Hematopoietic stem cell transplantation for CTLA-4 insufficiency across Europe: A European Society for Blood and Marrow Transplantation Inborn Errors Working Party study.

BACKGROUND: Cytotoxic T-lymphocyte antigen 4 (CTLA-4) insufficiency causes a primary immune regulatory disorder characterized by lymphoproliferation, dysgammaglobulinemia, and multiorgan autoimmunity including cytopenias and colitis. OBJECTIVE: We examined the outcome of hematopoietic stem cell transplantation (HSCT) for CTLA-4 insufficiency and study the impact of pre-HSCT CTLA-4 fusion protein (CTLA-4-Ig) therapy and pre-HSCT immune dysregulation on survival and immunologic outcome. METHODS: This was a retrospective study of HSCT for CTLA-4 insufficiency and 2q33.2-3 deletion from the European Society for Blood and Marrow Transplantation Inborn Errors Working Party. Primary end points were overall survival (OS) and disease- and chronic graft-versus-host disease-free survival (DFS). Secondary end point was immunologic outcome assessed by immune dysregulation disease activity (IDDA) score. RESULTS: Forty patients were included over a 25-year period. Before HSCT, 60% received CTLA-4-Ig, and median (range) IDDA score was 23.3 (3.9-84.0). Median (range) age at HSCT was 14.2 (1.3-56.0) years. Patients received peripheral blood stem cell (58%) or marrow (43%) from a matched unrelated donor (75%), mismatched unrelated donor (12.5%), or matched family donor (12.5%). Median (range) follow-up was 3 (0.6-15) years, and 3-year OS was 76.7% (58-87%) and DFS was 74.4% (54.9-86.0%). At latest follow-up, disease of 28 of 30 surviving patients was in disease-free remission with median IDDA reduction of 16. Probability of OS and DFS was greater in patients with lower disease activity before HSCT (IDDA < 23, P = .002 and P = .006, respectively). CTLA-4-Ig receipt did not influence OS or DFS. Cause of death was transplant related in 7 of 8 patients. CONCLUSION: HSCT is an effective therapy to prevent ongoing disease progression and morbidity, with improving survival rates over time and in patients with lower pre-HSCT disease activity.

The degree of HLA matching determines the incidence of cytokine release syndrome and associated nonrelapse mortality in matched related and unrelated allogeneic stem cell transplantation with post-transplant cyclophosphamide.

Cytokine release syndrome (CRS) occurs frequently after haplo-identical allogeneic stem cell transplantation (alloSCT) with post-transplant cyclophosphamide (PTCy), increasing nonrelapse mortality (NRM) and decreasing survival. Data on CRS in HLA-matched alloSCT are limited and effects of specific HLA-mismatches on CRS development unknown. We hypothesized that in HLA-matched alloSCT increasing degrees of HLA-mismatching influence CRS incidence, NRM and survival. Retrospective analysis of 126 HLA-matched PTCy-alloSCT patients showed that higher degrees of HLA-mismatching significantly increased CRS incidence (26%, 75% and 90% CRS with 12/12, 10/10 and 9/10 matched donors, respectively). Maximum temperature during CRS increased with higher HLA-mismatch. Specific associations between HLA-mismatches and CRS could be determined. Grade 2 CRS and CRS-induced grade 3 fever were associated with significantly increased NRM (p < 0.001 and p = 0.003, respectively) and inferior survival (p < 0.001 and p = 0.005, respectively). NRM was mainly caused by disease conditions that may be considered CRS-induced inflammatory responses (encephalopathy, cryptogenic organizing pneumonia and multi-organ failure).

Quality of life after immune suppressive therapy in aplastic anemia.

Acquired aplastic anemia (AA) is a rare form of immune-mediated bone marrow failure, which can result in life-threatening infections or bleeding if left untreated. Treatment consists of either immune suppressive therapy (IST) or allogeneic stem cell transplantation (alloHSCT). While considerable research has been published regarding survival, response rate and toxicity of both treatments, knowledge on the impact on quality of life (QoL) is scarce. We used the recently developed AA-specific QoL questionnaire (QLQ-AA/PNH-54) to evaluate QoL in a single center cohort of AA patients who were successfully treated with IST. The 54 questions represent 12 different QoL domains. Results were analyzed for all patients and grouped based on hematologic response (complete response (CR) or partial response (PR)). Thirty-six successfully treated adult patients (15 in CR, 21 in PR) completed the questionnaire (median age 54 years, range 21-71; median time since last IST 5 years, range 0-41). Fatigue was experienced by 83% of patients. Even though total QoL scores did not significantly differ between patients with PR and CR (105 vs 92, p-value 0,17) there appeared to be a trend towards higher scores in patients with PR, especially in domains concerning psychological wellbeing. This trend was most clear in the domains fear of progression (2,12 in PR patients vs 1,73 in CR patients; p-value 0,08) and role functioning (2,22 vs 1,88; p-value 0,07). In conclusion, patients with AA continue to experience psychological and physical effects despite successful IST.

