Background: Natural killer cells (NK) are a component of innate immunity and are capable of cytotoxic lysis and cytokine secretion without prior antigen presentation. They are among the first to recover after transplantation, so they can play a decisive role in preventing early relapses and infectious complications. The killer immunoglobulin-like receptor (KIR), which is expressed on NK cells, mediates the immune response, both anti-tumor and directed against infectious agents. In the case of haploidentical hematopoietic stem cell transplantation (haplo-HSCT), if the donor has inhibitory KIR, to which the recipient has no ligand, donor NK

Introduction. Chronic graft versus host disease (GVHD) is a frequently occurring complication after transplantation of allogeneic hematopoietic cells associated with a decrease in the quality of life and long-term administration of immunosuppressive drugs. Extracorporeal photopheresis (ECP) is a second line of therapy after treatment failure with glucocorticoids. Aim — to evaluate the effects of ECP treatment in patients with glucocorticosteroids (GCS)-refractory, GCS-dependent or GCS -intolerant chronic GVHD. Materials and methods. 24 patients with GCS-refractory, GCS-dependent or GCS-intolerant chronic GVHD were included in the therapy with ECP. Nine patients had moderate chronic GVHD, and 15 had a severe chronic GVHD. Skin and mucous membranes were the most frequently targeted organs, 21 and 20 of 24 patients respectively, liver damage was detected in 8 patients. The maximum duration of treatment was 33 months (median — 8.5 months). The number of procedures of ECP ranged from 6 to 48 (median — 22). Results. A response was determined in 23 patients with one patient being excluded from the assessment due to a relapse of acute leukemia. 16 (69.5 %) patients achieved an overall response to ECP treatment. Three patients had complete response and full reduction of immunosuppressive therapy. When assessing organ-specific response, the most notable improvement was observed in the mucous membranes of 17 patients (89.4 %), skin — 17 (85 %), and liver — 7 (75 %). Along with achieving a general overall response, 78.2 % of patients partly reduced or completely canceled immunosuppressive therapy. Conclusion. ECP is an effective and promising second line treatment method for chronic GVHD in patients with GCS-refractory, GCS-dependent or GCS-intolerant forms.

Background: Natural killer cells (NK) can play a decisive role in preventing early relapses and infectious complications after haploidentical hematopoietic stem cell transplantation (haplo-HSCT) due to their comparatively fast reconstitution after HSCT. And when performing haplo-HSCT with depletion of TCRαβ-positive T-lymphocytes, the graft versus leukemia (GVL) effect is carried out only at the expense of donor NK cells. NK differentiate from immature (CD56bright) to mature (CD56dim) and terminally differentiated. NK differentiation to the subpopulation CD56dimCD16+ is necessary to implement an alloreactive NK cell immune response. Aims: To study the rate and features of NK cell reconstitution after haplo-HSCT. Methods: The study included 16 patients with acute myeloid leukemia (AML) and 13 patients with acute lymphoblastic leukemia (ALL) who underwent haplo-HSCT at the National Medical Research Center for Hematology from April 2020 to June 2021. 15 patients - with the use of post-transplant cyclophosphamide (PT-Cy), 14 - with previous TCRαβ-depletion. To assess the reconstitution of NK, all patients underwent immunophenotypic studies of peripheral blood samples at +14, +30, +60, +90 and +180 days after haplo-HSCT using a 12-color flow cytometer (CytoFlex Beckman Coulter, USA). Monoclonal antibodies to human differentiation antigens CD3, CD8, CD158a, CD158b, CD158e, CD159a, CD57, CD16, CD56, CD14, CD45, CD62L were used in the analysis of subpopulations of NK cells. The SAS 9.4 software (Sas institute inc., Cary, NC, USA) was used for statistical data processing. Differences were considered statistically significant at p<0.05. Results: NK reached a mature immunophenotype faster in the case of haplo-HSCT with TCRαβ-depletion: by day +30 after HSCT, the median proportion of CD56dimCD16+ NK cells with TCRαβ-depletion was 57% versus 9% when using PT-Cy, p= 0.0045. Significant differences persisted up to +60 (74% vs. 41% (p=0.0079)) and +90 days after haplo-HSCT (81% vs. 46% (p=0.0076)) (Fig. 1). Image:Summary/Conclusion: The rate of reconstitution and the acquisition of an alloreactive phenotype by NK depends on the variant of T-cell depletion; faster recovery occurs when performing haplo-HSCT with TCRαβ-depletion. When using PT-Cy, the recovery of the mature NK cell population occurs at a later time after haplo-HSCT (from +3 to + 6 months) and such patients may benefit from transfusions of donor NK cells at the post-transplant stage. This approach can be considered as a worthy alternative to donor.

