Presence of innate lymphoid cells in allogeneic hematopoietic grafts correlates with reduced graft-versus-host disease.

BACKGROUND Allogeneic hematopoietic cell transplantation (HCT) can be devastating when graft-versus-host disease (GvHD) develops. GvHD is characterized by mucosal inflammation due to breaching of epithelial barriers. Innate lymphoid cells (ILCs) are immune modulatory cells that are important in the maintenance of epithelial barriers, via their production of interleukin (IL)-22 and their T cell suppressive properties. After chemo- and radiotherapy, ILCs are depleted, and recovery after remission-induction therapy and after allogeneic HCT is slow and incomplete in a significant number of patients, which is associated with an increased risk to develop acute GvHD. OBJECTIVE To investigate whether the presence of mature ILCs within G-CSF-mobilized HCT grafts is correlated with the development of acute GvHD after allogeneic HCT. STUDY DESIGN We analyzed ILCs in a cohort of 36 patients who received allogeneic HCT for a hematologic malignancy, by flow-cytometric immune-phenotyping of prospectively collected, cryopreserved peripheral blood mononuclear cells (PBMCs) and donor-derived HCT grafts collected for the same patients. Biased analysis, with ILCs defined as CD3-lineage-CD45+CD127+CD161+ lymphocytes, was performed using FlowJo version 10 software. Unbiased analysis was done using FlowSOM, which uses a self-organizing map (SOM) with a minimal spanning tree (MST) to define and visualize different clusters present in the samples. RESULTS Remission-induction therapy significantly depleted ILCs from the blood, and patients who had a relatively low percentage of ILCs before allogeneic HCT were significantly more prone to develop acute GvHD, confirming previous findings in a separate cohort. Allogeneic HCT grafts, which were all obtained from the blood of G-CSF-mobilized healthy donors, contained ILCs at a frequency very similar to the peripheral blood of healthy individuals. The ILC subset composition was also comparable to that of the blood of healthy individuals, with the exception of NKp44+ ILC3s, which were significantly more abundant in HCT grafts. The relative ILC content of the graft tended to correlate with ILC reconstitution after allogeneic HCT, suggesting that peripheral expansion of transplanted mature ILCs may contribute to early ILC reconstitution after allogeneic HCT. Patients who received a relatively ILC-poor HCT graft had a significantly increased risk to develop acute GvHD, compared with patients who received relatively ILC-rich allogeneic HCT grafts. Unbiased phenotypic analysis with the FlowSOM algorithm confirmed that allogeneic HCT grafts of patients who developed acute GvHD contained a lower frequency of ILCs that clustered in NKp44+ ILC3 signature groups. CONCLUSION The presence of ILCs in allogeneic HCT grafts is associated with a reduced risk to develop acute GvHD. These data suggest that enhancement of ILC reconstitution of ILC3s in particular, for example via adoptive transfer of ILCs, may prevent acute GvHD and has the potential to improve outcome of allogeneic HCT recipients

Exploratory Study of Predicted Indirectly ReCognizable HLA Epitopes in Mismatched Hematopoietic Cell Transplantations

HLA-mismatches in hematopoietic stem-cell transplantation are associated with an impaired overall survival (OS). The aim of this study is to explore whether the Predicted Indirectly ReCognizable HLA-Epitopes (PIRCHE) algorithm can be used to identify HLA-mismatches that are related to an impaired transplant outcome. PIRCHE are computationally predicted peptides derived from the patient’s mismatched-HLA molecules that can be presented by donor-patient shared HLA. We retrospectively scored PIRCHE numbers either presented on HLA class-I (PIRCHE-I) or class-II (PIRCHE-II) for a Dutch multicenter cohort of 103 patients who received a single HLA-mismatched (9/10) unrelated donor transplant in an early phase of their disease. These patients were divided into low and high PIRCHE-I and PIRCHE-II groups, based on their PIRCHE scores, and compared using multivariate statistical analysis methods. The high PIRCHE-II group had a significantly impaired OS compared to the low PIRCHE-II group and the 10/10 reference group (HR: 1.86, 95%-CI: 1.02–3.40; and HR: 2.65, 95%-CI: 1.53–4.60, respectively). Overall, PIRCHE-II seem to have a more prominent effect on OS than PIRCHE-I. This impaired OS is probably due to an increased risk for severe acute graft-vs.-host disease. These data suggest that high PIRCHE-II scores may be used to identify non-permissible HLA mismatches within single HLA-mismatched hematopoietic stem-cell transplantations

