate of DC was associated with total body irradiation-based conditioning and with acute GVHD grades II and IV.

Rapid salvage treatment with virus-specific T cells for therapy-resistant disease.

BACKGROUND: Viral infections are major complications after allogeneic hematopoietic stem cell transplantation (HSCT). During posttransplant immunosuppression the regular T-cell control is compromised. Even if treatment strategies against infections caused by herpes viruses such as cytomegalovirus, Epstein-Barr virus, and adenovirus have improved, the mortality rate is still considerable. If primary antiviral therapy fails or cannot be tolerated, adoptive therapy with virus-specific cytotoxic T cells (CTL) can be utilized. METHODS: In this study, we used virus-specific CTLs to treat 8 patients suffering from severe viral infections after allogeneic HSCT. Using positive selection with HLA multimers and magnetic beads, we isolated CTLs from both frozen donor material as well as third-party donors within hours. RESULTS: At 90 days after CTL infusions 7 out of 8 patients were still living. CTLs infused from third-party donors were detected in 5 of 6 patients up to 76 days after infusion. No graft-versus-host disease associated with CTL infusions was observed. CONCLUSIONS: Our separation approach offers a rapid alternative for adoptive CTL therapy if primary antiviral treatment strategies fail. Because no prolonged expansion steps are needed, this method may be used for early treatment of patients suffering from life-threatening infectious complications.

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.

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.

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.

Stable mixed double donor chimerism: Absence of war doesn't necessarily mean peace.

Double cord blood transplantation has successfully been introduced to remedy the obstacle of a limited stem cell dose in a single cord blood graft. After a short initial period, the sustained hematopoiesis is derived almost exclusively from one of the donated units. In a recent publication in Clinical and Experimental Immunology we investigated two rare individuals in which both cord blood units co-existed for more than two years after transplantation.

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.

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.

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.

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 Cr-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.