Risk factors for graft-versus-host-disease after donor lymphocyte infusion following T-cell depleted allogeneic stem cell transplantation.

Introduction: Unmodified donor lymphocyte infusions (DLI) after allogeneic stem cell transplantation (alloSCT) can boost the beneficial Graft-versus-Leukemia (GvL) effect but may also induce severe Graft-versus-Host-Disease (GvHD). To improve the balance between GvL and GvHD, it is crucial to identify factors that influence the alloreactivity of DLI. Methods: We investigated the effects of the presence of patient-derived antigen-presenting cells at time of DLI as estimated by the bone marrow (BM) chimerism status, lymphopenia as measured by the absolute lymphocyte count (ALC) at time of DLI, and the presence of a viral infection (de novo or reactivation) close to DLI on the risk of GvHD after DLI. The cohort consisted of patients with acute leukemia or myelodysplastic syndrome who prophylactically or pre-emptively received DLI as standard care after alemtuzumab-based alloSCT. In patients at high risk for relapse, DLI was administered at 3 months after alloSCT (n=88) with a dose of 0.3x106 or 0.15x106 T cells/kg in case of a related or unrelated donor, respectively. All other patients (n=76) received 3x106 or 1.5x106 T cells/kg, respectively, at 6 months after alloSCT. Results: For both DLIs, patients with reduced-intensity conditioning and an unrelated donor had the highest risk of GvHD. For DLI given at three months, viral infection within 1 week before and 2 weeks after DLI was an additional significant risk factor (hazard ratio (HR) 3.66 compared to no viral infection) for GvHD. At six months after alloSCT, viral infections were rare and not associated with GvHD. In contrast, mixed BM chimerism (HR 3.63 for ≥5% mixed chimerism compared to full donor) was an important risk factor for GvHD after DLI given at six months after alloSCT. ALC of <1000x106/l showed a trend for association with GvHD after this DLI (HR 2.05 compared to ≥1000x106/l, 95% confidence interval 0.94-4.45). Furthermore, the data suggested that the presence of a viral infection close to the DLI at three months or ≥5% mixed chimerism at time of the DLI at six months correlated with the severity of GvHD, thereby increasing their negative impact on the current GvHD-relapse-free survival. Conclusion: These data demonstrate that the risk factors for GvHD after DLI depend on the setting of the DLI.

Mass cytometric analysis unveils a disease-specific immune cell network in the bone marrow in acquired aplastic anemia.

Idiopathic acquired aplastic anemia (AA) is considered an immune-mediated syndrome of bone marrow failure since approximately 70% of patients respond to immunosuppressive therapy (IST) consisting of a course of anti-thymocyte globulin (ATG) followed by long-term use of ciclosporin. However, the immune response that underlies the pathogenesis of AA remains poorly understood. In this study, we applied high-dimensional mass cytometry on bone marrow aspirates of AA patients pre-ATG, AA patients post-ATG and healthy donors to decipher which immune cells may be implicated in the pathogenesis of AA. We show that the bone marrow of AA patients features an immune cell composition distinct from healthy donors, with significant differences in the myeloid, B-cell, CD4+ and CD8+ T-cells lineages. Specifically, we discovered that AA pre-ATG is characterized by a disease-specific immune cell network with high frequencies of CD16+ myeloid cells, CCR6++ B-cells, Th17-like CCR6+ memory CD4+ T-cells, CD45RA+CCR7+CD38+ CD8+ T-cells and KLRG1+ terminally differentiated effector memory (EMRA) CD8+ T-cells, compatible with a state of chronic inflammation. Successful treatment with IST strongly reduced the levels of CD16+ myeloid cells and showed a trend toward normalization of the frequencies of CCR6++ B-cells, CCR6+ memory CD4+ T-cells and KLRG1+EMRA CD8+ T-cells. Altogether, our study provides a unique overview of the immune landscape in bone marrow in AA at a single-cell level and proposes CCR6 as a potential new therapeutic target in AA.