Lyse or not to lyse: Clinical significance of red blood cell autoantibodies

Autoimmune hemolytic anemia (AIHA) is a rare autoimmune disease characterized by a hemolytic anemia caused by autoantibodies against red blood cells (RBCs). These autoantibodies are routinely detected via the direct antiglobulin test (DAT). As expected, the DAT score correlates with the presence of clinical symptoms, but this correlation is far from perfect. Regularly, strongly positive DAT scores are encountered with no sign of hemolysis, while severe hemolysis can be seen even in patients with a negative DAT score. Apparently, the mere amount of antibody is not the sole predictor of disease. In this paper, we review the current literature on aspects of both the autoantibodies and the patients' clearance system that together determine the clinical significance of an anti-RBC autoantibody, ranging from antibody isotype to antibody Fc-glycosylation to alternative clearance mechanisms. From this, the ensemble of tests is inferred that in our view best assesses the main determinants for pathogenicity of autoantibodie

Evaluation of a Platelet Function Analyser (PFA-100) in patients with a bleeding tendency

To investigate pre-analytical variables and the diagnostic performance of the platelet function analyser (PFA-100), a new device to test primary haemostasis in vitro by simulating platelet adhesion and aggregation under high shear stress. Venous whole citrated blood is aspirated through a capillary towards an aperture of a collagen coated membrane containing either adenosine diphosphate (ADP) or epinephrine (EPI). The time needed for occluding this aperture by plug formation is called closure time (CT) and was assessed in 70 healthy subjects and 43 patients with a suspected mild bleeding disorder. The reference range for the PFA-100 was found to be 82-159 s for EPI-CT and 62.5-120.5 s for ADP-CT. Duplicate analyses revealed a mean coefficient of variations of 7.1% (EPI-CT) and 5.7% (ADP-CT). The EPI- and ADP-CT of blood samples collected in the evening were significantly longer (p = 0.002 and p = 0.004, respectively) than the CT of blood samples collected in the morning. Acetylsalicylic acid(100 mg, 300 mg or 500 mg) administered as a single dose or daily on 10 consecutive days resulted in a prolongation of the EPI-CT, whereas the ADP-CT was not affected. EPI-CT was more sensitive in detecting acetylsalicylic acid (ASA) ingestion than was the bleeding time (BT). Sensitivity and specificity of the PFA-100 to detect von Willebrand disease (vWD) were comparable to the results obtained with the BT. The PFA-100 represents a simple and easy to use test for investigation of primary haemostasis. Limitations of the system are: special citrated whole blood has to be proceeded within 0.5 to 4 h after sampling, duplicate measurements are necessary, and the results differ between blood sampled in the morning or in the afternoon. The data indicate that the test is sensitive to ASA intake and vWD. Its use is preferable to BT determination, because it is less invasive and more sensitive to abnormalities of primary haemostasi

DAMPS and complement activation in platelet concentrates that induce adverse reactions in patients.

BACKGROUND Patients with severe thrombocytopenia due to bone marrow failure and after chemotherapy are still treated with platelet transfusions. Platelet concentrates (PC) are associated with a high incidence of adverse reactions (AR). Platelet-derived damage-associated molecular patterns (DAMPS) and complement were proposed to play a role in the pathology of AR. STUDY DESIGN AND METHODS Single donor apheresis platelet concentrates (SDA PCs) were produced in a regional setting of the French Blood Establishment. After transfusion samples were collected from PC and possible AR in patients were recorded. Platelet activation markers, High mobility group box 1 (HMGB1) and complement activation products (CAP) were measured. The correlation between platelet activation, and HMGB1 and complement activation was analyzed. RESULTS A total of 56 PC were included in the study. 30 PC induced no AR, and 26 induced AR (Febrile non-hemolytic transfusion reaction n = 16; Atypical Allergic Transfusion Reactions n = 11; hemodynamic instability n = 5) in the patients. The levels of P-selectin, sCD40L, HMGB1, C3b/c, and C4b/c were all significantly increased in PC that induced AR following transfusion in patients. Additionally, HMGB1, C3b/c, and C4b/c were positively correlated with P-selectin and sCD40L. CONCLUSION In this study, we observed an association between HMGB1 and CAP and the incidence of AR. Furthermore, we demonstrated that both HMGB1 and complement activation were correlated to platelet activation