Background:Programmed cell death protein 1 (PD‐1) is a surface receptor expressed normally on distinct immune cells. It's well known PD‐1 pathway blockade is a negative immune regulatory mechanism that has detrimental effects on anti‐tumor immunity. Currently some studies have revealed different immune cells overexpressed PD‐1 as a predictable marker of leukemia relapse. On the other hand its assay after allo‐HSCT could be controversial due to alternative GVHD prophylaxis regimens that might affect PD‐1 expression, such as post‐transplant high dose Cyclophosphamide (PT‐Cy) or graft‐manipulated TCR αβ‐depletion are taken place if mismatched or haploidentical donors are used.Aims:To evaluate PD‐1 expression on bone marrow (BM) resident CD8+ memory T‐cell subsets in leukemia patients after allo‐HSCT and its input on immunosuppressive mechanisms of different GVHD prophylaxis regimens after allo‐HSCT.Methods:BM samples were collected from 56 leukemia patients with a median age of 33 years on day +30, +60 and +90 after allo‐HSCT. Detailed patients characteristics are presented in Table 1. Flow cytometry analysis was performed on BD FACS Canto II (Becton Dickinson, USA) to define CD8+ T‐memory subsets with PD‐1 expression: T‐naive and T‐stem cell memory (Tnv+Tscm) –CD45R0‐CCR7+CD28+; T‐central memory (Tcm) ‐ CD45R0+CCR7+CD28+; T‐transitional memory (Ttm) ‐ CD45R0+CCR7‐CD28+; T‐effector memory (Tem) ‐ CD45R0+CCR7‐CD28‐; T‐terminal effector (Tte) ‐ CD45R0‐CCR7‐CD28‐. Sysmex XE‐2100 was used to calculate absolute count of different CD8+ T‐ memory cell subsets with PD‐1. Mann–Whitney U test was used for nonparametric data analysis. A p‐value less than 0.05 was considered as significant.Table 1. Patients characteristics.Results:During all follow‐up period PD‐1 expression remains increased on all CD8+ T‐memory cell subsets after TCR αβ‐depletion compared to PT‐Cy–based and classical immunosuppressive regimen based on Cyclosporine+MMF. On day +30 we observe significantly higher expression of PD‐1 on Tnv+scm, Tcm, Ttm after TCR αβ‐depletion compared to PT‐Cy or classical regimen (p < 0.05). On day +60 PD‐1 is still overexpressed on Tnv+scm after TCR αβ‐depletion compared to PT‐Cy or classical regimen (p = 0.008). Moreover on day +60 and +90 PD‐1 expression is elevated on terminally differentiated CD8+ T cells (Ttm, Tem) after TCR αβ‐depletion comparing PT‐Cy or classical regimen (р<0.05). (Figure 1).imageSummary/Conclusion:According to our data PD‐1 is overexpressed on distinct subsets of BM resident CD8+ T‐memory cells after TCR αβ‐depletion during follow‐up period in first 3 months after allo‐HSCT. Since other immune signaling pathways are inhibited due to the other immunosupressive agents had been used, PD‐1 pathway seems to be a key mechanism of immunological tolerance after haploidentacal allo‐HSCT with TCR αβ‐depletion.image