Joint models quantify associations between immune cell kinetics and allo-immunological events after allogeneic stem cell transplantation and subsequent donor lymphocyte infusion.

Alloreactive donor-derived T-cells play a pivotal role in alloimmune responses after allogeneic hematopoietic stem cell transplantation (alloSCT); both in the relapse-preventing Graft-versus-Leukemia (GvL) effect and the potentially lethal complication Graft-versus-Host-Disease (GvHD). The balance between GvL and GvHD can be shifted by removing T-cells via T-cell depletion (TCD) to reduce the risk of GvHD, and by introducing additional donor T-cells (donor lymphocyte infusions [DLI]) to boost the GvL effect. However, the association between T-cell kinetics and the occurrence of allo-immunological events has not been clearly demonstrated yet. Therefore, we investigated the complex associations between the T-cell kinetics and alloimmune responses in a cohort of 166 acute leukemia patients receiving alemtuzumab-based TCD alloSCT. Of these patients, 62 with an anticipated high risk of relapse were scheduled to receive a prophylactic DLI at 3 months after transplant. In this setting, we applied joint modelling which allowed us to better capture the complex interplay between DLI, T-cell kinetics, GvHD and relapse than traditional statistical methods. We demonstrate that DLI can induce detectable T-cell expansion, leading to an increase in total, CD4+ and CD8+ T-cell counts starting at 3 months after alloSCT. CD4+ T-cells showed the strongest association with the development of alloimmune responses: higher CD4 counts increased the risk of GvHD (hazard ratio 2.44, 95% confidence interval 1.45-4.12) and decreased the risk of relapse (hazard ratio 0.65, 95% confidence interval 0.45-0.92). Similar models showed that natural killer cells recovered rapidly after alloSCT and were associated with a lower risk of relapse (HR 0.62, 95%-CI 0.41-0.93). The results of this study advocate the use of joint models to further study immune cell kinetics in different settings.

Eltrombopag Added to Immunosuppression in Severe Aplastic Anemia.

BACKGROUND: A single-group, phase 1-2 study indicated that eltrombopag improved the efficacy of standard immunosuppressive therapy that entailed horse antithymocyte globulin (ATG) plus cyclosporine in patients with severe aplastic anemia. METHODS: In this prospective, investigator-led, open-label, multicenter, randomized, phase 3 trial, we compared the efficacy and safety of horse ATG plus cyclosporine with or without eltrombopag as front-line therapy in previously untreated patients with severe aplastic anemia. The primary end point was a hematologic complete response at 3 months. RESULTS: Patients were assigned to receive immunosuppressive therapy (Group A, 101 patients) or immunosuppressive therapy plus eltrombopag (Group B, 96 patients). The percentage of patients who had a complete response at 3 months was 10% in Group A and 22% in Group B (odds ratio, 3.2; 95% confidence interval [CI], 1.3 to 7.8; P = 0.01). At 6 months, the overall response rate (the percentage of patients who had a complete or partial response) was 41% in Group A and 68% in Group B. The median times to the first response were 8.8 months (Group A) and 3.0 months (Group B). The incidence of severe adverse events was similar in the two groups. With a median follow-up of 24 months, a karyotypic abnormality that was classified as myelodysplastic syndrome developed in 1 patient (Group A) and 2 patients (Group B); event-free survival was 34% and 46%, respectively. Somatic mutations were detected in 29% (Group A) and 31% (Group Β) of the patients at baseline; these percentages increased to 66% and 55%, respectively, at 6 months, without affecting the hematologic response and 2-year outcome. CONCLUSIONS: The addition of eltrombopag to standard immunosuppressive therapy improved the rate, rapidity, and strength of hematologic response among previously untreated patients with severe aplastic anemia, without additional toxic effects. (Funded by Novartis and others; RACE ClinicalTrials.gov number, NCT02099747; EudraCT number, 2014-000363-40.).

Anti Thymocyte Globulin-Based Treatment for Acquired Bone Marrow Failure in Adults.

