Arwen Stikvoort

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.

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.

Frontline Science: Placenta‐derived decidual stromal cells alter IL‐2R expression and signaling in alloantigen‐activated T cells

This study investigated how stromal cells affect the IL‐2 pathway in alloantigen‐activated T cells. We found that decidual stromal cells (DSCs) from term placentas promoted a high production of IL‐2 in cultures with alloantigen‐activated T cells. The intensity of expression of cluster of differentiation 25 (CD25; IL‐2Rα) on T cells was increased by DSCs, whereas the frequency and intensity of expression of the signaling subunits CD122 (IL‐2Rβ) and CD132 (IL‐2Rγc) were reduced. Consequently, uptake of IL‐2 and STAT5 phosphorylation (pSTAT5) was abrogated. DSCs also decreased the proportion of pSTAT5+ T cells in response to IL‐15, which also use CD122 for signaling. Addition of DSCs to the allogeneic cultures did not increase the expression of programmed death 1 (PD‐1) or CD95, indicating that they did not promote T cell exhaustion. However, exogenous recombinant (r)IL‐2 in similar concentrations in the same setting increased the expression of CD95 and down‐regulated CD122 in T cells. The antiproliferative effect of sirolimus (SRL) and cyclosporine A (CsA), which target the IL‐2 signaling pathway, was diminished by DSCs in vitro. To conclude, DSCs affect IL‐2 production and IL‐2R expression and signaling, which may contribute to the stromal cell‐mediated immune modulation and phenotype shift seen in activated T cells. Altered proliferation in cultures when combining DSCs and SRL or CsA may be of clinical importance, as stromal cells are used in trials for acute inflammation and are often used in combination with conventional immunosuppressive therapies.

Novel method to characterize immune cells from human prostate tissue

Benign prostatic hyperplasia (BPH) is the most common benign adenoma and prostate cancer is the most frequent malignancy in men over 50 years of age in the Western world, where it remains a significant health problem. Prostate lesions are known to contain immune cells, which may contribute to the immune control of tumor progression. However, due to their low numbers and restricted access to necessary material it is difficult to isolate immune cells from prostate tissue to characterize their immunological features.

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.

T Cell Receptor Excision Circle (TREC) Monitoring after Allogeneic Stem Cell Transplantation; a Predictive Marker for Complications and Clinical Outcome

Allogeneic hematopoietic stem cell transplantation (HSCT) is a well-established treatment modality for a variety of malignant diseases as well as for inborn errors of the metabolism or immune system. Regardless of disease origin, good clinical effects are dependent on proper immune reconstitution. T cells are responsible for both the beneficial graft-versus-leukemia (GVL) effect against malignant cells and protection against infections. The immune recovery of T cells relies initially on peripheral expansion of mature cells from the graft and later on the differentiation and maturation from donor-derived hematopoietic stem cells. The formation of new T cells occurs in the thymus and as a byproduct, T cell receptor excision circles (TRECs) are released upon rearrangement of the T cell receptor. Detection of TRECs by PCR is a reliable method for estimating the amount of newly formed T cells in the circulation and, indirectly, for estimating thymic function. Here, we discuss the role of TREC analysis in the prediction of clinical outcome after allogeneic HSCT. Due to the pivotal role of T cell reconstitution we propose that TREC analysis should be included as a key indicator in the post-HSCT follow-up.

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.

Lymphocytes in Placental Tissues: Immune Regulation and Translational Possibilities for Immunotherapy

Immune modulation at the fetomaternal interface is crucial to ensure that the fetal allograft is not rejected. In the present review, the focus is to describe basic functions of lymphocyte populations and how they may contribute to fetomaternal immune regulation, as well as determining what proportions and effector functions of these cells are reported to be present in placental tissues in humans. Also explored is the possibility that unique cell populations at the fetomaternal interface may be targets for adoptive cell therapy. Increasing the understanding of immune modulation during pregnancy can give valuable insight into other established fields such as allogeneic hematopoietic stem cell transplantation and solid organ transplantation. In these settings, lymphocytes are key components that contribute to inflammation and rejection of either patient or donor tissues following transplantation. In contrast, an allogeneic fetus eludes rejection by the maternal immune system.

Risk Factors for Severe Acute Graft-versus-Host Disease in Donor Graft Composition

Acute graft-versus-host disease (aGVHD) is 1 of the main major complications of post-hematopoietic stem cell transplantation (HSCT). Identifying patients at risk of severe aGVHD may lead to earlier intervention and treatment, resulting in increased survival and a better quality of life. We aimed to identify biomarkers in donor grafts and patient plasma around the time of transplantation that might be predictive of aGVHD development. We build on our previously published methods by using multiplex assays and multicolor flow cytometry. We identified 5 easily assessable cellular markers in donor grafts that combined could potentially be used to calculate risk for severe aGVHD development. Most noteworthy are the T cell subsets expressing IL-7 receptor-α (CD127) and PD-1. Additionally, we identified a potential role for elevated tumor necrosis factor-α levels in both graft and patient before HSCT in development of aGVHD.