Aim. To assess the rehospitalization data of patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT), to determine possible risk factors for rehospitalization, and to work out a strategy of post-transplantation follow-up for this category of patients. Materials & Methods. From 2009 to 2019 at the National Research Center for Hematology 418 patients received allo-HSCT. The final analysis included 374 patients who were discharged from hospital after allo-HSCT. The reasons for rehospitalizations of patients with allo-HSCT within 30 days after their hospital discharge were subjected to analysis. Independent risk factors for rehospitalizations were identified by the Cox model. Risk density was visually estimated within 365 days after hospital discharge with the purpose of working out the optimal strategy of post-transplantation follow-up for this category of patients. Results. The probability of rehospitalization within 30 days after hospital discharge was 30.7 % for all patients with allo-HSCT. The data assessment showed that the majority of rehospitalizations (55.7 %) were associated with infectious complications. Acute graft-versus-host disease (GVHD) during the first hospitalization, i.e. immediately after allo-HSCT during the hospital stay, proved to enhance the probability of rehospitalizations within 30 days after hospital discharge by 1.7 times compared with the patients without acute GVHD. Conclusion. The leading cause of rehospitalizations of patients with allo-HSCT within 30 days after hospital discharge was acute GVHD which occurred before, i.e. during the first hospital stay. The data obtained demonstrate the necessity of close monitoring of a patient’s status within the first 120 days after discharge from the hospital where allo-HSCT was performed.

Background:Primary and secondary graft failure (GF) following allogeneic hematopoietic stem cell transplantation (allo‐HSCT) leads to prolonged neutropenia, infection complications and as a consequence lowered overall survival. Bone marrow as a graft source, HLA‐mismatched donor is a main factors which can influence GF in acute leukemia patients (pts) during first six months after allo‐HSCT. Nevertheless not all GF episodes can be explained by this factors. Here we report data about influence of spleen size (length width) assessed by transabdominal ultrasound on graft failure in first 6 months in acute leukemia patients in CR who underwent allo‐HSCT.Aims:Identify risk factors for GF in patients with acute leukemia remission.Methods:We analyzed medical records of 143 pts with acute leukemia (AML n = 100 (69.9%), ALL n = 43 (30.1%)) who underwent allo‐HSCT between 2013 and 2018 at National Research Center for Hematology and was alive for more than 28 days after allo‐HSCT. A median age of patients was 34 (19‐66) years, 66 (46.2%) males and 77 (53.8%) females. At the time of transplantation 124 (86.7%) pts were in complete remission and 19 (13.3%) pts had advanced disease. Most pts (n = 112; 78.3%) received reduced intensity conditioning regimen (RIC) and 31pts (21.7%) underwent myeloablative conditioning (MAC). Donors were: HLA‐matched unrelated (MRD) – 43.4% (n = 62); mismatched unrelated donor (MMUD) 27.3% (n = 39); matched related donor (MRD) ‐25.9% (n = 37); haploidentical (excluding TCR ab‐depletion) – 3.4% (n = 5), Bone marrow (BM) was used as a graft source in 60.1% (86 pts) and peripheral blood stem cell (PBSC) ‐ in 39.9% (57 pts). Spleen size was routinely evaluated before allo‐HSCT using transabdominal sonography. Cox Proportional‐Hazards model was used to identify influence of spleen size (≥3600 mm2 vs <3600 mm2) as independent prognostic factor (conditioning regimen, donor type, graft source, HLA‐disparity were also entered as covariates as well known risk factors for GF).Results:Results of Cox Proportional‐Hazards model are shown on Figure 1. According to our data spleen size (length width) ≥3600 mm2 assessed by transabdominal sonography before allo‐HSCT was associated with high risk of GF within six months of follow up period after allo‐HSCT.HR‐3.67 (95% Cl, 1.05‐12.74, p = 0.04).Figure 1. Results of Cox Proportional‐Hazards modelSummary/Conclusion:HLA‐disparity, BM as a graft source is characterized by increased probability of GF. In addition we identify that spleen size ≥3600 mm2 assessed by transabdominal sonography before allo‐HSCT also increased GF (up to 3.67 times). Due to that fact it's not recommended to use BM as a graft source for acute leukemia patients who underwent allo‐HSCT especially from MMUD.image