Idiopathic acquired aplastic anemia can be successfully treated with Anti Thymocyte Globulin (ATG)-based immune suppressive therapy and is therefore considered a T cell-mediated auto immune disease. Based on this finding, several other forms of idiopathic acquired bone marrow failure are treated with ATG as well. For this review, we extensively searched the present literature for evidence that ATG can lead to enduring remissions in different forms of acquired multi- or single-lineage bone marrow failure. We conclude that ATG-based therapy can lead to an enduring hematopoietic response and increased overall survival (OS) in patients with acquired aplastic aplasia. In patients with hypocellular myelodysplastic syndrome, ATG can lead to a hematological improvement without changing the OS. ATG seems less effective in acquired single-lineage failure diseases like Pure Red Cell Aplasia, Amegakaryocytic Thrombocytopenia and Pure White Cell Aplasia, suggesting a different pathogenesis in these bone marrow failure states compared to aplastic anemia. T cell depletion is hypothesized to play an important role in the beneficial effect of ATG but, as ATG is a mixture of polyclonal antibodies binding to different antigens, other anti-inflammatory or immunomodulatory effects could play a role as well.

Dendritic cells promote expansion and survival of aberrant TCR-negative intraepithelial lymphocyte lines from refractory celiac disease type II patients.

Celiac disease (CD) patients who fail to respond to a gluten-free diet suffer from refractory celiac disease (RCD). A marked expansion of intraepithelial lymphocytes (IEL) lacking surface TCR/CD3 expression characterizes the RCD subtype II. In up to 50% of RCDII patients these so-called aberrant IEL (a-IEL) develop into lymphoma and can disseminate into other tissues. Elevated levels of Interleukin-15 (IL-15) in the intestine of CD and RCD patients likely contribute to the expansion of a-IEL. Here, we investigated if interactions with other cells might also influence a-IEL expansion. Similar to IL-15, cells from the monocyte lineage, particularly mature dendritic cells (DCs), promoted proliferation, prevented apoptosis and induced IFNγ secretion of a-IEL derived from RCDII biopsies (RCDII cell lines), which in turn induced CXCL10. In contrast to IL-15, mature DCs did not induce proliferation of regular TCR(+)IEL lines, generated from CD biopsies and IL-15-blocking antibodies did not inhibit DC-induced proliferation of RCDII cell lines. Furthermore, proliferation was dependent on cell-cell contact, but independent of the HLA-genotype of the stimulating cells. Our results suggest that contact with DC, either in the epithelium or upon dissemination, contributes to uncontrolled expansion of a-IEL in RCDII, independent of HLA-genotype and IL-15.

Identification of a potential physiological precursor of aberrant cells in refractory coeliac disease type II.

OBJECTIVE: Refractory coeliac disease type II (RCDII) is a severe complication of coeliac disease (CD) characterised by aberrant intraepithelial lymphocytes (IELs) of unknown origin that display an atypical CD3(-)CD7(+)icCD3(+) phenotype. In approximately 40% of patients with RCDII these lymphocytes develop into an invasive lymphoma. In the current study we aimed to identify the physiological counterpart of these cells. DESIGN: RCDII cell lines were compared with T-cell receptor positive (TCR(+)) IEL (T-IEL) lines by microarray analysis, real-time quantitative PCR and flow cytometry. This information was used to identify cells with an RCDII-associated phenotype in duodenal biopsies from non-refractory individuals by multicolour flow cytometry. RESULTS: RCDII lines were transcriptionally distinct from T-IEL lines and expressed higher levels of multiple natural killer (NK) cell receptors. In addition to the CD3(-)CD7(+)icCD3(+) phenotype, the RCDII lines were distinguishable from other lymphocyte subsets by the absence of CD56, CD127 and CD34. Cells matching this surface lineage-negative (Lin(-)) CD7(+)CD127(-)CD34(-) phenotype expressed a functional interleukin-15 (IL-15) receptor and constituted a significant proportion of IELs in duodenal specimens of patients without CD, particularly children, and were also found in the thymus. In patients without CD, the Lin(-)CD7(+)CD127(-)CD34(-) subset was one of four subsets within the CD3(-)CD7(+)icCD3(+) population that could be distinguished on the basis of differential expression of CD56 and/or CD127. CONCLUSION: Our studies indicate that the CD3(-)CD7(+)icCD3(+) population is heterogeneous and reveal the existence of a Lin(-) subset that is distinct from T, B, NK and lymphoid tissue inducer cells. We speculate that this IL-15 responsive population represents the physiological counterpart of aberrant cells expanded in RCDII and transformed in RCDII-associated lymphoma.

Origin and immunophenotype of aberrant IEL in RCDII patients.

OBJECTIVES: Aberrant intra-epithelial lymphocytes (IELs) are the hallmark of refractory coeliac disease type II RCDII and considered a premalignant cell population from which aggressive enteropathy-associated T cell lymphoma (EATL) can evolve. The aim of this study was to gain further insight in the origin and characteristics of aberrant IELs by analysing T-cell receptor (TCR) rearrangements, and by immunophenotypic analysis of aberrant IELs. DESIGN: Duodenal biopsies from 18 RCDII patients and three RCDII cell lines were analysed for the presence of TCR delta, gamma, and beta rearrangements. In addition, IELs isolated from biopsies derived from RCDII patients were phenotypically analysed. RESULTS: Aberrant IELs showed an upregulated expression of granzyme B and decreased expression of PCNA. TCR rearrangements in the aberrant IEL population in biopsies of RCDII patients were heterogenic, which is most likely due to a variation in maturity. Similarly, RCDII cell lines displayed a heterogenic TCR rearrangement pattern. CONCLUSION: Aberrant IELs originate from deranged immature T lymphocytes and display clear differentiation to a cytotoxic phenotype. Aberrant IELs displayed different stages of maturity between RCDII patients, of which only the patients harbouring the most mature aberrant IEL population developed an EATL.

DNAM-1 mediates epithelial cell-specific cytotoxicity of aberrant intraepithelial lymphocyte lines from refractory celiac disease type II patients.

In refractory celiac disease (RCD), intestinal epithelial damage persists despite a gluten-free diet. Characteristic for RCD type II (RCD II) is the presence of aberrant surface TCR-CD3(-) intraepithelial lymphocytes (IELs) that can progressively replace normal IELs and eventually give rise to overt lymphoma. Therefore, RCD II is considered a malignant condition that forms an intermediate stage between celiac disease (CD) and overt lymphoma. We demonstrate in this study that surface TCR-CD3(-) IEL lines isolated from three RCD II patients preferentially lyse epithelial cell lines. FACS analysis revealed that DNAM-1 was strongly expressed on the three RCD cell lines, whereas other activating NK cell receptors were not expressed on all three RCD cell lines. Consistent with this finding, cytotoxicity of the RCD cell lines was mediated mainly by DNAM-1 with only a minor role for other activating NK cell receptors. Furthermore, enterocytes isolated from duodenal biopsies expressed DNAM-1 ligands and were lysed by the RCD cell lines ex vivo. Although DNAM-1 on CD8(+) T cells and NK cells is known to mediate lysis of tumor cells, this study provides, to our knowledge, the first evidence that (pre)malignant cells themselves can acquire the ability to lyse epithelial cells via DNAM-1. This study confirms previous work on epithelial lysis by RCD cell lines and identifies a novel mechanism that potentially contributes to the gluten-independent tissue damage in RCD II and RCD-associated lymphoma.

Defective synthesis or association of T-cell receptor chains underlies loss of surface T-cell receptor-CD3 expression in enteropathy-associated T-cell lymphoma.

Enteropathy-associated T-cell lymphoma, an often fatal complication of celiac disease, can result from expansion of aberrant intraepithelial lymphocytes in refractory celiac disease type II (RCD II). Aberrant intraepithelial lymphocytes and lymphoma cells are intracellularly CD3epsilon(+) but lack expression of the T-cell receptor (TCR)-CD3 complex on the cell surface. It is unknown what causes the loss of TCR-CD3 expression. We report the isolation of a cell line from an RCD II patient with the characteristic phenotype of enteropathy-associated T-cell lymphoma. We demonstrate that in this cell line the TCR-alpha and -beta chains as well as the CD3gamma, CD3delta, CD3epsilon, and zeta-chains are present intracellularly and that assembly of the CD3gammaepsilon, CD3deltaepsilon, and zetazeta-dimers is normal. However, dimerization of the TCR chains and proper assembly of the TCR-CD3 complex are defective. On introduction of exogenous TCR-beta chains, but not of TCR-alpha chains, assembly and functional cell surface expression of the TCR-CD3 complex were restored. Defective synthesis of both TCR chains was found to underlie loss of TCR expression in similar cell lines isolated from 2 additional patients. (Pre)malignant transformation in RCD II thus correlates with defective synthesis or defective association of the TCR chains, resulting in loss of surface TCR-CD3 expression.