Background:Cytomegalovirus (CMV) is the opportunistic infection that persists in healthy individuals asymptomatically and reactivates in immunodeficient host, e.g. after allo‐HSCT. The main cause of viral reactivation is the lack of CMV‐specific T cells. The adoptive cellular therapy with CMV‐specific T cells seems very attractive clinical option to accelerate virus‐specific T‐cell reconstitution after allo‐HSCT. Here we report the preliminary data about CMV‐specific T‐cell reconstitution in allo‐HSCT patients.Aims:To identify a group of patients after allo‐HSCT who are in high‐risk of CMV reactivation and could be eligible for prophylaxis with CMV‐specific T cells.Methods:To detect CMV‐specific CD8+ cells we utilized MHC I tetramers loaded with the two immunodominant epitopes of viral pp65 protein: NLVPMVATV (NLV) and TPRVTGGGAM (TPR) presented in HLA‐A∗02 and ‐B∗07 respectively.We analyzed CD3+CD8+ cytotoxic T‐lymphocytes (CTLs) in peripheral blood and bone marrow of 7 patients on day +30 after allo‐HSCT. All patients were CMV‐seropositive. 5 patients were HLA‐A∗02 positive and 2 HLA‐B∗07 positive.Leukocyte cell suspension (5∗106 white blood cells per test) after preliminary red blood cells lysing with Lysing Buffer (BD Pharm Lyse™) were incubated with the protein tyrosine kinase inhibitor dasatinib for 1 hr at 37°С to increase surface expression of both TCR and CD8. NLV/TPR‐ specific cytotoxic T‐lymphocytes (CTLs) were identified by flow cytometry using PE‐Cy7 labeled anti‐CD45 antibody (Ab), Alexa Fluor 700 labeled anti‐CD3 Ab and PerCP‐Cy5.5 labeled anti‐CD8 Ab (Sony Biotechnology Inc.) and in house produced MHC class I tetramers conjugated with Phycoerythrin and loaded with NLV or TPR peptides. Alexa Fluor™ 750 NHS Ester (Succinimidyl Ester) was used (Invitrogen™) to exclude dead cells. Flow cytometry was performed on a BD FACSCanto™ II Flow Cytometer using BD FACSDiva™ software. Two‐platform method was used to calculate absolute CMV‐specific CD3+CD8+ count in peripheral blood.Results:According to our data, the lowest rates of CMV‐specific CTLs are presented in patients who underwent allo‐HSCT with post‐transplant high‐dose cyclophosphamide (PT‐Cy, 50 mg/kg on +3, +4 day) as GVHD prophylaxis.Results are presented in Table 1.Summary/Conclusion:Clinical option to accelerate virus‐specific T‐cell reconstitution using adoptive cellular therapy with CMV‐specific T cells seems very attractive. Modern technologies make this possible but first we should identify the patients after allo‐HSCT who benefit from this effective but very expensive therapy. Patients with using PT‐Cy as GVHD prophylaxis are the possible candidates for this treatment. Further studies CMV‐specific T‐cell reconstitution will help to expand the group of patients in high risk.image

Incidence, severity, and risk factors for hemorrhagic cystitis (HC) were assessed in 267 patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). HC was diagnosed in 14.6% (39 patients) within 1-139 days after allo-HSCT (median duration – 39 days). Chemotherapy-related HC was diagnosed in 4 patients only. The majority (19⁄35) of patients developed late HC of viral aetiology. Median time from a day of HC diagnosis to clinical symptoms resolution was 25 days (range: 6 to 133 days). Using a multivariate analysis, allo-HSCT from mismatched unrelated/haploidentical donor was found to be a risk factor of HC (р=0.01). The analysis also showed that 82.1% of patients with HC received cyclophosphamide as a part of conditioning regimen or +3/+4 days after allo-HSCT.

Introduction: Hematopoetic stem cell transplantation (HSCT) is the only curative therapy for many patients with hematologic malignancies. Occurrence of complications and mortality after allo HSCT is still high and it's strongly associated with immune reconstitution. Despite the wide-spread of Post-Transplant High-Dose Cyclophosphamide (PTCy) immune reconstitution and immunological safety of this method is still poorly understood.